WO2011154503A1 - Microbiological production of c4 bodies from saccharose and carbon dioxide - Google Patents

Microbiological production of c4 bodies from saccharose and carbon dioxide Download PDF

Info

Publication number
WO2011154503A1
WO2011154503A1 PCT/EP2011/059619 EP2011059619W WO2011154503A1 WO 2011154503 A1 WO2011154503 A1 WO 2011154503A1 EP 2011059619 W EP2011059619 W EP 2011059619W WO 2011154503 A1 WO2011154503 A1 WO 2011154503A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
encoded
catalyzes
enzyme
coa
Prior art date
Application number
PCT/EP2011/059619
Other languages
German (de)
French (fr)
Inventor
Steffen Schaffer
Nicolas Rudinger
Liv Reinecke
Original Assignee
Evonik Degussa Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Evonik Degussa Gmbh filed Critical Evonik Degussa Gmbh
Publication of WO2011154503A1 publication Critical patent/WO2011154503A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1223—Phosphotransferases with a nitrogenous group as acceptor (2.7.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2431—Beta-fructofuranosidase (3.2.1.26), i.e. invertase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01038—Malate dehydrogenase (oxaloacetate-decarboxylating) (1.1.1.38)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/0104—Malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+) (1.1.1.40)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01083—D-Malate dehydrogenase (decarboxylating) (1.1.1.83)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/05—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with a quinone or similar compound as acceptor (1.2.5)
    • C12Y102/05001—Pyruvate dehydrogenase (quinone) (1.2.5.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01054—Formate C-acetyltransferase (2.3.1.54), i.e. pyruvate formate-lyase or PFL
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/01004—Fructokinase (2.7.1.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/01069—Protein-Npi-phosphohistidine-sugar phosphotransferase (2.7.1.69), i.e. sucrose phosphotransferase system II
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/03—Phosphotransferases with a nitrogenous group as acceptor (2.7.3)
    • C12Y207/03009—Phosphoenolpyruvate-protein phosphotransferase (2.7.3.9)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01026—Beta-fructofuranosidase (3.2.1.26), i.e. invertase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01031—Phosphoenolpyruvate carboxylase (4.1.1.31)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01032—Phosphoenolpyruvate carboxykinase (GTP) (4.1.1.32)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01038—Phosphoenolpyruvate carboxykinase (diphosphate) (4.1.1.38)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01049—Phosphoenolpyruvate carboxykinase (ATP) (4.1.1.49)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y604/00—Ligases forming carbon-carbon bonds (6.4)
    • C12Y604/01—Ligases forming carbon-carbon bonds (6.4.1)
    • C12Y604/01001—Pyruvate carboxylase (6.4.1.1)

Definitions

  • the present invention relates to a cell which has been genetically engineered with respect to its wild type such that it produces more C 4 bodies and / or over these C 4 bodies compared to their wild type of sucrose and carbon dioxide as the carbon source
  • the invention further relates to a process for the production of such a genetically modified cell and to a process for the production of C 4 bodies and / or of secondary compounds produced via these C 4 bodies with the aid of these cells. Finally, the invention also relates to the use of the cells for the production of C 4 bodies bodies and / or derived from these C 4 body compounds of sucrose and carbon dioxide.
  • C 4 -body such as succinate, malate, fumarate, oxaloacetate, aspartate, asparagine, threonine, tetrahydrofuran, pyrrolidone, acetoin, 4,4-Bionell, hydroxysuccinate, epoxy-v-butyrolactone, butenoic acid, butyrate, butanediol, 1, 2 Butanediol, 1, 3-butanediol, 1, 4-butanediol, 2,3-butanediol,
  • C 4 bodies Feed industry, agriculture and the pharmaceutical industry of importance.
  • these C 4 bodies are also used as raw materials of the chemical industry for mass-produced products, such as polymers and solvents, or specialty chemicals. Due to the foreseeable shortage of petrochemical raw materials and increasing demand for products based on renewable raw materials, it is desirable alternatives to the existing petrochemical processes for the production of C 4 bodies
  • the currently pursued approaches are based mainly on microorganisms that produce C 4 bodies in fermentative processes.
  • glucose is used as carbon source.
  • the necessary glucose is recovered from starch, which z. From corn,
  • the present invention is therefore based on the object to overcome the disadvantages resulting from the prior art.
  • the invention has the object to replace glucose as a raw material by a cheaper and available in sufficient quantities alternative.
  • sucrose is used as a raw material for the production of C 4 bodies.
  • sucrose is mostly made from sugar cane (about% of annual production) and to a lesser extent from sugar beet (about% of annual production). Due to the high sucrose production from sugarcane in South America and Asia and less competition with the food industry, sucrose is available in large quantities and cheaper than glucose.
  • carbon dioxide CO 2
  • carbon dioxide is a by-product of a number of chemical processes and is therefore comparatively inexpensive to obtain.
  • carbon dioxide is a by-product of a number of chemical processes and is therefore comparatively inexpensive to obtain.
  • Carbon dioxide reduce the pollution of the atmosphere with greenhouse gases.
  • Anaerobiospirillum succiniciproducens or Actinobacillus succinogenes which are able to convert sucrose into C 4 bodies, are not suitable for biotechnological production because the yields are low and these organisms require complex nutrient media which, due to the associated costs and effort, render the process uneconomic ,
  • Sucrose and C0 2 as carbon sources can produce large quantities of C 4 bodies.
  • the inventors of the present invention have surprisingly found that the yield of C 4 bodies is fixed by genetic manipulation of suitable cells, in particular an enhancement of carboxylation reactions, the carbon dioxide and formation lead from C 4 -Körpern, a suppression of metabolic pathways that cause the carbon flux of sucrose, to fermentation products that are not C 4 -body, and increasing uptake of sucrose and the sucrose metabolism can be greatly increased.
  • This approach allows i) organisms that are usually unable
  • the present invention relates to a recombinant cell which has been genetically engineered with respect to its wild type to produce more C 4 bodies and / or over these C 4 bodies from sucrose and carbon dioxide as carbon sources compared to their wild-type Can form secondary connections.
  • C 4 body refers to a chemical
  • Compound containing four carbon atoms includes corresponding carboxylic acids, aldehydes, alcohols, sugars, alkanes, alkenes, amines and derivatives thereof.
  • Exemplary C 4 bodies within the meaning of the invention include, but are not limited to, succinate, malate, fumarate, oxaloacetate, aspartate, asparagine, threonine, tetrahydrofuran, pyrrolidone, acetoin, 4,4-bionell, hydroxysuccinate, epoxy-v-butyrolactone, Butenoic acid, butyrate, butanediol, 1, 2-butanediol, 1, 3-butanediol, 1, 4-butanediol, 2,3-butanediol, butene, n-butene, cis-2-butene, trans-2-butene, isobutene, Butadiene, 1,2-butadiene, 1,3-butad
  • the C 4 body is selected from the group consisting of: malate and oxaloacetate and / or secondary compounds prepared by enzymatic synthesis from these C 4 bodies, for example succinate, aspartate, asparagine, threonine, tetrahydrofuran, Butyrate, butanediol, 1, 2-butanediol, 1, 3-butanediol, 1, 4-butanediol, 2,3-butanediol, 3- and 4-hydroxybutyrolactone, 1-, 2-, and tert-butanol, isobutanol, 2- , 3- and 4-hydroxybutyric acid and 2- and 3-hydroxyisobutyric acid, methionine and lysine as well as malate, oxaloacetate and secondary compounds produced by chemical means compounds.
  • succinate aspartate
  • asparagine threonine
  • tetrahydrofuran Butyrate
  • butanediol 1, 2-butan
  • carboxylic acids include both the base form and the acid form, as well as mixtures of these forms.
  • succinate, malate, oxaloacetate, fumarate, aspartate, butyrate, 2-, 3- or 4-hydroxybutyric acid and 2- or 3-hydroxyisobutyric acid also each include succinic, malic, oxalacetic, fumaric, aspartic, butyric, 2-, 3- or 4-hydroxybutyrate and 2- or 3-hydroxyisobutyrate.
  • wild-type of the genetically modified cell does not form any C 4 bodies at all or does not form the desired C 4 body or that this compound (s) can not be produced in a detectable amount and only after the genetic modification verifiable amounts of these compounds are formed.Furthermore, this expression detects that the corresponding cell in a fixed period, for example within 2, 4 , 8, 12, 24 or 48 hours, at least the 2-, 5-, 10-, 100- or 1000-fold amount of C 4 bodies or one or more desired C 4 bodies forms like the wild type.
  • a "wild-type” cell is referred to herein as a cell whose genome is in a state as naturally evolved. The term is used for both the entire cell and for individual genes. The term “wild-type” therefore does not include, in particular, those cells or genes whose gene sequences have been altered at least in part by humans by means of recombinant methods.
  • the cells of the invention may be prokaryotes or eukaryotes. These may be mammalian cells (such as human cells), other animal cells (e.g.
  • Insect cells plant cells or microorganisms such as yeasts, fungi or bacteria, with microorganisms being preferred and bacteria and yeasts being particularly preferred.
  • bacteria, yeasts or fungi are those bacteria, yeasts or fungi which are deposited in the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ), Braunschweig, Germany, as bacterial, yeast or fungal strains.
  • Bacteria suitable according to the invention belong to the genera which can be found at http://www.dsmz.de/microorganisms/bacteria_catalogue.php are listed.
  • Yeasts which are suitable according to the invention belong to the genera listed under http://www.dsmz.de/microorganisms/yeast_catalogue.php.
  • Fungi suitable according to the invention are those listed at http://www.dsmz.de/microorganisms/fungus_catalogue.php.
  • Preferred cells according to the invention are those of the genera Aspergillus,
  • Fibrobacter succinogenes Ruminococcus flavefaciens, A. aerobiospirillum succiniciproducens, Mannheimia succiniciproducens, Actinobacillus succinogenes, Saccharomyces cerevisiae, Kluveromyces lactis, Candida blankii, Candida rugosa, Corynebacterium glutamicum,
  • Paracoccus versutus Pseudomonas aeruginosa, Acinetobacter calcoaceticus, Pichia pastoris, Thermoanaerobacter kivui, Acetobacterium woodii, Acetoanaerobium notera, Clostridium aceticum, acetobutylicum Butyribacterium methylotrophicum, Clostridium saccharoperbutylacetonicum Clostridium, Clostridium beijerinckii, butyricum Clostridium, Moor Ella thermoacetica, Eubacterium limosum, Peptostreptococcus productus, Clostridium ljungdahlii , Clostridium carboxidivorans, Clostridium scatalogenes, Rhodospirillum rubrum, Burkholderia thailandensis and Pseudomonas putida are particularly preferred.
  • carbon dioxide and “C0 2 " as used herein refer to both the gas C0 2 , the carbonic acid (H 2 C0 3 ) which is in equilibrium with dissolved carbon dioxide in aqueous solution, and the two carbonic acid deprotonation products , Bicarbonate (HC0 3 " ) and carbonate (C0 3 2" ) and their salts.
  • the C 4 bodies are selected from the group consisting of:
  • the C 4 bodies are selected from the group consisting of: malate, oxaloacetate and derived compounds produced by enzymatic or chemical synthesis via malate or oxalacetate.
  • the secondary compounds produced by enzymatic synthesis via malate or oxalacetate can be selected, for example, from the group consisting of: succinate, aspartate, asparagine, threonine, tetrahydrofuran, butyrate, butanediol, 1,2-butanediol, 1,3-butanediol, 1,4 Butanediol, 2,3-butanediol, 3- and 4-
  • Hydroxybutyrolactone 1-, 2- and tert-butanol, isobutanol, 2-, 3- and 4-hydroxybutyric acid, 2- and 3-hydroxyisobutyric acid, methionine and lysine.
  • the C 4 bodies are C 4 carboxylic acids, preferably C 4 dicarboxylic acids, more preferably succinate.
  • the C 4 bodies are hydroxycarboxylic acids, more preferably 2-, 3- and / or 4-hydroxybutyric acid and 2- and / or 3-hydroxyisobutyric acid.
  • the C 4 bodies are preferably malate and / or oxaloacetate.
  • the recombinant cell is a microbial cell, in particular an Escherichia coli, Alcaligenes latus, Bacillus megaterium, Bacillus subtilis, Brevibacterium flavum, Brevibacterium lactofermentum, Escherichia coli, Basfia succiniciproducens, Wollinella succinogenes, Fibrobacter succinogenes, Ruminococcus flavefaciens , Anaerobiospirillum succiniciproducens, Mannheimia succiniciproducens, Actinobacillus succinogenes, Corynebacterium glutamicum, Corynebacterium efficiens,
  • Zymonomas mobilis Methylobacterium extorquens, Ralstonia eutropha, Saccharomyces cerevisiae, Rhodobacter sphaeroides, Paracoccus versutus, Pseudomonas aeruginosa, Acinetobacter calcoaceticus, Clostridium acetobutylicum, Clostridium
  • saccharoperbutylacetonicum Clostridium beijerinckii, Rhodospirillum rubrum, Burkholderia thailandensis or Pseudomonas putida cell.
  • the recombinant cell of the present invention may be genetically engineered to accommodate more sucrose compared to its wild-type.
  • Another possibility, which may alternatively or additionally be used, is a genetic modification of the cell which allows the recombinant cell to fix more carbon dioxide compared to its wild-type.
  • the increase in sucrose uptake is achieved by virtue of the cell having an increased activity of at least one enzyme which catalyzes the transport of sucrose into the cell compared to its wild-type.
  • At least one refers to amounts of> 1, ie 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 22, 24, 26, 28, 30 or more, in particular 1, 2, 3, 4 or 5.
  • the recombinant cell of the invention comprises an enzyme E-i, which catalyzes the transport of sucrose into the cell and the conversion to sucrose-6-phosphate.
  • enzyme or "E x " as used herein, wherein x is an integer, is meant a protein or protein complex that catalyzes one or more biochemical reactions and / or the transport of certain compounds , For example, by a membrane, is used.
  • enhanced activity of an enzyme or “decreased activity of an enzyme” as used herein preferably refer to increased / decreased intracellular or membrane-bound activity.
  • increased or decreasing the enzyme activity in cells apply both to the increase / decrease in the activity of the enzyme E-1 and to all the enzymes mentioned below, the activity of which may optionally be increased or decreased.
  • an increase in enzymatic activity can be achieved by increasing the copy number of the gene sequence or gene sequences which code for the enzyme, using a strong promoter, changing the codon usage of the gene, in various ways the half-life of the mRNA or of the enzyme increases, eliminates repression, prevents inhibition or infiltrates a gene or allele, or manipulates the species to encode a corresponding enzyme with enhanced activity. If necessary, these measures will be combined.
  • Genetically engineered cells according to the invention are produced for example by transformation, transduction, conjugation or a combination of these methods with a vector which contains the desired gene, an allele of this gene or parts thereof and a vector which enables expression of the gene.
  • heterologous expression is achieved by integration of the gene or alleles into the chromosome of the cell or an extrachromosomally replicating vector.
  • Protein separations between wild type and genetically engineered cell can be determined.
  • a common method for preparing the protein gels in coryneform bacteria and for identifying the proteins is that described by Hermann et al. (Electrophoresis, 22: 1712.23 (2001)).
  • the protein concentration can also be determined by Western Blot hybridization with an antibody specific for the protein to be detected (Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY USA, 1989) and subsequent optical evaluation with
  • DNA-binding proteins can be measured by DNA band shift assays (also referred to as gel retardation) (Wilson et al., (2001) Journal of Bacteriology, 183: 2151-2155).
  • DNA band shift assays also referred to as gel retardation
  • the effect of DNA-binding proteins on the expression of other genes can be demonstrated by various well-described methods of the reporter gene assay (Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd Ed Cold Spring Harbor Laboratory Press, Cold Spring Harbor). NY USA, 1989).
  • the intracellular enzymatic activities can be determined by various methods described (Donahue et al., (2000) Journal of Bacteriology 182 (19): 5624-5627, Ray et al., (2000) Journal of Bacteriology 182 (8): 2277-2284, Freedberg et (1973) Journal of Bacteriology 1 15 (3): 816-823). Unless specific methods for determining the activity of a specific enzyme are specified in the following, the determination of the increase in the enzyme activity and also the determination of the reduction of an enzyme activity are preferably carried out by means of the methods described in Hermann et al. (Electophoresis, 22: 1712-23 (2001)), Lohaus et al.
  • Nucleotide substitution results in genetically engineered cells.
  • Particularly preferred mutants of enzymes are, in particular, also those enzymes which no longer diminish or at least in comparison with the wild-type enzyme
  • the increase in enzyme activity is accomplished by increasing the expression of an enzyme, for example, one increases the copy number of the corresponding genes or mutates the promoter and regulatory region or the ribosome binding site, which is upstream of the structural gene.
  • expression cassettes act, which are installed upstream of the structural gene.
  • Inducible promoters also make it possible to increase expression at any time.
  • enzyme activity is also enhanced.
  • the genes or gene constructs are either present in plasmids with different copy numbers or are integrated and amplified in the chromosome. Alternatively, a further
  • Vectors can, for. B. the brochures of the companies Novagen, Promega, New England Biolabs, Clontech or Gibco BRL be removed. Further preferred plasmids and vectors can be found in: Glover, DM (1985), DNA cloning: a practical approach, Vol. I-III, IRL Press Ltd., Oxford; Rodriguez, RL and Denhardt, D.T. (eds) (1988), Vectors: a survey of molecular cloning vectors and their uses, 179-204, Butterworth, Stoneham; Goeddel, DV (1990), Systems for heterologous gene expression, Methods Enzymol. 185, 3-7; Sambrook, J .; Fritsch, EF and Maniatis, T.
  • plasmid vectors such as pZ1 (Menkel et al., Applied and Environmental Microbiology 64: 549-554 (1989)), pEKEx1 (Eikmanns et al., Gene 107: 69-74 (1991)) or pHS2-l ( Sonnen et al., Gene 107: 69- 74 (1991)) are based on the cryptic plasmids pHM1519, pBL1 or pGA1.
  • plasmid vectors such as those based on pCG4 (US 4,489,160) or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66: 1 19-124 (1990)) or pAG1 (US 5,158,891) can be used in the same way be used. Also suitable are those plasmid vectors with the aid of which one can apply the method of gene amplification by integration into the chromosome, as described for example by Reinscheid et al. (Applied and Environmental Microbiology 60: 126-132 (1994)) for duplication or amplification of the homodB operon.
  • the complete gene is cloned into a plasmid vector which can be replicated in a host (typically Escherichia coli) but not in Corynebacterium glutamicum.
  • vectors include pSUP301 (Simon et al., Bio / Technology 1: 784-791 (1983)), pK18mob or pK19mob (Schäfer et al., Gene 145: 69-73 (1994)), pGEM-T (Promega Corporation , Madison, Wisconsin, USA), pCR2.1 -TOPO (Shuman, Journal of Biological Chemistry 269: 32678-84 (1994)), pCR ® Blunt (Invitrogen, Groningen, The Netherlands), pEM1 (shrink et al, Journal of. Bacteriology 173: 4510-4516)) or pBGS8 (Spratt et al., Gene 41: 337-342 (1986)).
  • the above-mentioned methods can analogously achieve a reduction in the enzymatic activity. This is also divided into two strategies, the reduction of expression and / or the inhibition of enzyme activity. To reduce the expression, for example, the corresponding gene can be completely or partially deleted. In addition, the
  • Transcription for example, by the manipulation of the promoter region or enhancement of repression (genetically or chemically) or the reduction of the mRNA half-life, be inhibited or reduced.
  • RNA level translation can be disturbed or reduced.
  • Numerous techniques are known to the person skilled in the art, for example the RNAi technology or the modification of the DNA sequence in that it is intended for Secondary structure formation occurs at the mRNA level, which inhibit or reduce translation.
  • the enzyme activity can be reduced, for example, by adding inhibitors, by introducing directional or non-directional mutations.
  • an activity of an enzyme E x which is increased with respect to its wild type is preferably always a factor greater than or equal to 2, particularly preferably at least 10, more preferably at least 100, and even more preferably of at least 1, 000 and most preferably of at least 10,000 increased activity of the respective enzyme E x
  • the cell according to the invention comprises "an activity of an enzyme E x increased compared to its wild type, in particular also a cell whose Wild type has no or at least no detectable activity of this enzyme E x and only after increasing the enzyme activity, for example by overexpression, a
  • detectable activity of this enzyme E x shows.
  • the term "overexpression” or the expression “increase in expression” used in the following also encompasses the case that a starting cell, for example a wild-type cell, has no or at least no detectable expression and only by recombinant methods a detectable Expression of the enzyme E x is induced.
  • the genetically modified cell is genetically modified in such a way that it is metabolized in a defined time interval, preferably within 2 hours, more preferably within a defined time interval 8 hours n and most preferably within 24 hours, at least 2 times, more preferably at least 10 times, more preferably at least 100 times, even more preferably at least I.OOO times, and most preferably at least 10,000 times more C 4 body forms than the wild type of the cell.
  • the increase in product formation can be determined, for example, by culturing the cell according to the invention and the wild-type cell separately under the same conditions (same cell density, same nutrient medium, same culture conditions) for a specific time interval in a suitable nutrient medium and then the amount Target product (C 4 body) is determined in the nutrient medium.
  • the enzyme E-1 can, for example, a phosphoenolpyruvate (PEP) -dependent
  • PPS Phosphotransferase system
  • the enzyme E-1 can be coded, for example, by the scrA gene.
  • the coding nucleotide sequence and the associated protein sequence can be found, for example, in the "Kyoto Encyclopedia of Genes and Genomes" (KEGG database), the National Library of Biotechnology Information (NCBI) databases of the National Library of Medicine (Bethesda, MD, USA), the protein database UniProt (cooperation of the European Bioinformatics Institute (EBI), the Swiss Institute of Bioinformatics (SI B) and the Protein Information Resource (PI R)) or the nucleotide sequence database of the European Molecular Biology Laboratories (EMBL, Heidelberg, Germany and Cambridge, In a particular case, egg is the scrA gene (SEQ ID NO: 41) derived from E.
  • the enzyme Ei is preferably encoded by genes derived from the Group of those are selected which encode gene products whose amino acid sequence over a range of at least 100, preferably at least 200, in particular minde at least 300 amino acids, at least 60%, preferably at least 80%, more preferably at least 95%, most preferably at least 99%, in particular 100% to SEQ ID NO: 58 is identical.
  • the enzyme E-1 is encoded by genes which are selected from the group of those encoding gene products in whose amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (Version 2.20) by means of RPS BLAST the presence of the conserved domain "PTS-II BC-sucr" (TIGR01996 or PssmI D 131051) with an E value (English “e-value”) less than 1 x 10 "5 detected (English” domain hit ”) , In addition to the enzyme egg, which facilitates the transport of sucrose into the cell and the
  • the recombinant cell may further have an increased compared to their wild type activity of at least one enzyme E 2 , E 3 and E 4 or a combination of these enzymes.
  • E 2 can be an enzyme that the
  • E 3 may be an enzyme that catalyzes the conversion of D-fructose to D-fructose-6-phosphate
  • E 4 may be a channel that allows diffusion-dependent sucrose transport into the cell.
  • the invention therefore relates to recombinant cells which have an increased activity of at least one of the enzymes E 2 , E 3 and E 4 in comparison to their wild type.
  • the activity of the enzyme Ei and at least one of the enzymes E 2 , E 3 and E 4 is increased compared to the wild type.
  • the activity of the enzymes compared to the wild-type is i) Ei, E 2 and E 3 , ii) E 2 , E 3 and E 4 , iii) E 2 and E 3 , iv) E 3 and E 4 , v) E 2 and E 4 , or vi) Ei, E 3 and E 4 increased.
  • the recombinant cell according to the invention has an increased activity of the enzymes Ei, E 2 , E 3 and E 4 in comparison with the wild type.
  • the enzyme E 2 may be a sucrose-6-phosphate fructohydrolase (EC 3.2.1 .26), the enzyme E 3 may be a fructokinase (EC 2.7.1 .4) and / or the Enzyme E 4 is a sucrose porin (TCDB Classification 1 .B.3.1 .2).
  • E 2 is encoded by the scrB (E. coli scrB: SEQ ID NO: 42), bfrA, sacA, sacB, cscA, fruA or susH gene (Streptococcus pneumoniae susH: SEQ ID NO: 52), E 3 is derived from the mac, yajF, mtlZ, rbsK, glcK, pfkB, frcK, frk, sacK, ydhR, kdgK, suk, ydjE, gmuE, ydjE, scrK (E coli scrK: SEQ ID NO: 43) or cscK gene (£ coli ⁇ S ⁇ K. SEQ ID NO: 46) encodes and e 4 is of the ScrY gene (£ coli ScrY. 44: SEQ ID NO) coded.
  • Protein sequences as well as other genes for the enzymes E 2 to E 4 can also be taken from the KEGG, NCBI, UniProt or EMBL database.
  • E 2 , E 3 and / or E 4 are genes derived from E. coli or the proteins encoded thereby.
  • the enzyme E 2 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, particularly preferably of at least 95%, very particularly preferably of at least 99%, in particular of 100%, to SEQ ID NO: 59.
  • the enzyme E 2 is encoded by genes which are selected from the group of those encoding gene products in their amino acid sequence in a search for conserved contained in the relevant amino acid sequence
  • the enzyme E 3 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, particularly preferably of at least 95%, very particularly preferably of at least 99%, in particular of 100%, to SEQ ID NO: 60 or SEQ ID NO: 63.
  • the enzyme E 3 is encoded by genes which are selected from the group of those encoding gene products whose amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (Version 2.20) by means of RPS-BLAST Presence of the conserved domain
  • the enzyme E 4 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, more preferably at least 95%, most preferably at least 99%, especially 100% to SEQ ID NO: 61.
  • the enzyme E 4 is encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST the presence of the conserved domain
  • the recombinant cell is characterized by the fact that the increased sucrose uptake compared to the wild type is brought about by the increased activity of a channel E 5 which sorbs sucrose into the cell.
  • the present invention also covers embodiments in which this increased sucrose symport is combined with the aforementioned increased activities of the enzymes Ei, E 2 , E 3 and / or E 4 .
  • the channel E 5 may be, for example, a sucrose permease.
  • E 5 is encoded by the cscB gene (for example, E. coli cscB: SEQ ID NO: 45).
  • the recombinant cell may have an increased activity compared to its wild type of at least one of the following enzymes: E 6 , which catalyzes the conversion of sucrose to D-glucose and D-fructose; and E 3 , which catalyzes the conversion of D-fructose to D-fructose-6-phosphate.
  • E 6 which catalyzes the conversion of sucrose to D-glucose and D-fructose
  • E 3 which catalyzes the conversion of D-fructose to D-fructose-6-phosphate.
  • the enzyme E 5 is a sucrose permease (TCDB classification 2.A.1 .5.3), the enzyme E 3 is a fructokinase (EC 2.7.1 .4). and the enzyme E 6 is a -D-fructofuranoside fructohydrolase (EC 3.2.1 .26).
  • the enzyme E 5 may be replaced by cscB (for example, E. coli cscB: SEQ ID NO: 45), lamB or scrY (for example, E. coli scrY: SEQ ID NO: 44), E 3 by mac, yajF, mtlZ, rbsK , glcK, pfkB, frcK, frk, sacK, ydhR, kdgK, suk, ydjE, gmuE, ydjE, scrK (for example, E. coli scrK: SEQ ID NO: 43) or cscK (for example, E.
  • coli cscK SEQ ID NO: 46
  • cscA for example E. coli cscA: SEQ ID NO: 47
  • susH for example Streptococcus pneumoniae susH: SEQ ID NO: 52
  • scrB for example E. coli scrB: SEQ ID NO: 42
  • rafD sacA, fruA or bfrA.
  • the nucleotide sequences of the abovementioned genes, their corresponding protein sequence and other genes for the enzymes E 3 , E 5 and E 6 can be taken from among others the KEGG, the NCBI, the UniProt or EMBL database. In a particular case, E 3 , E 5 and / or E 6 are of £. coli-derived genes and the proteins encoded thereby.
  • the enzyme E 5 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, particularly preferably of at least 95%, very particularly preferably of at least 99%, in particular of 100%, to SEQ ID NO: 62.
  • the enzyme E 5 is encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) using RPS-BLAST the presence of the conserved domain "LacY_symp” (PFAM domain 01306 or PssmID 1 10319) with an E value (English “e-value”) is smaller than 1 x 10 "5 found (English” domain hit ").
  • the enzyme E 6 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, particularly preferably of at least 95%, very particularly preferably of at least 99%, in particular of 100%, to SEQ ID NO: 64 or SEQ ID NO: 51.
  • the enzyme E 6 is encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST the presence of the conserved domain "SacC” (COG1621 or PssmID 31808) with an E value (English “e-value") smaller 1 x 10 "5 is found (English” domain hit ").
  • the increased sucrose uptake of the recombinant cell according to the invention is effected by having at least one increased activity of an enzyme complex which transports sucrose into the cell compared to its wild-type.
  • This sucrose-transporting enzyme complex can be, for example, from the
  • Enzymes E 7 , E 8 and E 9 and includes, for example, a sucrose-specific ABC transporter (TCDB classification 3.A.1 .1.-).
  • the enzyme E 7 can be replaced by a susT1 gene (for example
  • Streptococcus pneumoniae susT1 SEQ ID NO: 48
  • E 8 by a susT2 gene
  • E 9 by a susX gene
  • the cell having increased sucrose transporter activity may have an increased activity of at least one enzyme E 10 , E 2, E 3 or E 6 or any combination of these enzymes as compared to its wild-type.
  • the enzyme E 10 catalyzes the
  • sucrose-6-phosphate Conversion of sucrose to sucrose-6-phosphate, the enzyme E 2 the conversion of sucrose-6-phosphate to D-glucose-6-phosphate and D-fructose, the enzyme E 3 the conversion of D-fructose to - D-fructose 6-phosphate and the enzyme E 6 the conversion of sucrose to D-glucose and D-fructose.
  • the recombinant cell has an increased activity of the enzymes E 10 , E 2 , E 3 and E 6 compared to their wild type.
  • Enzyme E 10 may be a sucrose kinase, enzyme E 2 is a sucrose-6-phosphate fructohydrolase (EC 3.2.1.26), enzyme E 3 is a fructokinase (EC 2.7.1.4) and / or enzyme E 6 is a -D- Fructofuranoside fructohydrolase (EC 3.2.1 .26).
  • the enzyme E 10 can be replaced by a sucrose kinase gene, E 2 by a scrB (for example E. coli scrB: SEQ ID NO: 42), bfrA, sacA, sacB, cscA (for example E.
  • coli cscA SEQ ID NO : 47
  • fruA or susH gene and / or E 3 by a mac, yajF, mtlZ, rbsK, glcK, pfkB, frcK, frk, sacK, ydhR, kdgK, suk, ydjE, gmuE, ydjE, scrK for example E. coli scrK: SEQ ID NO: 43
  • cscK for example, E. coli cscK: SEQ ID NO: 46
  • E 2 , E 3 , E 7 , E 8 , E 9 and E 10 can also be found in the KEGG, NCBI, UniProt or EMBL database.
  • E 2 , E 3 , and / or E 10 are E. coli and E 7 , E 8 and / or E 9 are Streptococcus pneumoniae genes and the proteins encoded thereby.
  • the enzyme E 7 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, more preferably at least 95%, most preferably at least 99%, especially 100% to SEQ ID NO: 65.
  • the enzyme E 7 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has been searched for conserved in the relevant amino acid sequence
  • Protein domains in the NCBI CDD (Version 2.20) by means of RPS-BLAST the presence of the conserved domain "LplB” (COG4209 or PssmID 33938) with an E-value (English “e-value") smaller 1 x 10 "5 is found (English "Domain hit”).
  • the enzyme E 8 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, more preferably at least 95%, most preferably at least 99%, especially 100% to SEQ ID NO: 66.
  • the enzyme E 9 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, especially preferably of at least 95%, very particularly preferably of at least 99%, in particular of 100%, to SEQ ID NO: 67.
  • the enzyme E 9 is encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST the presence of the conserved domain "UgpB” (COG1653 or PssmID 31839) with an E value (English “e-value”) is less than 1 x 10 "5 is found (English" domain hit ").
  • embodiments in a recombinant cell according to the invention increase the activity of the following enzymes over the wild-type:
  • the activity is at least one, preferably
  • the recombinant cell is genetically engineered to have an increased activity of at least one enzyme that catalyzes the fixation of C0 2 to a C 3 body compared to its wild-type.
  • This heightened Carbon dioxide fixation may be alternative or in addition to an increase in sucrose transport into the cell.
  • the increase in sucrose transport can be accomplished by the techniques outlined above.
  • the at least one enzyme that catalyzes the fixation of C0 2 to a C 3 body may, in certain embodiments of the invention, be selected from the group consisting of the following enzymes:
  • E 12 which catalyzes the conversion of pyruvate, ATP and C0 2 to oxaloacetate, ADP and phosphate;
  • E 15 , E 16 , E 17 and E 18 which catalyze the reaction of pyruvate NAD (P) H + and C0 2 to malate and NAD (P) + .
  • Also encompassed by the present invention are any combination of increased activities of the enzymes E 12 -E 18 and the increased activity of all enzymes E 12 -E 18 .
  • any combination of increased activities of the enzymes E 12 -E 18 and the increased activity of all enzymes E 12 -E 18 are also encompassed by the present invention.
  • E 13 is a phosphoenolpyruvate carboxylase (phosphate: oxaloacetate carboxylase) (EC 4.1 .1 .31);
  • E 14 is a phosphoenolpyruvate carboxykinase (ATP / GTP / PPi: oxaloacetate carboxylase) (EC 4.1 .1 .32, EC 4.1 .1 .38 or EC 4.1 .1 .49);
  • E 15 a malate dehydrogenase ((S) -malate: NAD + oxidoreductase) (EC 1 .1 .1 .38);
  • E 16 a malate dehydrogenase ((S) -malate: NAD + oxidoreductase) (EC 1 .1 .1 .39);
  • E 17 a malate dehydrogenase ((S) -malate: NADP + oxidoreductase) (EC 1 .1 .1 .40);
  • E 18 is a D-malate dehydrogenase ((R) -malate: NAD + oxidoreductase) (EC 1 .1 .1 .83).
  • the enzyme E 12 may preferably be encoded by a gene selected from the group comprising cgl516, aarl62Cppyrl, pca, cgl0689, pc, pcx, pyc-1, pyc-2, accC-2, pycA, pycA2, pyc, pycB, pycB1, pycB2, accC, accA, oadA, pyr, acc and accC1, with the pyc gene (for example, E. coli pyc: SEQ ID NO: 5) being particularly preferred.
  • a gene selected from the group comprising cgl516, aarl62Cppyrl, pca, cgl0689, pc, pcx, pyc-1, pyc-2, accC-2, pycA, pycA2, pyc, pycB, pyc
  • Pyruvate carboxylases preferred according to the invention are also described in particular in US Pat. Nos. 6,455,284, 6,171,833, 6,884,606, 6,403,351, 6,852,516 and 6,861,246.
  • a pyruvate carboxylase pyc which is particularly preferred in this context is that mutant which is known in the novel methodology employing Corynebacterium glutamicum genome
  • pyc is derived
  • the enzyme E 13 may preferably be encoded by a gene selected from the group comprising ppc, capP, pepC and clpA, with the ppc gene being preferred.
  • ppc is from E. coli (SEQ ID NO: 1).
  • the enzyme E 14 may preferably be encoded by a gene selected from the group comprising pck, pckG, pckA, pckI, pck2 and pck, the pckA gene being particularly preferred.
  • pckA is from E. coli (SEQ ID NO: 2).
  • Phosphoenolpyruvate carboxylases preferred according to the invention are also described in particular in US Pat. Nos. 4,757,009, 4,980,285, 5,573,945, 6,872,553 and 6,599,732.
  • the disclosure of these references for phosphoenolpyruvate carboxylases is hereby incorporated by reference and forms part of the disclosure of the present invention.
  • the malate dehydrogenases E 15 , E 16 and E 17 may preferably be encoded by a gene selected from the group comprising me, me1, me2, me3, mae, mael, mae2, sfcA, sfcA1, maeA, maeB, maeB1, maeB2 , tme, yqkJ, ywkA, yqkJ, malS, ytsJ, mleA, mleS, mez, sce59.10c, 2sc7gll.23, malSI, malS2, dme, maeBl, maeB2, mdh, mdh1, mdh2, dmel cgi 0120, dmel cgi 0120 , dme1 -cg5889, fl9kl6.27, f6f22.7, t22p22.60, fl8al7.1, mod1, tme, mao, cgl300
  • E. coli maeA SEQ ID NO: 3
  • E 16 mmel for example Chlamydomonas reinhardti mmel: SEQ ID NO: 55
  • E 17 maeB for example E. coli maeB : SEQ ID NO: 4
  • mouse and mouse are from E. coli.
  • the malate dehydrogenase E 18 may preferably be encoded by a gene selected from the group comprising yeaU, ycsA, ttuC, ttuC1, ttuC2, ttuC3, tdh, leuB, leuB1 and dmlA, with yeaU and dmlA being particularly preferred.
  • dmlA is derived from £. coli (SEQ ID NO: 57).
  • the aforementioned enzymes may be individually, all or in any combination increased in their activity.
  • at least one, at least 2, 3, 4, 5, 6, or 7 enzymes of the group E 12 -E 18 can be increased in their activity.
  • Protein sequence and other genes for the enzymes E 12 -E 18 can also be found in the KEGG, the NCBI, the UniProt or EMBL database.
  • the enzyme E 12 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, more preferably at least 95%, most preferably at least 99%, especially 100% to SEQ ID NO: 68.
  • the enzyme E 12 is encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST the presence of the conserved domain "DRE_TIM_PC_TC_5S” (cd07937) or the conserved domain "pyruvate carboxylase” (PRK12999) with an E value (English “e-value") smaller than 1 x 10 "5 is found (English” domain hit ”) ,
  • the enzyme E 13 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, more preferably at least 95%, most preferably at least 99%, especially 100% to SEQ ID NO: 69.
  • the enzyme E 13 is encoded by genes selected from the group of those encoding gene products in an amino acid sequence in a search for conserved protein domains contained in the subject amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST Presence of the conserved domain "phosphoenolpyruvate carboxylase” (PssmID 166715 or COG3252 or c14574 or PRK00009) with an E value (English “e-value") less than 1 x 10 "5 is found (English” domain hit ").
  • the enzyme E 14 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, more preferably at least 95%, most preferably at least 99%, especially 100% to SEQ ID NO: 70.
  • the enzyme E 14 is encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST the presence of the conserved domain "PEPCK_ATP” (cd00484) with an E value (English “e-value") smaller 1 x 10 "5 is found (English” domain hit ").
  • the enzyme E 15 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, more preferably at least 95%, most preferably at least 99%, especially 100% to SEQ ID NO: 71.
  • the enzyme E 15 is encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST the presence of the conserved domain "NAD (P) binding domain of malic enzyme (ME), subgroup 1" (cd05312) with an E value (English “e-value") smaller 1 x 10 "5 is found (English” domain hit”).
  • the enzyme E 16 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, more preferably at least 95%, most preferably at least 99%, especially 100% to SEQ ID NO: 56.
  • the enzyme E 16 is encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST the presence of the conserved domain "malate dehydrogenase” (PRK13529) with an E value (English “e-value") smaller 1 x 10 "5 is found (English” domain hit ").
  • the enzyme E 17 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a range of at least 100, preferably at least 200, in particular at least 300 amino acids have a sequence identity of at least 60%, preferably of at least 80%, more preferably of at least 95%, most preferably of at least 99%, in particular of 100% to SEQ ID NO: 54.
  • the enzyme E 17 is encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST the presence of the conserved domain "NAD (P) binding domain of malic enzyme (ME), subgroup 2" (cd0531 1) with an E value (English “e-value”) smaller 1 x 10 "5 is found (English” domain hit ").
  • the enzyme E 18 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, particularly preferably of at least 95%, very particularly preferably of at least 99%, in particular of 100%, to SEQ ID NO: 53.
  • the enzyme E 18 is encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST the presence of the conserved domain "lso_dh super-family" (c00445 or PssmID 174206) with an E-value (English “e-value") smaller 1 x 10 "5 is found (English” domain hit ").
  • the recombinant cell can additionally be genetically engineered such that it has a reduced carbon flux from sucrose to fermentation products which are not C 4 bodies compared to their wild type.
  • This embodiment encompasses recombinant cells according to the invention which, in addition to their wild type, are characterized by producing less d, C 2 and / or C 3 by- products from sucrose.
  • the requisite genetic modification may be by manipulation of the recombinant cell, thereby reducing or disrupting the metabolism that produces the fermentation products.
  • the activity is at least one of the following
  • Ei9, E 2 o, E21 and E22 which are components of a glucose-specific phosphoenolpyruvate phosphotransferase system and, as such, catalyze the import of glucose and in sum the conversion of phosphoenolpyruvate and glucose to pyruvate and glucose B-phosphate;
  • E 2 3 and E 2 4 which catalyze the reaction of pyruvate and CoA to formate and acetyl-CoA;
  • E 2 which catalyzes the reaction of pyruvate, H 2 0 and ferricytochrome bi to acetate, C0 2 and ferrocytochrome bi;
  • E 2 6 which catalyzes the reaction of S-methylmalonyl-CoA to propanoyl-CoA and C0 2 ;
  • E 2 7 and E 2 8 which catalyze the reaction of acetyl phosphate and ADP / P, to acetate and ATP / PP;
  • E 42 which catalyzes the conversion of 2-methyl citrate to 2-methyl cis-aconitate or 2-methyl-trans aconitate and H 2 O;
  • E 43 which catalyzes the conversion of 2-methyl trans aconitate to 2-methyl cis aconitate;
  • E 44 which catalyzes the conversion of 2-methyl cis aconitate to succinate and pyruvate;
  • E 45 which catalyzes the conversion of propionyl phosphate and ADP to propionate and ATP;
  • E 48 which catalyzes the reaction of pyruvate and CoA 2 oxidized ferredoxins to acetyl CoA, C0 2 , and 2 reduced ferredoxins and 2 H + ;
  • E51 which catalyzes the reaction of (S) -methylmalonyl-CoA to succinyl-CoA
  • E 52 which catalyzes the conversion of 2 pyruvate to 2-acetolactate and C0 2 ;
  • E 6 o which catalyzes the reaction of acetoacetate and H + to acetone and C0 2
  • E 6 i which catalyzes the reaction of acetone and NAD (P) H + to propanol and NAD (P) + ;
  • E 6 which catalyzes the conversion of pyruvate and NADH to L-lactate and NAD + .
  • also several, ie at least 2, or all of the aforementioned enzymes may be reduced in their activity.
  • E 19 and E 2 o a PEP-dependent phosphotransferase system enzyme II (EC 2.7.1 .69);
  • E 2 i is a PEP-dependent phosphotransferase system Enzyme I (EC 2.7.3.9);
  • E 2 2 is a phosphohistidine protein (HPr) -hexose phosphotransferase component of the
  • E 2 3 and E 2 4 a formate C-acetyltransferase (EC 2.3.1.54);
  • E 26 is a methylmalonyl-CoA decarboxylase (EC 4.1 .1 .41);
  • E 27 an ATP / PPi: acetate phosphotransferase (EC 2.7.2.1 or EC 2.7.2.1);
  • E 28 is a propionate / acetate kinase (EC 2.7.2.15);
  • E 29 an acetyl-CoA: phosphate acetyltransferase (EC 2.3.1 .8);
  • E 30 is a D-lactate dehydrogenase (EC 1 .1 .1 .28);
  • E 31 an acetaldehyde dehydrogenase (CoA-acetylating) (EC 1 .2.1 .10);
  • E 32 an NAD-dependent alcohol dehydrogenase (EC 1 .1 .1 .1);
  • E 33 is a glycerone phosphate phospholyase (EC 4.2.3.3);
  • E 34 , E 35 , E 36 , E 37 , E 38 , E 39 and E 40 formate dehydrogenases (EC 1 .2.1 .2);
  • E 41 is a 2-methylcitrate synthase (EC 2.3.3.5);
  • E 42 a 2-methylcitrate dehydratase (EC 4.2.1 .79 or EC 4.2.1 .1 17);
  • E 43 is a 2-methylaconitate isomerase
  • E 44 is a methyl isocitrate lyase (EC 4.1 .3.30);
  • E 45 is a propionate kinase (EC 2.7.2.15);
  • E 46 a phosphate-propionyltransferase (EC 2.3.1 .8)
  • E 47 is a pyruvate decarboxylase (EC 4.1 .1 .1)
  • E 48 is a pyruvate: ferredoxin oxidoreductase (EC 1 .2.7.1);
  • E 49 is a phosphoketolase (EC 4.1 .2.9);
  • E 50 is a methylmalonyl-CoA-carboxytransferase (EC 2.1 .3.1);
  • E 52 an acetolactate synthase (EC 2.2.1.6);
  • E 53 an acetolactate decarboxylase (EC 4.1 .1 .5)
  • E 54 a butanediol dehydrogenase (EC 1 .1 .1 .4 or EC 1 .1 .1 .76);
  • E 55 a thiolase (EC 2.3.1 .9);
  • E 56 is a 3-hydroxybutyryl-CoA dehydrogenase (EC 1 .1 .1.157, EC 1 .1.1.35, EC 1.1 .1 .36 or EC 1.1 .1.21 1);
  • E 58 is a butanol dehydrogenase (EC 1.1 .1.1 or EC 1.1.1.2);
  • E 59 a butyraldehyde dehydrogenase (EC 1.2.1.3, EC 1 .2.1.4 or EC 1.2.1.5);
  • E 6 i is a propanol dehydrogenase (EC 1 .1 .1.1 or EC 1 .1 .1.2);
  • E 6 2 an acyl-CoA: CoA transferase (EC 2.8.3.-); and or
  • E 6 3 an L-lactate dehydrogenase (EC 1.1 .1 .27).
  • the cells according to the invention have a reduced activity compared to the wild type of at least one, preferably at least 2, more preferably at least 3, most preferably at least 5 of said enzymes.
  • the activity of at least one of the enzymes E19-E22 is reduced.
  • Such a reduction in enzyme activity may be beneficial as it eliminates the energy-consuming import and activation of glucose, thereby providing the cell with more energy to import and activate sucrose and to fix carbon dioxide.
  • the enzyme E 2 6 is preferably encoded by a gene selected from the group comprising ygfG, mmdA, oadB, oadB2, oadB3, SC1C2.16, SC1G7.10, pCCBI, mmdB, mmdC and ppcB, where the ygfG Gene is particularly preferred.
  • ygfG is from £. coli.
  • the cell has a reduced activity of at least one enzyme E 19 -E 45 compared to its wild type, wherein:
  • E 19 is encoded by a ptsG gene
  • E 2 o is encoded by a ptsl gene
  • E21 is encoded by a ptsH gene
  • E22 is encoded by a crr gene
  • E 2 3 is encoded by a tdcE gene
  • E 24 is encoded by a pflA or pflB gene
  • E 2 5 is encoded by a poxB gene
  • E 26 is encoded by a ygfG gene
  • E 27 is encoded by a ackA gene
  • E 28 is encoded by an ackA or tdcD gene
  • E 2 9 is encoded by a pta gene
  • E 30 is encoded by an IdhA gene
  • E is encoded by a gene adhE 31;
  • E 32 is encoded by an adhE gene
  • E 33 is encoded by a mgsA gene
  • E 34 is encoded by a fdnG gene
  • E is encoded by a gene fdnH 35;
  • E 36 is encoded by a fdnl gene
  • E 37 is encoded by a fdhF gene
  • E 38 is encoded by a fdoG gene
  • E 39 is encoded by a fdoH gene
  • E is encoded by a gene fdol 40;
  • E 41 is encoded by a prpC gene
  • E 42 is encoded by a prpD or acnD gene
  • E 43 is encoded by a prpF gene
  • E 44 is encoded by a prpB gene
  • E 45 is encoded by a tdcD gene
  • E 46 is encoded by a pta gene
  • E 47 is encoded by a pdc gene
  • E 48 is encoded by a porA, porB, porC or porD gene
  • E 49 is encoded by an xpkl or xpk2 gene
  • E 50 is encoded by a methylmalonyl CoA carboxytransferase gene
  • E 51 is encoded by a sbm or a mcmA and a mcmB gene;
  • E 52 is encoded by a gene as, ilvB, ilvM, ilvN, ilvG, ilvl or ilvH gene;
  • E 53 is encoded by a alsD gene
  • E 54 is encoded by a butBGen
  • E 55 is encoded by a thI, thIA, thIB or phaA gene
  • E 56 is encoded by a phaB gene
  • E 57 is encoded by a crt gene
  • E 58 is encoded by an adhE gene
  • E 59 is encoded by an adhE, bdhA or bdhB gene;
  • E 6 o is encoded by an ade gene;
  • E 6 i is encoded by an adh gene
  • E 6 2 is encoded by a ctfA and a ctfB or an atoA and an atoD gene and / or E 6 3 is encoded by an IdhL gene.
  • Protein sequence and other genes for the enzymes E 19 -E 6 3 can be found, inter alia, the KEGG, the NCBI, the UniProt or EMBL database.
  • the recombinant cell which, starting from sucrose and C0 2 as carbon sources, can produce more C 4 bodies than the wild type, has a reduced activity of at least one, at least two, at least 3, or their wild-type at least 4 of the enzymes E 19 , E 2 6, E 2 7, E 29 , E 30 , E 31 , E 32 and E 46 on.
  • the enzymes E 19 , E 26 , E 27 , E 29 / E 46 , E 30 and E 31 / E 32 are IdhA, adhE, ack, pta, ygfG and ptsG.
  • the cell according to the invention can also have a reduced activity of all the enzymes E 19 , E 26 , E 27 , E 29 , E 30 , E 31 , E 32 and E 46 or of all 6 enzymes which are represented by the genes IdhA, adhE, ack, pta, ygfG and ptsG.
  • the recombinant cell of sucrose and C0 2 can produce 4 -body than wild type as carbon sources more C, increased compared with its wild type activity of at least one of the enzymes EE 18 and, optionally, a reduced activity of at least one of the enzymes E 19 -E 63 .
  • Certain embodiments of the invention relate to cells in which, compared to the wild type, the activity:
  • the enzymes E-1 to E 10 and E 12 to E 18 may be represented by the genes scrA, scrB, scrK, scrY, cscB, cscA, cscK, susT1, susT2, susX or pyc, ppc, pckA, maeA, mm, maeB and dmlA and the enzymes E 19 , E 20 , E 2 i, E 22 , E 23 , E 24 , E 25 , E 27 , E 28 , E 29 , E 30 , E 3 i, E 32 , E 33 , E 45 , E 4 6, E 47, E 4 8, E 49, E 5 O, E 55 , E 5 6, E 57, E 5 8, E 59, EQO, E ⁇ i and ⁇ 3 ptsG, ptsl, ptsH, crr, tdcE, pflA, pflB, poxB, ack, pta,
  • the invention relates to a method for producing a genetically modified cell according to the invention.
  • the increase of the activity of at least one of the above-described enzymes EE 18 and, optionally, the reduction of the activity of at least one of the above-described enzymes E 19 -E 63 can be carried out by one of the methods described above.
  • Preferred combinations of enzymes with increased or decreased activity are the above-mentioned combinations.
  • the invention relates to a process for producing at least one C 4 body and / or at least one prepared over this C 4 body
  • Sequential compound the method comprising incubating a cell according to the invention with a nutrient medium containing sucrose under conditions which include the production of at least one C 4 body and / or at least one sucrose and C0 2 secondary compound produced via this C 4 body allow, includes.
  • the medium contains carbon dioxide.
  • the process according to the invention for producing the at least one C 4 body and / or at least one secondary compound prepared via this C 4 body may additionally also comprise isolating the at least one C 4 body and / or at least one secondary compound produced via this C 4 body from the nutrient medium.
  • the invention is directed to the use of the cells according to the invention for the production of at least one C 4 body and / or at least one sucrose and carbon dioxide secondary compound produced via this C 4 body as carbon sources.
  • the C 4 bodies are selected from the following group: succinate, malate, fumarate, oxaloacetate, aspartate, asparagine, threonine, tetrahydrofuran, pyrrolidone, acetoin, 4,4-bionell, hydroxysuccinate, Epoxy-v-butyrolactone, butenoic acid, butyrate, butanediol, 1, 2-butanediol, 1, 3-butanediol, 1, 4-butanediol, 2,3-butanediol, butene, n-butene, cis-2-butene, trans- 2-butene, isobutene
  • the C 4 bodies are selected from the group consisting of: malate, oxaloacetate and the derivatives produced via these C 4 bodies are selected from the group consisting of secondary compounds prepared by enzymatic or chemical synthesis via malate or oxalacetate.
  • the secondary compounds produced by enzymatic synthesis via malate or oxalacetate can be selected, for example, from the group consisting of: succinate, aspartate, asparagine, threonine,
  • the genetically modified cells of the invention can be used continuously or discontinuously in the batch process (batch culturing) or in the fed-batch process
  • sucrose and / or carbon dioxide serve as carbon sources in the nutrient media used.
  • nitrogen sources organic nitrogen-containing compounds such as peptones,
  • Yeast extract meat extract, malt extract, corn steep liquor, soybean meal and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate.
  • the nitrogen sources can be used singly or as a mixture.
  • a phosphorus source can phosphoric acid, potassium dihydrogen phosphate or
  • Dipotassium hydrogen phosphate or the corresponding sodium-containing salts are used.
  • the culture medium may further contain metal salts, such as. As magnesium sulfate or iron sulfate, which are necessary for the growth of the cells.
  • the medium may contain other substances, e.g. Amino acids and / or vitamins are added.
  • suitable precursors can be added to the culture medium.
  • the stated feedstocks can be used for culture in the form of a unique approach
  • Phosphoric acid or sulfuric acid used in a suitable manner.
  • Foaming can anti-foaming agents such.
  • B. fatty acid polyglycol esters are used.
  • the medium suitable selective substances such. B. antibiotics are added.
  • the temperature of the culture is usually 20 ° C to 45 ° C, and preferably 25 ° C to 40 ° C.
  • the purification of the C 4 body (s) or secondary products from the nutrient solution is preferably carried out continuously, it being further preferred in this context to carry out the production of the C 4 body by fermentation continuously, so that the entire process from the production of the C 4 body to its purification from the fermentation broth can be carried out continuously.
  • this is continuously passed through a device for separating the microorganisms used in the fermentation, preferably via a filter with an exclusion size in a range of 20 to 200 kDa, in which a solid / liquid Separation takes place. It is also conceivable to use a centrifuge, a suitable sedimentation device or a combination of these devices, it being particularly preferred that at least some of the microorganisms pass through first
  • the enriched in terms of its C 4 body portion fermentation product is fed after separation of the microorganisms of a preferably multi-stage separation plant.
  • a separation plant In this separation plant a plurality of series-connected separation stages are provided, from which each return lines lead, which are returned to the fermentation tank. Furthermore lead out of the respective separation stages derivatives.
  • Separation stages can operate on the principle of electrodialysis, reverse osmosis, ultrafiltration or nanofiltration. As a rule, these are membrane separation devices in the individual separation stages. The selection of the individual separation stages results from the nature and extent of the fermentation by-products and substrate residues.
  • the C 4 body can also be separated by extraction from the freed from microorganisms fermentation solution in which case, ultimately, the pure C 4 body can be obtained.
  • ammonium compounds or amines may be added to the fermentation solution to form an ammonium salt of C 4 carboxylic acid.
  • Ammonium salt can then be separated from the fermentation solution by adding an organic extractant and then heating the mixture thus obtained, whereby the ammonium salt accumulates in the organic phase. From this phase, the C 4 carboxylic acid can then be isolated to obtain the pure C 4 carboxylic acid, for example by further extraction steps. More details regarding this
  • C 4 -carboxylic acids prepared by the process according to the invention can still be neutralized before, during or after the purification, it being possible to use bases such as calcium hydroxide or sodium hydroxide for this purpose.
  • glutamicum SEQ ID NO: 5
  • these genes are made from chromosomal DNA of £. coli MG1655 or C. glutamicum ATCC 13032 were amplified by PCR and introduced simultaneously via the oligonucleotides used an interface upstream of the respective ribosome binding site and an interface downstream of the stop codon.
  • Chromosomal DNA of E. coli MG 1655 or C. glutamicum ATCC 13032 is carried out using DNeasy Blood & Tissue Kit (Qiagen, Hilden) according to the manufacturer's instructions.
  • the following oligonucleotides are used in the amplification of the genes ppc, pck, maeA and maeB from E. coli and pyc from C. glutamicum with chromosomal DNA of E. coli MG 1655 or C. glutamicum ATCC 13032 as template.
  • E. coli pck pck-fw: 5'-ATA GGA TCC TTA CTA TTC AGG CAA TAC ATA TTG GCT AAG
  • PCR fragments of the expected size can be amplified. For ppc this is 2710 bp, for pck 1686 bp, for maeA 1745 bp, for maeB 3220 bp and for pyc 3466 bp.
  • PCR products are digested with Sac ⁇ and Sbfi ⁇ ppc, maeA, maeB and pyc) and / or bamHI and Sbfi ⁇ pck) according to the recommendations of the manufacturer of the Restriktiosendonukleasen (New England Biolabs, bath Schwalbach) and in those with Sac ⁇ and Sbfi ⁇ ppc, maeA, maeB and pyc) or ßamHI and Sbfi ⁇ pck) cut vector pEC-XC99E (SEQ ID NO: 16) ligated.
  • pEC-XC99E is a coli coli C. g / yfam / ci / m shuttle vector present in both organisms
  • Chloramphenicol resistance and a ColE1 origin of replication (high
  • the correct insertion of the ppc, pck, maeA, maeB or pyc fragments is checked by a restriction with Sac ⁇ and Sbfi ⁇ ppc, maeA, maeB and pyc) or ßamHI and Sbfi ⁇ pck).
  • the authenticity of the inserted fragments is checked by DNA sequencing.
  • the completed £ co // expression vectors are identified as pEC-XC99E-ppc (SEQ ID NO: 17), pEC-XC99E-pck (SEQ ID NO: 18), pEC-XC99E-maeA (SEQ ID NO: 19), pEC XC99E-maeB (SEQ ID NO: 20) and pEC-XC99E-pyc (SEQ ID NO: 21).
  • SEQ ID NO: 17 The completed £ co // expression vectors are identified as pEC-XC99E-ppc (SEQ ID NO: 17), pEC-XC99E-pck (SEQ ID NO: 18), pEC-XC99E-maeA (SEQ ID NO: 19), pEC XC99E-maeB (SEQ ID NO: 20) and pEC-XC99E-pyc (SEQ ID NO: 21).
  • Coli cscA-cscKB csc-fw: 5'-ATA CAT ATG TTA TTA ACC CAG TAG CCA GAG TGC TCC AT GT-
  • Coli coli scrK-scrYAB scr-fw 5'-ATA CCA TGG TCC GCC AGT TCA TCC GGG AAC GG-3 '(SEQ ID NO: 5'-ATA CCA TGG TCC GCC AGT TCA TCC GGG AAC GG-3 '(SEQ ID NO: 5'-ATA CCA TGG TCC GCC AGT TCA TCC GGG AAC GG-3 '(SEQ ID NO: 5'-ATA CCA TGG TCC GCC AGT TCA TCC GGG AAC GG-3 '(SEQ ID NO: 5'-ATA CCA TGG TCC GCC AGT TCA TCC GGG AAC GG-3 '(SEQ ID NO: 5'-ATA CCA TGG TCC GCC AGT TCA TCC GGG AAC GG-3 '(SEQ ID NO: 5'-ATA CCA TGG TCC GCC AGT TCA TCC GGG AAC GG-3 '(SEQ ID NO: 5'-ATA CCA T
  • PCR products are labeled with ⁇ / col and Not ⁇ (scrK-scrYAB) or Nde ⁇ and Xho ⁇ (cscA-cscKB) according to the
  • pCOLADuet-1 (SEQ ID NO : 28; Merck Biosciences; Nottigham, UK).
  • pCOLADuet-1 is a £. co // vector of low copy number (20-40 copies per cell) that mediates kanamycin resistance and carries a ColA origin of replication.
  • the chromatographic quantification of sucrose is carried out in the following way: The quantification of sucrose is carried out by means of high-performance liquid chromatography carried out.
  • the quantification of sucrose is carried out by means of high-performance liquid chromatography carried out.
  • For the separation of sugars is an anion exchange column with
  • the mobile phase consists of a mixture of acetonitrile (56% v / v), acetone (26% v / v) and water (16% v / v).
  • the samples are sterile filtered and measured undiluted.
  • the injection volume is 10 ⁇ _, the mobile phase flow 2 mL / min and the column temperature 30 ° C.
  • Sucrose is quantified by refractive index detector (Agilent 1200 Series RID, Agilent Technologies, Böblingen). The
  • Reference substance (Sigma-Aldrich, Steinheim) is measured dissolved in water. There is a linear dependence between the peak area and the substance concentration up to a concentration of 100 g / L. The limit of quantification for sucrose is 1 g / L.
  • Ion exclusion column Aminex® HPX 87H with the dimensions 300 mm x 7.8 mm (Bio Rad Laboratories, Kunststoff) as a stationary phase use. As the mobile phase becomes 10 mM
  • Sulfuric acid used.
  • the column temperature is 40 ° C, the flow rate 0.6 ml / min.
  • the sample is acidified with 0.5 M sulfuric acid to a pH of 4 to 5 and injected into the column with a volume of 20 ⁇ .
  • Detection is performed using a diode array detector (Agilent 1200 Series DAD, Agilent Technologies, Böblingen) at a wavelength of 190 to 400 nm and a refractive index detector (Agilent 1200 Series RID, Agilent Technologies, Böblingen).
  • the reference substances are measured in concentrations of 0.1 g / L to 20 g / L dissolved in water.
  • lactate, acetate, succinate and formate are 0.8 g / L
  • ethanol can be determined up to a concentration of 1 g / L.
  • concentration of 1 g / L In the range of 0.8 g / L to 20 g / L there is a linear dependence of the peak areas of the
  • the electrocompetent cells are prepared by washing with a sterile 10% (w / v) glycerol solution as follows: The 100 mL culture is harvested by centrifugation at 4 ° C and 5500 xg for 10 minutes and washed by resuspension in 10% glycerol solution. After another centrifugation and washing step, the pelleted E. coli MG1655 cells are taken up in 0.5 mL 10% glycerol solution and stored in aliquots of 50 L at -80 ° C until electroporation.
  • Coli coli 1655 (pCOLADuet-1, pEC-XC99E)
  • Coli coli 1655 (pCOLA-csc, pEC-XC99E)
  • Coli coli 1655 (pCOLA-csc, pEC-XC99E-ppc)
  • Coli coli 1655 (pCOLA-csc, pEC-XC99E-pck)
  • Coli coli 1655 (pCOLA-csc, pEC-XC99E-maeA)
  • Coli coli 1655 (pCOLA-csc, pEC-XC99E-maeB)
  • Coli coli 1655 (pCOLA-csc, pEC-XC99E-pyc)
  • Coli coli 1655 (pCOLA-scr, pEC-XC99E)
  • Coli coli 1655 (pCOLA-scr, pEC-XC99E-ppc)
  • Coli coli 1655 (pCOLA-scr, pEC-XC99E-maeA)
  • Coli coli 1655 (pCOLA-scr, pEC-XC99E-maeB)
  • E. coli MG1655 (pCOLA-scr, pEC-XC99E-pyc)
  • strains are then used to analyze their ability to produce succinate under anaerobic conditions.
  • the procedure is as follows: The strains are subjected to a multi-stage cultivation process.
  • the preculture for the production of biomass is carried out aerobically in a modified M9 medium which additionally contains yeast extract as complex component.
  • the medium consisting of 38 mM disodium hydrogen phosphate dihydrate (Merck, Darmstadt), 22 mM potassium dihydrogen phosphate (Merck, Darmstadt), 8.6 mM sodium chloride (Merck, Darmstadt), 18.7 mM ammonium chloride (Merck, Darmstadt), 1% ( w / v) yeast extract (Merck, Darmstadt), 2% (w / v) sucrose (Sigma-Aldrich, Steinheim), 0.1mM calcium chloride dihydrate (Sigma-Aldrich, Steinheim), 1mM
  • modified M9 medium with 0.2 mg / L biotin and 0.5 mM IPTG in a 1000 ml Erlenmeyer flask with chicane inocultivated with the preculture so that an optical density (600 nm) of 0.2 is achieved.
  • the culture broth is harvested by centrifugation at 3300 g and 4 ° C for 10 minutes.
  • the sedimented bacterial cells are washed with sterile 0.9% sodium chloride solution and then taken up in anaerobic culture medium.
  • the main anaerobic culture is carried out in modified M9 medium without complex components (38 mM disodium hydrogen phosphate dihydrate, 22 mM potassium dihydrogen phosphate, 8.6 mM
  • Oxygen indicator 50 mL of medium are placed in 100 mL laboratory glass bottles (Schott, Mainz) and inoculated with the cell suspension so that an optical density (600 nm) of 20 is achieved.
  • the bottles are gas-tight with a cap with a stopper closed, the cultivation is carried out at 37 ° C and 250 rev / min in incubation shaker for a period of 24 hours.
  • Anaerobiosis occurs within the first hour of cultivation and is detected by decolorization of resazurin.
  • samples of 1 ml_ sterile are withdrawn through the stopper with a cannula which, after sterile filtration, is analyzed for its sucrose content and organic acid content by means of chromatographic methods described in Example 3.
  • Example 5 was the production of succinate by E. co // 'strains with deletions in the genes ack-pta, adhE, IDHA and ygfG, in which the gene is replaced by the ptsG SCRk-scrYAB locus from the plasmid pUR400 and which the Gene pyc, coding for a
  • a pound. co // strain with deletions in the genes ack-pta (SEQ ID NO: 31), adhE (SEQ ID NO: 32), IdhA (SEQ ID NO: 33) and ygfG (SEQ ID NO: 34) and an exchange of the pisG gene (SEQ ID NO: 35) is constructed by the scrK scrYAB locus.
  • the E. coli strain MG1655 ack-pta adhE IdhAggfG ptsG :: scrK-scrYAB was generated by methods known to the person skilled in the art (eg see Datsenko KA, Wanner BL Proc Natl Acad Sei USA 2000. 97 ( 12): 6640-5)..
  • SEQ ID NO: 36 (ack-pta), SEQ ID NO: 37 (adhE), SEQ ID NO: 38 (IdhA), SEQ ID NO: 39 (ygfG ) and SEQ ID NO: 40 ⁇ ptsGwscrK-scrYAB).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention relates to a cell which has been genetically modified with respect to the wild type thereof such that it, in comparison with the wild type, is able to form more C4 bodies and/or more subsequent compounds produced by means of said C4 bodies from saccharose and carbon dioxide as the carbon source. The invention further relates to a method for producing such a genetically modified cell and to a method for producing C4 bodies and/or subsequent compounds produced by means of said C4 bodies with the aid of said cells. Finally, the invention also relates to the use of the cells for producing C4 bodies and/or subsequent compounds produced by means of said C4 bodies from saccharose and carbon dioxide.

Description

Mikrobiologische Herstellung von Cd-Körpern aus Saccharose und Kohlendioxid  Microbiological production of Cd bodies from sucrose and carbon dioxide
Die vorliegende Erfindung betrifft eine Zelle, die gegenüber ihrem Wildtyp derart gentechnisch verändert wurde, dass sie im Vergleich zu ihrem Wildtyp aus Saccharose und Kohlendioxid als Kohlenstoffquelle mehr C4-Körper und/oder über diese C4-Körper hergestellte The present invention relates to a cell which has been genetically engineered with respect to its wild type such that it produces more C 4 bodies and / or over these C 4 bodies compared to their wild type of sucrose and carbon dioxide as the carbon source
Folgeverbindungen zu bilden vermag. Die Erfindung betrifft ferner ein Verfahren zur Herstellung einer solchen gentechnisch veränderten Zelle und ein Verfahren zur Herstellung von C4- Körpern und/oder von über diese C4-Körper hergestellte Folgeverbindungen mit Hilfe dieser Zellen. Schließlich betrifft die Erfindung noch die Verwendung der Zellen zur Herstellung von C4-Körpern Körpern und/oder von über diese C4-Körper hergestellte Folgeverbindungen aus Saccharose und Kohlendioxid. Can form secondary connections. The invention further relates to a process for the production of such a genetically modified cell and to a process for the production of C 4 bodies and / or of secondary compounds produced via these C 4 bodies with the aid of these cells. Finally, the invention also relates to the use of the cells for the production of C 4 bodies bodies and / or derived from these C 4 body compounds of sucrose and carbon dioxide.
C4-Körper, wie etwa Succinat, Malat, Fumarat, Oxalacetat, Aspartat, Asparagin, Threonin, Tetrahydrofuran, Pyrrolidon, Acetoin, 4,4-Bionell, Hydroxysuccinat, Epoxy-v-Butyrolacton, Butensäure, Butyrat, Butandiol, 1 ,2-Butandiol, 1 ,3-Butandiol, 1 ,4-Butandiol, 2,3-Butandiol,C 4 -body, such as succinate, malate, fumarate, oxaloacetate, aspartate, asparagine, threonine, tetrahydrofuran, pyrrolidone, acetoin, 4,4-Bionell, hydroxysuccinate, epoxy-v-butyrolactone, butenoic acid, butyrate, butanediol, 1, 2 Butanediol, 1, 3-butanediol, 1, 4-butanediol, 2,3-butanediol,
Buten, n-Buten, cis-2-Buten, trans-2-Buten, Isobuten, Butadien, 1 ,2- Butadien, 1 ,3- Butadien, 3- und 4-Hydroxybutyrolacton, 1 -, 2-, und tert-Butanol, Isobutanol, 2-, 3- und 4-Hydroxybuttersäure sowie 2- und 3-Hydroxyisobuttersäure werden heutzutage im Wesentlichen aus Butene, n-butene, cis-2-butene, trans-2-butene, isobutene, butadiene, 1,2-butadiene, 1,3-butadiene, 3- and 4-hydroxybutyrolactone, 1-, 2-, and tert-butyl Butanol, isobutanol, 2-, 3- and 4-hydroxybutyric acid as well as 2- and 3-hydroxyisobutyric acid are essentially eliminated today
petrochemischen Rohstoffen hergestellt und sind als Ausgangsstoffe für Produkte der petrochemical raw materials and are used as raw materials for the products
Futterindustrie, Agrarwirtschaft und der Pharmazeutischen Industrie von Bedeutung. Zusätzlich werden diese C4-Körper auch als Rohstoff der Chemieindustrie für Massenprodukte, wie zum Beispiel Polymere und Lösungsmittel, oder SpezialChemikalien eingesetzt. Aufgrund der absehbaren Verknappung petrochemischer Rohstoffe und zunehmender Nachfrage nach Produkten, die auf nachwachsenden Rohstoffen basieren, ist es wünschenswert, Alternativen zu den existierenden petrochemischen Verfahren zur Herstellung von C4-Körpern Feed industry, agriculture and the pharmaceutical industry of importance. In addition, these C 4 bodies are also used as raw materials of the chemical industry for mass-produced products, such as polymers and solvents, or specialty chemicals. Due to the foreseeable shortage of petrochemical raw materials and increasing demand for products based on renewable raw materials, it is desirable alternatives to the existing petrochemical processes for the production of C 4 bodies
bereitzustellen, die auf nachwachsenden Rohstoffen basieren. provide based on renewable resources.
Die zurzeit verfolgten Ansätze basieren mehrheitlich auf Mikroorganismen, die in fermentativen Prozessen C4-Körper herstellen. Dabei wird insbesondere Glucose als Kohlenstoffquelle verwendet. Die notwendige Glucose wird aus Stärke gewonnen, welche z. B. aus Mais,The currently pursued approaches are based mainly on microorganisms that produce C 4 bodies in fermentative processes. In particular, glucose is used as carbon source. The necessary glucose is recovered from starch, which z. From corn,
Kartoffeln, Weizen oder anderen Getreidearten hergestellt wird. Nachteilig an diesen Ansätzen ist, dass Glucose vergleichsweise teuer ist und die entsprechende Herstellung und Verwendung von Glucose mit der Verwendung oder Verarbeitung der Ausgangsstoffe zu Nahrungsmitteln konkurriert. Der vorliegenden Erfindung liegt daher die Aufgabe zugrunde, die sich aus dem Stand der Technik ergebenden Nachteile zu überwinden. Insbesondere liegt der Erfindung die Aufgabe zugrunde Glucose als Rohstoff durch eine günstigere und in ausreichenden Mengen verfügbare Alternative zu ersetzen. Potatoes, wheat or other cereals. A disadvantage of these approaches is that glucose is relatively expensive and the corresponding production and use of glucose competes with the use or processing of the starting materials to foods. The present invention is therefore based on the object to overcome the disadvantages resulting from the prior art. In particular, the invention has the object to replace glucose as a raw material by a cheaper and available in sufficient quantities alternative.
Erfindungsgemäß wird diese Aufgabe dadurch gelöst, dass Saccharose als Rohstoff für die Herstellung von C4-Körpern verwendet wird. Weltweit betrachtet wird Saccharose zum größten Teil aus Zuckerrohr (ca. % der Jahresproduktion) und zum geringeren Teil aus Zuckerrüben (ca. % der Jahresproduktion) gewonnen. Durch die hohe Saccharose Produktion aus Zuckerrohr in Südamerika und Asien und einer geringeren Konkurrenz zur Nahrungsmittelindustrie, ist Saccharose in großen Mengen und günstiger als Glucose erhältlich. Eine weitere Verbesserung der bestehenden Prozesse liegt darin, dass erfindungsgemäß zusätzlich zu Saccharose Kohlendioxid (C02) als Kohlenstoffquelle verwendet wird. According to the invention this object is achieved in that sucrose is used as a raw material for the production of C 4 bodies. Worldwide, sucrose is mostly made from sugar cane (about% of annual production) and to a lesser extent from sugar beet (about% of annual production). Due to the high sucrose production from sugarcane in South America and Asia and less competition with the food industry, sucrose is available in large quantities and cheaper than glucose. A further improvement of the existing processes is that according to the invention, in addition to sucrose, carbon dioxide (CO 2 ) is used as carbon source.
Kohlendioxid fällt beispielsweise als Abfallprodukt einer Reihe chemischer Prozesse an und ist daher vergleichsweise günstig erhältlich. Zusätzlich kann durch die Verwertung von For example, carbon dioxide is a by-product of a number of chemical processes and is therefore comparatively inexpensive to obtain. In addition, through the utilization of
Kohlendioxid die Belastung der Atmosphäre mit Treibhausgasen reduziert werden. Carbon dioxide reduce the pollution of the atmosphere with greenhouse gases.
Im Stand der Technik sind bisher keine Technologien für die Herstellung von C4-Körpern aus Saccharose und Kohlendioxid verfügbar, die mit den bestehenden petrochemischen Prozessen konkurrieren könnten. Wildtyporganismen, wie etwa Basfia succiniciproducens, The prior art has not yet provided technologies for the production of C 4 bodies from sucrose and carbon dioxide which could compete with existing petrochemical processes. Wild-type organisms, such as Basfia succiniciproducens,
Anaerobiospirillum succiniciproducens oder Actinobacillus succinogenes, die Saccharose zu C4- Körpern umsetzen können, sind für die biotechnologische Herstellung nicht geeignet, da die Ausbeuten gering sind und diese Organismen komplexe Nährmedien benötigen, die durch die damit verbundenen Kosten und den Aufwand den Prozess unwirtschaftlich werden lassen. Anaerobiospirillum succiniciproducens or Actinobacillus succinogenes, which are able to convert sucrose into C 4 bodies, are not suitable for biotechnological production because the yields are low and these organisms require complex nutrient media which, due to the associated costs and effort, render the process uneconomic ,
Erfindungsgemäß wird dieser Nachteil dadurch überwunden, dass rekombinante Zellen bereitgestellt werden, die unter entsprechenden Kulturbedingungen, ausgehend von According to the invention, this disadvantage is overcome by providing recombinant cells which, under appropriate culture conditions, are prepared from
Saccharose und C02 als Kohlenstoffquellen, große Mengen an C4-Körpern herstellen können. Sucrose and C0 2 as carbon sources, can produce large quantities of C 4 bodies.
Die Erfinder der vorliegenden Erfindung haben überraschenderweise herausgefunden, dass die Ausbeute an C4-Körpern durch gentechnische Manipulation geeigneter Zellen, insbesondere eine Verstärkung von Carboxylierungs-Reaktionen, die Kohlendioxid fixieren und zur Bildung von C4-Körpern führen, eine Unterdrückung von Stoffwechselwegen, die den Kohlenstofffluss von Saccharose hin zu Gärungsprodukten verursachen, welche nicht C4-Körper sind, und die Steigerung der Saccharose-Aufnahme und des Saccharose-Metabolismus stark erhöht werden kann. Dieser Ansatz erlaubt es i) Organismen, die üblicherweise nicht in der Lage sind The inventors of the present invention have surprisingly found that the yield of C 4 bodies is fixed by genetic manipulation of suitable cells, in particular an enhancement of carboxylation reactions, the carbon dioxide and formation lead from C 4 -Körpern, a suppression of metabolic pathways that cause the carbon flux of sucrose, to fermentation products that are not C 4 -body, and increasing uptake of sucrose and the sucrose metabolism can be greatly increased. This approach allows i) organisms that are usually unable
Saccharose zu metabolisieren, die aber im Hinblick auf die erforderlichen Kulturbedingungen günstig sind und ii) Organismen, die auch natürlicherweise in der Lage sind Saccharose zu metabolisieren, aber nur geringe Effizienz im Sinne von Raum-Zeit- und/oder Metabolize sucrose, but which are favorable in view of the required culture conditions, and ii) organisms which are naturally capable of metabolizing sucrose, but have low efficiency in terms of space-time and / or
Kohlenstoffausbeute in Bezug auf die Synthese von C4-Körpers besitzen, als effiziente Have carbon yield with respect to the synthesis of C 4 bodies, as efficient
Ganzzellkatalysatoren zur Herstellung von C4-Körpern einzusetzen. To use whole-cell catalysts for the production of C 4 bodies.
In einem ersten Aspekt betrifft die vorliegende Erfindung daher eine rekombinante Zelle, welche gegenüber ihrem Wildtyp derart gentechnisch verändert wurde, dass sie aus Saccharose und Kohlendioxid als Kohlenstoffquellen im Vergleich zu ihrem Wildtyp mehr C4-Körper und/oder über diese C4-Körper hergestellte Folgeverbindungen zu bilden vermag. In a first aspect, therefore, the present invention relates to a recombinant cell which has been genetically engineered with respect to its wild type to produce more C 4 bodies and / or over these C 4 bodies from sucrose and carbon dioxide as carbon sources compared to their wild-type Can form secondary connections.
Der Begriff„C4-Körper", wie er hierin verwendet wird, bezieht sich auf eine chemische The term "C 4 body" as used herein refers to a chemical
Verbindung, die vier Kohlenstoffatome enthält, und umfasst entsprechende Carbonsäuren, Aldehyde, Alkohole, Zucker, Alkane, Alkene, Amine sowie deren Derivate. Beispielhafte C4- Körper im Sinne der Erfindung umfassen, sind aber nicht beschränkt auf Succinat, Malat, Fumarat, Oxalacetat, Aspartat, Asparagin, Threonin, Tetrahydrofuran, Pyrrolidon, Acetoin, 4,4- Bionell, Hydroxysuccinat, Epoxy-v-Butyrolacton, Butensäure, Butyrat, Butandiol, 1 ,2-Butandiol, 1 ,3-Butandiol, 1 ,4-Butandiol, 2,3-Butandiol, Buten, n-Buten, cis-2-Buten, trans-2-Buten, Isobuten, Butadien, 1 ,2- Butadien, 1 ,3- Butadien, 3- und 4-Hydroxybutyrolacton, 1 -, 2- und tert- Butanol, Isobutanol, 2-, 3- und 4-Hydroxybuttersäure sowie 2- und 3-Hydroxyisobuttersäure. In einer Ausführungsform der Erfindung wird der C4-Körper ausgewählt aus der Gruppe bestehend aus: Malat und Oxalacetat und/oder aus durch enzymatische Synthese aus diesen C4-Körpern hergestellte Folgeverbindungen, wie zum Beispiel Succinat, Aspartat, Asparagin, Threonin, Tetrahydrofuran, Butyrat, Butandiol, 1 ,2-Butandiol, 1 ,3-Butandiol, 1 ,4-Butandiol, 2,3-Butandiol, 3- und 4-Hydroxybutyrolacton, 1 -, 2-, und tert-Butanol, Isobutanol, 2-, 3- und 4- Hydroxybuttersäure sowie 2- und 3-Hydroxyisobuttersäure, Methionin und Lysin sowie über Malat, Oxalacetat und Folgeverbindungen auf chemischem Wege hergestellten Verbindungen. Wie hierin verwendet umfassen die Bezeichnungen von Carbonsäuren, sowohl die Basenform als auch die Säureform sowie Mischungen dieser Formen. Das bedeutet, dass Begriffe wie Succinat, Malat, Oxalacetat, Fumarat, Aspartat, Butyrat, 2-, 3- oder 4-Hydroxybuttersäure und 2- oder 3-Hydroxyisobuttersäure jeweils auch Bernsteinsäure, Äpfelsäure, Oxalessigsäure, Fumarsäure, Asparaginsäure, Buttersäure, 2-, 3- oder 4-Hydroxybutyrat und 2- oder 3- Hydroxyisobutyrat umfassen. Der Ausdruck„dass sie im Vergleich zu ihrem Wildtyp mehr C4-Körper zu bilden vermag" umfasst auch den theoretischen Fall, dass der Wildtyp der gentechnisch veränderten Zelle überhaupt keine C4-Körper, bzw. nicht den gewünschten C4-Körper zu bilden vermag, bzw. diese Verbindung(en) nicht in nachweisbarer Menge erzeugen kann und erst nach der gentechnischen Veränderung nachweisbare Mengen dieser Verbindungen gebildet werden. Ferner ist durch diesen Ausdruck erfasst, dass die entsprechende Zelle in einem festgelegten Zeitraum, beispielsweise innerhalb von 2, 4, 8, 12, 24 oder 48 Stunden, mindestens die 2-, 5-, 10-, 100- oder 1000-fache Menge an C4-Körpern bzw. einem oder mehreren gewünschten C4- Körpern bildet wie der Wildtyp. Mit„Wildtyp" einer Zelle wird hierin eine Zelle bezeichnet, deren Genom in einem Zustand vorliegt, wie er natürlicherweise durch die Evolution entstanden ist. Der Begriff wird sowohl für die gesamte Zelle als auch für einzelne Gene verwendet. Unter den Begriff„Wildtyp" fallen daher insbesondere nicht solche Zellen bzw. solche Gene, deren Gensequenzen zumindest teilweise durch den Menschen mittels rekombinanter Verfahren verändert worden sind. Compound containing four carbon atoms, and includes corresponding carboxylic acids, aldehydes, alcohols, sugars, alkanes, alkenes, amines and derivatives thereof. Exemplary C 4 bodies within the meaning of the invention include, but are not limited to, succinate, malate, fumarate, oxaloacetate, aspartate, asparagine, threonine, tetrahydrofuran, pyrrolidone, acetoin, 4,4-bionell, hydroxysuccinate, epoxy-v-butyrolactone, Butenoic acid, butyrate, butanediol, 1, 2-butanediol, 1, 3-butanediol, 1, 4-butanediol, 2,3-butanediol, butene, n-butene, cis-2-butene, trans-2-butene, isobutene, Butadiene, 1,2-butadiene, 1,3-butadiene, 3- and 4-hydroxybutyrolactone, 1-, 2- and tert-butanol, isobutanol, 2-, 3- and 4-hydroxybutyric acid and 2- and 3-hydroxyisobutyric acid. In one embodiment of the invention, the C 4 body is selected from the group consisting of: malate and oxaloacetate and / or secondary compounds prepared by enzymatic synthesis from these C 4 bodies, for example succinate, aspartate, asparagine, threonine, tetrahydrofuran, Butyrate, butanediol, 1, 2-butanediol, 1, 3-butanediol, 1, 4-butanediol, 2,3-butanediol, 3- and 4-hydroxybutyrolactone, 1-, 2-, and tert-butanol, isobutanol, 2- , 3- and 4-hydroxybutyric acid and 2- and 3-hydroxyisobutyric acid, methionine and lysine as well as malate, oxaloacetate and secondary compounds produced by chemical means compounds. As used herein, the terms of carboxylic acids include both the base form and the acid form, as well as mixtures of these forms. This means that terms such as succinate, malate, oxaloacetate, fumarate, aspartate, butyrate, 2-, 3- or 4-hydroxybutyric acid and 2- or 3-hydroxyisobutyric acid also each include succinic, malic, oxalacetic, fumaric, aspartic, butyric, 2-, 3- or 4-hydroxybutyrate and 2- or 3-hydroxyisobutyrate. The expression "that it is able to form more C 4 bodies compared to its wild type" also encompasses the theoretical case that the wild-type of the genetically modified cell does not form any C 4 bodies at all or does not form the desired C 4 body or that this compound (s) can not be produced in a detectable amount and only after the genetic modification verifiable amounts of these compounds are formed.Furthermore, this expression detects that the corresponding cell in a fixed period, for example within 2, 4 , 8, 12, 24 or 48 hours, at least the 2-, 5-, 10-, 100- or 1000-fold amount of C 4 bodies or one or more desired C 4 bodies forms like the wild type. A "wild-type" cell is referred to herein as a cell whose genome is in a state as naturally evolved. The term is used for both the entire cell and for individual genes. The term "wild-type" therefore does not include, in particular, those cells or genes whose gene sequences have been altered at least in part by humans by means of recombinant methods.
Die erfindungsgemäßen Zellen können Prokaryoten oder Eukaryoten sein. Dabei kann es sich um Säugetierzellen (wie etwa Zellen aus dem Menschen), andere tierische Zellen (z.B. The cells of the invention may be prokaryotes or eukaryotes. These may be mammalian cells (such as human cells), other animal cells (e.g.
Insektenzellen), um pflanzliche Zellen oder um Mikroorganismen wie Hefen, Pilze oder Bakterien handeln, wobei Mikroorganismen bevorzugt und Bakterien und Hefen besonders bevorzugt sind. Insect cells), plant cells or microorganisms such as yeasts, fungi or bacteria, with microorganisms being preferred and bacteria and yeasts being particularly preferred.
Als Bakterien, Hefen oder Pilze sind insbesondere diejenigen Bakterien, Hefen oder Pilze geeignet, die bei der Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Braunschweig, Deutschland, als Bakterien-, Hefe- oder Pilz-Stämme hinterlegt sind. Erfindungsgemäß geeignete Bakterien gehören zu den Gattungen, die unter http://www.dsmz.de/microorganisms/bacteria_catalogue.php aufgeführt sind. Erfindungsgemäß geeignete Hefen gehören zu denjenigen Gattungen, die unter http://www.dsmz.de/microorganisms/yeast_catalogue.php aufgeführt sind. Erfindungsgemäß geeignete Pilze sind diejenigen, die unter http://www.dsmz.de/microorganisms/fungus_catalogue.php aufgeführt sind. Particularly suitable bacteria, yeasts or fungi are those bacteria, yeasts or fungi which are deposited in the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ), Braunschweig, Germany, as bacterial, yeast or fungal strains. Bacteria suitable according to the invention belong to the genera which can be found at http://www.dsmz.de/microorganisms/bacteria_catalogue.php are listed. Yeasts which are suitable according to the invention belong to the genera listed under http://www.dsmz.de/microorganisms/yeast_catalogue.php. Fungi suitable according to the invention are those listed at http://www.dsmz.de/microorganisms/fungus_catalogue.php.
Erfindungsgemäß bevorzugte Zellen sind diejenigen der Gattungen Aspergillus, Preferred cells according to the invention are those of the genera Aspergillus,
Corynebacterium, Brevibacterium, Bacillus, Acinetobacter, Alcaligenes, Actinobacillus, Corynebacterium, Brevibacterium, Bacillus, Acinetobacter, Alcaligenes, Actinobacillus,
Anaerobiospirillum, Basfia, Wollinella, Fibrobacter, Ruminococcus, Mannheimia, Lactobacillus, Lactococcus, Paracoccus, Lactococcus, Candida, Pichia, Hansenula, Kluveromyces, Anaerobiospirillum, Basfia, Wollinella, Fibrobacter, Ruminococcus, Mannheimia, Lactobacillus, Lactococcus, Paracoccus, Lactococcus, Candida, Pichia, Hansenula, Kluveromyces,
Saccharomyces, Escherichia, Zymomonas, Yarrowia, Methylobacterium, Ralstonia, Saccharomyces, Escherichia, Zymomonas, Yarrowia, Methylobacterium, Ralstonia,
Pseudomonas, Rhodospirillum, Rhodobacter, Burkholderia, Clostridium Cupriavidus sowie carboxydotrophe Mikroorganismen, wobei Aspergillus nidulans, Aspergillus niger, Alcaligenes latus, Bacillus megaterium, Bacillus subtilis, Brevibacterium flavum, Brevibacterium Pseudomonas, Rhodospirillum, Rhodobacter, Burkholderia, Clostridium Cupriavidus and carboxydotrophic microorganisms, wherein Aspergillus nidulans, Aspergillus niger, Alcaligenes latus, Bacillus megaterium, Bacillus subtilis, Brevibacterium flavum, Brevibacterium
lactofermentum, Escherichia coli, Basfia succiniciproducens, Wollinella succinogenes, lactofermentum, Escherichia coli, Basfia succiniciproducens, Wollinella succinogenes,
Fibrobacter succinogenes, Ruminococcus flavefaciens, .Anaerobiospirillum succiniciproducens, Mannheimia succiniciproducens, Actinobacillus succinogenes, Saccharomyces cerevisiae, Kluveromyces lactis, Candida blankii, Candida rugosa, Corynebacterium glutamicum,  Fibrobacter succinogenes, Ruminococcus flavefaciens, A. aerobiospirillum succiniciproducens, Mannheimia succiniciproducens, Actinobacillus succinogenes, Saccharomyces cerevisiae, Kluveromyces lactis, Candida blankii, Candida rugosa, Corynebacterium glutamicum,
Corynebacterium efficiens, Zymonomas mobilis, Yarrowia lipolytica, Methylobacterium extorquens, Hansenula polymorpha, Ralstonia eutropha, Rhodobacter sphaeroides, Corynebacterium efficiens, Zymonomas mobilis, Yarrowia lipolytica, Methylobacterium extorquens, Hansenula polymorpha, Ralstonia eutropha, Rhodobacter sphaeroides,
Paracoccus versutus, Pseudomonas aeruginosa, Acinetobacter calcoaceticus, Pichia pastoris, Thermoanaerobacter kivui, Acetobacterium woodii, Acetoanaerobium notera, Clostridium aceticum, Butyribacterium methylotrophicum, Clostridium acetobutylicum, Clostridium saccharoperbutylacetonicum, Clostridium beijerinckii, Clostridium butyricum, Moorella thermoacetica, Eubacterium limosum, Peptostreptococcus productus, Clostridium ljungdahlii, Clostridium carboxidivorans, Clostridium scatalogenes, Rhodospirillum rubrum, Burkholderia thailandensis und Pseudomonas putida besonders bevorzugt sind. Die Begriffe„Kohlendioxid" und„C02", wie sie hierin verwendet werden, beziehen sich sowohl auf das Gas C02, die in wässriger Lösung mit gelöstem Kohlendioxid im Gleichgewicht stehende Kohlensäure (H2C03) als auch die beiden Deprotonierungsprodukte der Kohlensäure, Hydrogencarbonat (HC03 ") und Carbonat (C03 2") sowie deren Salze. Paracoccus versutus, Pseudomonas aeruginosa, Acinetobacter calcoaceticus, Pichia pastoris, Thermoanaerobacter kivui, Acetobacterium woodii, Acetoanaerobium notera, Clostridium aceticum, acetobutylicum Butyribacterium methylotrophicum, Clostridium saccharoperbutylacetonicum Clostridium, Clostridium beijerinckii, butyricum Clostridium, Moor Ella thermoacetica, Eubacterium limosum, Peptostreptococcus productus, Clostridium ljungdahlii , Clostridium carboxidivorans, Clostridium scatalogenes, Rhodospirillum rubrum, Burkholderia thailandensis and Pseudomonas putida are particularly preferred. The terms "carbon dioxide" and "C0 2 " as used herein refer to both the gas C0 2 , the carbonic acid (H 2 C0 3 ) which is in equilibrium with dissolved carbon dioxide in aqueous solution, and the two carbonic acid deprotonation products , Bicarbonate (HC0 3 " ) and carbonate (C0 3 2" ) and their salts.
In einer Ausführungsform der Erfindung werden die C4-Körper aus der Gruppe, die aus: In one embodiment of the invention, the C 4 bodies are selected from the group consisting of:
Succinat, Malat, Fumarat, Oxalacetat, Aspartat, Asparagin, Threonin, Tetrahydrofuran, Succinate, malate, fumarate, oxaloacetate, aspartate, asparagine, threonine, tetrahydrofuran,
Pyrrolidon, Acetoin, 4,4-Bionell, Hydroxysuccinat, Epoxy-v-Butyrolacton, Butensäure, Butyrat, Butandiol, 1 ,2-Butandiol, 1 ,3-Butandiol, 1 ,4-Butandiol, 2,3-Butandiol, Buten, n-Buten, cis-2- Buten, trans-2-Buten, Isobuten, Butadien, 1 ,2-Butadien, 1 ,3-Butadien, 3-Hydroxybutyrolacton, 4-Hydroxybutyrolacton, 1 -Butanol, 2-Butanol, tert-Butanol, Isobutanol, 2-Hydroxybuttersäure, 3- Hydroxybuttersäure, 4-Hydroxybuttersäure, 2-Hydroxyisobuttersäure und 3- Hydroxyisobuttersäure besteht, ausgewählt. In einer Ausführungsform werden die C4-Körper ausgewählt aus der Gruppe bestehend aus: Malat, Oxalacetat und aus durch enzymatische oder chemische Synthese über Malat oder Oxalacetat hergestellte Folgeverbindungen. Die durch enzymatische Synthese über Malat oder Oxalacetat hergestellte Folgeverbindungen können beispielsweise ausgewählt werden aus der Gruppe bestehend aus: Succinat, Aspartat, Asparagin, Threonin, Tetrahydrofuran, Butyrat, Butandiol, 1 ,2-Butandiol, 1 ,3-Butandiol, 1 ,4-Butandiol, 2,3-Butandiol, 3- und 4-Pyrrolidone, acetoin, 4,4-bionell, hydroxysuccinate, epoxy-v-butyrolactone, butenoic acid, butyrate, butanediol, 1, 2-butanediol, 1, 3-butanediol, 1, 4-butanediol, 2,3-butanediol, butene, n-butene, cis-2-butene, trans-2-butene, isobutene, butadiene, 1, 2-butadiene, 1, 3-butadiene, 3-hydroxybutyrolactone, 4-hydroxybutyrolactone, 1-butanol, 2-butanol, tert-butyl Butanol, isobutanol, 2-hydroxybutyric acid, 3-hydroxybutyric acid, 4-hydroxybutyric acid, 2-hydroxyisobutyric acid and 3-hydroxyisobutyric acid. In one embodiment, the C 4 bodies are selected from the group consisting of: malate, oxaloacetate and derived compounds produced by enzymatic or chemical synthesis via malate or oxalacetate. The secondary compounds produced by enzymatic synthesis via malate or oxalacetate can be selected, for example, from the group consisting of: succinate, aspartate, asparagine, threonine, tetrahydrofuran, butyrate, butanediol, 1,2-butanediol, 1,3-butanediol, 1,4 Butanediol, 2,3-butanediol, 3- and 4-
Hydroxybutyrolacton, 1 -, 2- und tert-Butanol, Isobutanol, 2-, 3- und 4-Hydroxybuttersäure, 2- und 3-Hydroxyisobuttersäure, Methionin und Lysin. Hydroxybutyrolactone, 1-, 2- and tert-butanol, isobutanol, 2-, 3- and 4-hydroxybutyric acid, 2- and 3-hydroxyisobutyric acid, methionine and lysine.
In einer Ausführungsform sind die C4-Körper C4-Carbonsäuren, vorzugsweise C4- Dicarbonsäuren, noch bevorzugter Succinat. In einer weiteren Ausführungsform sind die C4- Körper Hydroxycarbonsäuren, noch bevorzugter 2-, 3- und/oder 4-Hydroxybuttersäure sowie 2- und/oder 3-Hydroxyisobuttersäure. In einer anderen Ausführungsform sind die C4-Körper vorzugsweise Malat und/oder Oxalacetat. In weiteren Ausführungsformen handelt es sich bei der rekombinanten Zelle um eine mikrobielle Zelle, insbesondere um eine Escherichia coli, Alcaligenes latus, Bacillus megaterium, Bacillus subtilis, Brevibacterium flavum, Brevibacterium lactofermentum, Escherichia coli, Basfia succiniciproducens, Wollinella succinogenes, Fibrobacter succinogenes, Ruminococcus flavefaciens, Anaerobiospirillum succiniciproducens, Mannheimia succiniciproducens, Actinobacillus succinogenes, Corynebacterium glutamicum, Corynebacterium efficiens, In one embodiment, the C 4 bodies are C 4 carboxylic acids, preferably C 4 dicarboxylic acids, more preferably succinate. In another embodiment, the C 4 bodies are hydroxycarboxylic acids, more preferably 2-, 3- and / or 4-hydroxybutyric acid and 2- and / or 3-hydroxyisobutyric acid. In another embodiment, the C 4 bodies are preferably malate and / or oxaloacetate. In further embodiments, the recombinant cell is a microbial cell, in particular an Escherichia coli, Alcaligenes latus, Bacillus megaterium, Bacillus subtilis, Brevibacterium flavum, Brevibacterium lactofermentum, Escherichia coli, Basfia succiniciproducens, Wollinella succinogenes, Fibrobacter succinogenes, Ruminococcus flavefaciens , Anaerobiospirillum succiniciproducens, Mannheimia succiniciproducens, Actinobacillus succinogenes, Corynebacterium glutamicum, Corynebacterium efficiens,
Zymonomas mobilis, Methylobacterium extorquens, Ralstonia eutropha, Saccharomyces cerevisiae, Rhodobacter sphaeroides, Paracoccus versutus, Pseudomonas aeruginosa, Acinetobacter calcoaceticus, Clostridium acetobutylicum, Clostridium Zymonomas mobilis, Methylobacterium extorquens, Ralstonia eutropha, Saccharomyces cerevisiae, Rhodobacter sphaeroides, Paracoccus versutus, Pseudomonas aeruginosa, Acinetobacter calcoaceticus, Clostridium acetobutylicum, Clostridium
saccharoperbutylacetonicum, Clostridium beijerinckii, Rhodospirillum rubrum, Burkholderia thailandensis oder Pseudomonas putida Zelle. saccharoperbutylacetonicum, Clostridium beijerinckii, Rhodospirillum rubrum, Burkholderia thailandensis or Pseudomonas putida cell.
Die erfindungsgemäße rekombinante Zelle kann beispielsweise derart gentechnisch verändert sein, dass sie, im Vergleich zu ihrem Wildtyp, mehr Saccharose aufnehmen kann. Eine andere Möglichkeit, die alternativ oder auch zusätzlich Anwendung finden kann, ist eine gentechnische Veränderung der Zelle, die es der rekombinanten Zelle ermöglicht, im Vergleich zu ihrem Wildtyp, mehr Kohlendioxid zu fixieren. For example, the recombinant cell of the present invention may be genetically engineered to accommodate more sucrose compared to its wild-type. Another possibility, which may alternatively or additionally be used, is a genetic modification of the cell which allows the recombinant cell to fix more carbon dioxide compared to its wild-type.
In einer Ausführungsform der Erfindung wird die Steigerung der Saccharoseaufnahme dadurch erreicht, dass die Zelle eine im Vergleich zu ihrem Wildtyp gesteigerte Aktivität mindestens eines Enzyms aufweist, welches den Transport von Saccharose in die Zelle katalysiert. In one embodiment of the invention, the increase in sucrose uptake is achieved by virtue of the cell having an increased activity of at least one enzyme which catalyzes the transport of sucrose into the cell compared to its wild-type.
Der Ausdruck„mindestens eines", wie hierin verwendet, bezieht sich auf Mengen von >1 , d.h. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30 oder mehr, insbesondere 1 , 2, 3, 4 oder 5. The term "at least one" as used herein refers to amounts of> 1, ie 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 22, 24, 26, 28, 30 or more, in particular 1, 2, 3, 4 or 5.
In einer Ausführungsform umfasst die rekombinante Zelle der Erfindung ein Enzym E-i , das den Transport von Saccharose in die Zelle und die Umsetzung zu Saccharose-6-Phosphat katalysiert. In one embodiment, the recombinant cell of the invention comprises an enzyme E-i, which catalyzes the transport of sucrose into the cell and the conversion to sucrose-6-phosphate.
Unter dem Begriff„Enzym" oder„Ex", wie hierin verwendet, wobei x eine ganze Zahl bedeutet, wird ein Protein oder Proteinkomplex verstanden, das/der eine oder mehrere biochemische Reaktionen katalysiert und/oder das/der zum Transport von bestimmten Verbindungen, beispielsweise durch eine Membran, dient. By the term "enzyme" or "E x " as used herein, wherein x is an integer, is meant a protein or protein complex that catalyzes one or more biochemical reactions and / or the transport of certain compounds , For example, by a membrane, is used.
Die Begriffe„gesteigerte Aktivität eines Enzyms" oder„verminderte Aktivität eines Enzyms", wie hierin verwendet, beziehen sich vorzugsweise auf eine gesteigerte/verminderte intrazelluläre oder membranständige Aktivität. Die nun folgenden Ausführungen zur Erhöhung oder Verminderung der Enzymaktivität in Zellen gelten sowohl für die Erhöhung/Verminderung der Aktivität des Enzyms E-ι als auch für alle nachfolgend genannten Enzyme, deren Aktivität gegebenenfalls erhöht oder vermindert werden kann. The terms "enhanced activity of an enzyme" or "decreased activity of an enzyme" as used herein preferably refer to increased / decreased intracellular or membrane-bound activity. The following statements on increasing or decreasing the enzyme activity in cells apply both to the increase / decrease in the activity of the enzyme E-1 and to all the enzymes mentioned below, the activity of which may optionally be increased or decreased.
Grundsätzlich lässt sich eine Steigerung der enzymatischen Aktivität dadurch erzielen, dass man die Kopienzahl der Gensequenz bzw. der Gensequenzen erhöht, welche für das Enzym kodieren, einen starken Promotor verwendet, die Kodonnutzung des Gens verändert, auf verschiedene Art und Weise die Halbwertszeit der mRNA oder des Enzyms erhöht, Repression eliminiert, Inhibition verhindert oder ein Gen oder Allel einschleust oder der Art manipuliert, dass es für ein entsprechendes Enzym mit einer gesteigerten Aktivität kodiert. Diese Maßnahmen werden gegebenenfalls kombiniert. Erfindungsgemäß gentechnisch veränderte Zellen werden beispielsweise durch Transformation, Transduktion, Konjugation oder einer Kombination dieser Methoden mit einem Vektor erzeugt, der das gewünschte Gen, ein Allel dieses Gens oder Teile davon und einen die Expression des Gens ermöglichenden Vektor enthält. Die heterologe Expression wird insbesondere durch Integration des Gens oder der Allele in das Chromosom der Zelle oder einem extrachromosomal replizierenden Vektor erzielt. In principle, an increase in enzymatic activity can be achieved by increasing the copy number of the gene sequence or gene sequences which code for the enzyme, using a strong promoter, changing the codon usage of the gene, in various ways the half-life of the mRNA or of the enzyme increases, eliminates repression, prevents inhibition or infiltrates a gene or allele, or manipulates the species to encode a corresponding enzyme with enhanced activity. If necessary, these measures will be combined. Genetically engineered cells according to the invention are produced for example by transformation, transduction, conjugation or a combination of these methods with a vector which contains the desired gene, an allele of this gene or parts thereof and a vector which enables expression of the gene. In particular, heterologous expression is achieved by integration of the gene or alleles into the chromosome of the cell or an extrachromosomally replicating vector.
Einen Überblick über die Möglichkeiten zur Erhöhung der Enzym-Aktivität in Zellen am Beispiel der Pyruvat-Carboxylase gibt DE-A-100 31 999, die hiermit als Referenz eingeführt wird und deren Offenbarungsgehalt hinsichtlich der Möglichkeiten zur Erhöhung der Enzym-Aktivität in Zellen einen Teil der Offenbarung der vorliegenden Erfindung bildet. An overview of the possibilities for increasing the enzyme activity in cells using the example of the pyruvate carboxylase is given in DE-A-100 31 999, which is hereby incorporated by reference, and the disclosure of which relates to the possibilities of increasing the enzyme activity in cells of the disclosure of the present invention.
Die Expression der vorstehend und aller nachfolgend genannten Enzyme bzw. Gene ist mit Hilfe von 1 - und 2- dimensionaler Proteingelauftrennung und anschließender optischer The expression of the above and of all the enzymes and genes mentioned below is effected with the aid of 1- and 2-dimensional protein gel separation and subsequent optical
Identifizierung der Proteinkonzentration mit entsprechender Auswert-Software im Gel nachweisbar. Wenn die Erhöhung einer Enzymaktivität ausschließlich auf einer Erhöhung der Expression des entsprechenden Gens basiert, so kann die Quantifizierung der Erhöhung der Enzymaktivität in einfacher Weise durch einen Vergleich der 1 - oder 2- dimensionalen Identification of the protein concentration with appropriate evaluation software in the gel detectable. If the enhancement of an enzyme activity is based solely on an increase in the expression of the corresponding gene, the quantification of the increase in the enzyme activity can be easily determined by comparing the 1 - or 2 - dimensional
Proteinauftrennungen zwischen Wildtyp und gentechnisch veränderter Zelle bestimmt werden. Eine gebräuchliche Methode zur Präparation der Proteingele bei coryneformen Bakterien und zur Identifizierung der Proteine ist die von Hermann et al. (Electrophoresis, 22: 1712.23 (2001 )) beschriebene Vorgehensweise. Die Proteinkonzentration kann ebenfalls durch Western-Blot- Hybridisierung mit einem für das nachzuweisende Protein spezifischen Antikörper (Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. USA, 1989) und anschließender optische Auswertung mit Protein separations between wild type and genetically engineered cell can be determined. A common method for preparing the protein gels in coryneform bacteria and for identifying the proteins is that described by Hermann et al. (Electrophoresis, 22: 1712.23 (2001)). The protein concentration can also be determined by Western Blot hybridization with an antibody specific for the protein to be detected (Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY USA, 1989) and subsequent optical evaluation with
entsprechender Software zur Konzentrationsbestimmung (Lohaus und Meyer (1989) appropriate software for concentration determination (Lohaus and Meyer (1989)
Biospektrum, 5: 32-39; Lottspeich (1999), Angewandte Chemie 1 1 1 : 2630-2647) analysiert werden. Die Aktivität von DNA-bindenden Proteinen kann mittels DNA-Band-Shift-Assays (auch als Gelretardation bezeichnet) gemessen werden (Wilson et al. (2001 ) Journal of Bacteriology, 183: 2151 -2155). Die Wirkung von DNA- bindenden Proteinen auf die Expression anderer Gene kann durch verschiedene gut beschriebene Methoden des Reportergen-Assays nachgewiesen werden (Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. USA, 1989). Die intrazellulären enzymatischen Aktivitäten können nach verschiedenen beschriebenen Methoden (Donahue et al. (2000) Journal of Bacteriology 182 (19): 5624-5627; Ray et al. (2000) Journal of Bacteriology 182 (8) : 2277-2284; Freedberg et al. (1973) Journal of Bacteriology 1 15 (3): 816- 823) bestimmt werden. Sofern in den nachfolgenden Ausführungen keine konkreten Methoden zur Bestimmung der Aktivität eines bestimmten Enzyms angegeben werden, erfolgt die Bestimmung der Steigerung der Enzymaktivität und auch die Bestimmung der Verminderung einer Enzymaktivität vorzugsweise mittels der in Hermann et al. (Electophoresis, 22: 1712-23 (2001 )), Lohaus et al. (Biospektrum 5 32- 39 (1998)), Lottspeich (Angewandte Chemie 1 1 1 : 2630- 2647 (1999)) und Wilson et al. (Journal of Bacteriology 183: 2151 -2155 (2001 )) beschriebenen Methoden. Wird die Erhöhung der Enzymaktivität durch Mutation des endogenen Gens bewerkstelligt, so können derartige Mutationen entweder nach klassischen Methoden ungerichtet erzeugt werden, wie etwa durch UV-Bestrahlung oder durch mutationsauslösende Chemikalien, oder gezielt mittels gentechnologischer Methoden wie Deletion(en) Jnsertion(en) und/oder Bios spectrum, 5: 32-39; Lottspeich (1999), Angewandte Chemie 1 1 1: 2630-2647). The activity of DNA-binding proteins can be measured by DNA band shift assays (also referred to as gel retardation) (Wilson et al., (2001) Journal of Bacteriology, 183: 2151-2155). The effect of DNA-binding proteins on the expression of other genes can be demonstrated by various well-described methods of the reporter gene assay (Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd Ed Cold Spring Harbor Laboratory Press, Cold Spring Harbor). NY USA, 1989). The intracellular enzymatic activities can be determined by various methods described (Donahue et al., (2000) Journal of Bacteriology 182 (19): 5624-5627, Ray et al., (2000) Journal of Bacteriology 182 (8): 2277-2284, Freedberg et (1973) Journal of Bacteriology 1 15 (3): 816-823). Unless specific methods for determining the activity of a specific enzyme are specified in the following, the determination of the increase in the enzyme activity and also the determination of the reduction of an enzyme activity are preferably carried out by means of the methods described in Hermann et al. (Electophoresis, 22: 1712-23 (2001)), Lohaus et al. (Biospektrum 5 32-39 (1998)), Lottspeich (Angewandte Chemie 1 1 1: 2630- 2647 (1999)) and Wilson et al. (Journal of Bacteriology 183: 2151-2155 (2001)). If the increase in enzyme activity is accomplished by mutation of the endogenous gene, then such mutations can be generated either undirected by classical methods, such as by UV irradiation or by mutagenic chemicals, or specifically by genetic engineering methods such as deletion (s) Jnsertion (s) and /or
Nukleotidaustausch(e). Durch diese Mutationen werden gentechnisch veränderte Zellen erhalten. Besonders bevorzugte Mutanten von Enzymen sind insbesondere auch solche Enzyme, die nicht mehr oder zumindest im Vergleich zum Wildtyp- Enzym vermindert Nucleotide substitution (s). These mutations result in genetically engineered cells. Particularly preferred mutants of enzymes are, in particular, also those enzymes which no longer diminish or at least in comparison with the wild-type enzyme
Feedback-inhibierbar sind, wenn eine gesteigerte Enzymaktivität gewünscht ist. Feedback-inhibited, if an increased enzyme activity is desired.
Wird die Erhöhung der Enzymaktivität durch Erhöhung der Expression eines Enzyms bewerkstelligt, so erhöht man beispielsweise die Kopienzahl der entsprechenden Gene oder mutiert die Promotor- und Regulationsregion oder die Ribosomenbindungsstelle, die sich stromaufwärts des Strukturgens befindet. In gleicher weise wirken Expressionskassetten, die stromaufwärts des Strukturgens eingebaut werden. Durch induzierbare Promotoren ist es zusätzlich möglich, die Expression zu jedem beliebigen Zeitpunkt zu steigern. Des Weiteren können dem Enzym-Gen als regulatorische Sequenzen aber auch sogenannte "Enhancer" zugeordnet sein, die über eine verbesserte Wechselwirkung zwischen RNA-Polymerase und DNA ebenfalls eine erhöhte Genexpression bewirken. Durch Maßnahmen zur Verlängerung der Lebensdauer der m-RNA wird ebenfalls die Expression verbessert. Weiterhin wird durch Verhinderung des Abbaus des Enzymproteins ebenfalls die Enzymaktivität verstärkt. Die Gene oder Genkonstrukte liegen dabei entweder in Plasmiden mit unterschiedlicher Kopienzahl vor oder sind im Chromosom integriert und amplifiziert. Alternativ kann weiterhin eine If the increase in enzyme activity is accomplished by increasing the expression of an enzyme, for example, one increases the copy number of the corresponding genes or mutates the promoter and regulatory region or the ribosome binding site, which is upstream of the structural gene. In the same way, expression cassettes act, which are installed upstream of the structural gene. Inducible promoters also make it possible to increase expression at any time. Furthermore may be assigned to the enzyme gene as regulatory sequences but also so-called "enhancer", which also cause increased gene expression via an improved interaction between RNA polymerase and DNA. Measures to extend the lifetime of m-RNA also improve expression. Furthermore, by preventing degradation of the enzyme protein, enzyme activity is also enhanced. The genes or gene constructs are either present in plasmids with different copy numbers or are integrated and amplified in the chromosome. Alternatively, a further
Überexpression der betreffenden Gene durch Veränderung der Medienzusammensetzung und Kulturführung erreicht werden. Anleitungen hierzu findet der Fachmann unter anderem bei Martin et al. (Bio/Technology 5, 137-146 (1987)), bei Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya und Morinaga (Bio/Technology 6, 428-430 (1988)), bei Eikmanns et al. (Gene 102, 93- 98 (1991 )), in EP-A-0 472 869, in US 4,601 ,893, bei Schwarzer und Puhler (Bio/Technology 9, 84-87 (1991 )), bei Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), bei LaBarre et al. (Journal of Bacteriology 175, 1001 -1007 (1993)), in WO-A- 96/15246, bei Malumbres et al. (Gene 134, 15-24 (1993)), in JP-A-10-229891 , bei Jensen und Hammer (Biotechnology and Bioengineering 58, 191 -195 (1998)) und in bekannten Lehrbüchern der Genetik und Molekularbiologie. Die vorstehend beschriebenen Maßnahmen führen ebenso wie die Mutationen zu gentechnisch veränderten Zellen. Zur Erhöhung der Expression der jeweiligen Gene werden zum Beispiel episomale Plasmide eingesetzt. Als Plasmide bzw. Vektoren kommen im Prinzip alle dem Fachmann für diesen Zweck zur Verfügung stehenden Ausführungsformen in Frage. Derartige Plasmide und Overexpression of the genes in question can be achieved by changing the composition of the medium and culture. For instructions on this, the skilled person will find, inter alia, in Martin et al. (Bio / Technology 5, 137-146 (1987)), Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio / Technology 6, 428-430 (1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), in EP-A-0 472 869, in US 4,601,893, in Schwarzer and Puhler (Bio / Technology 9, 84-87 (1991)), in Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), WO-A-96/15246, Malumbres et al. (Gene 134, 15-24 (1993)), in JP-A-10-229891, in Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)) and in known textbooks of genetics and molecular biology. The measures described above as well as the mutations lead to genetically modified cells. To increase the expression of the respective genes, episomal plasmids are used, for example. In principle, all embodiments which are available to the person skilled in the art for this purpose are suitable as plasmids or vectors. Such plasmids and
Vektoren können z. B. den Broschüren der Firmen Novagen, Promega, New England Biolabs, Clontech oder Gibco BRL entnommen werden. Weitere bevorzugte Plasmide und Vektoren können gefunden werden in: Glover, D. M. (1985), DNA cloning: a practical approach, Vol. I-Ill, IRL Press Ltd., Oxford; Rodriguez, R.L. und Denhardt, D. T (eds) (1988), Vectors : a survey of molecular cloning vectors and their uses, 179-204, Butterworth, Stoneham; Goeddel, D. V. (1990), Systems for heterologous gene expression, Methods Enzymol. 185, 3-7; Sambrook, J.; Fritsch, E. F. und Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York; Casali, N. and Preston, A. (eds) (2003) E. coli Plasmid Vectors. Humana Press. Als Plasmide eignen sich insbesondere solche, die in coryneformen Bakterien repliziert werden. Zahlreiche bekannte Plasmidvektoren, wie zum Beispiel pZ1 (Menkel et al., Applied and Environmental Microbiology 64: 549-554 (1989)), pEKExl (Eikmanns et al., Gene 107: 69-74 (1991 )) oder pHS2-l (Sonnen et al., Gene 107: 69- 74 (1991 )) beruhen auf den kryptischen Plasmiden pHM1519, pBL1 oder pGA1 . Andere Plasmidvektoren, wie zum Beispiel solche, die auf pCG4 (US 4,489,160) oder pNG2 (Serwold- Davis et al. , FEMS Microbiology Letters 66: 1 19- 124 (1990)) oder pAG1 (US 5,158,891 ) beruhen, können in gleicher weise eingesetzt werden. Weiterhin eignen sich auch solche Plasmidvektoren, mit Hilfe derer man das Verfahren der Genamplifikation durch Integration in das Chromosom anwenden kann, so wie es beispielsweise von Reinscheid et al. (Applied and Environmental Microbiology 60: 126-132 (1994)) zur Duplikation bzw. Amplifikation des hom- thrB-Operons beschrieben wurde. Bei dieser Methode wird das vollständige Gen in einen Plasmidvektor kloniert, der in einem Wirt (typischerweise Escherichia coli), nicht aber in Corynebacterium glutamicum repliziert werden kann. Als Vektoren kommen beispielsweise pSUP301 (Simon et al., Bio/Technology 1 : 784-791 (1983)), pK18mob oder pK19mob (Schäfer et al., Gene 145: 69-73 (1994)), pGEM-T (Promega Corporation, Madison, Wisconsin, USA), pCR2.1 -TOPO (Shuman, Journal of Biological Chemistry 269: 32678-84 (1994)), pCR®Blunt (Invitrogen, Groningen, Niederlande), pEM1 (Schrumpf et al., Journal of Bacteriology 173: 4510-4516)) oder pBGS8 (Spratt et al., Gene 41 : 337-342 (1986)) in Frage. Der Plasmidvektor, der das zu amplifizierende Gen enthält, wird anschließend durch Konjugation oder Vectors can, for. B. the brochures of the companies Novagen, Promega, New England Biolabs, Clontech or Gibco BRL be removed. Further preferred plasmids and vectors can be found in: Glover, DM (1985), DNA cloning: a practical approach, Vol. I-III, IRL Press Ltd., Oxford; Rodriguez, RL and Denhardt, D.T. (eds) (1988), Vectors: a survey of molecular cloning vectors and their uses, 179-204, Butterworth, Stoneham; Goeddel, DV (1990), Systems for heterologous gene expression, Methods Enzymol. 185, 3-7; Sambrook, J .; Fritsch, EF and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York; Casali, N. and Preston, A. (eds) (2003) E. coli Plasmid Vectors. Humana Press. Suitable plasmids are in particular those which are replicated in coryneform bacteria. Numerous known plasmid vectors, such as pZ1 (Menkel et al., Applied and Environmental Microbiology 64: 549-554 (1989)), pEKEx1 (Eikmanns et al., Gene 107: 69-74 (1991)) or pHS2-l ( Sonnen et al., Gene 107: 69- 74 (1991)) are based on the cryptic plasmids pHM1519, pBL1 or pGA1. Other plasmid vectors, such as those based on pCG4 (US 4,489,160) or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66: 1 19-124 (1990)) or pAG1 (US 5,158,891) can be used in the same way be used. Also suitable are those plasmid vectors with the aid of which one can apply the method of gene amplification by integration into the chromosome, as described for example by Reinscheid et al. (Applied and Environmental Microbiology 60: 126-132 (1994)) for duplication or amplification of the homodB operon. In this method, the complete gene is cloned into a plasmid vector which can be replicated in a host (typically Escherichia coli) but not in Corynebacterium glutamicum. Examples of vectors include pSUP301 (Simon et al., Bio / Technology 1: 784-791 (1983)), pK18mob or pK19mob (Schäfer et al., Gene 145: 69-73 (1994)), pGEM-T (Promega Corporation , Madison, Wisconsin, USA), pCR2.1 -TOPO (Shuman, Journal of Biological Chemistry 269: 32678-84 (1994)), pCR ® Blunt (Invitrogen, Groningen, The Netherlands), pEM1 (shrink et al, Journal of. Bacteriology 173: 4510-4516)) or pBGS8 (Spratt et al., Gene 41: 337-342 (1986)). The plasmid vector containing the gene to be amplified is then purified by conjugation or
Transformation in den gewünschten Stamm von Corynebacterium glutamicum überführt. Die Methode der Konjugation ist beispielsweise bei Schäfer et al., Applied and Environmental Microbiology 60: 756-759 (1994) beschrieben. Methoden zur Transformation sind Transformation into the desired strain of Corynebacterium glutamicum transferred. The method of conjugation is described, for example, in Schäfer et al., Applied and Environmental Microbiology 60: 756-759 (1994). Methods for transformation are
beispielsweise bei Thierbach et al., Applied Microbiology and Biotechnology 29: 356-362 (1988), Dunican und Shivnan, Bio/Technology 7: 1067-1070 (1989) und Tauch et al., FEMS Microbiology Letters 123: 343-347 (1994) beschrieben. Nach homologer Rekombination mittels eines„cross-over"-Ereignisses enthalt der resultierende Stamm mindestens zwei Kopien des betreffenden Gens. for example, Thierbach et al., Applied Microbiology and Biotechnology 29: 356-362 (1988), Dunican and Shivnan, Bio / Technology 7: 1067-1070 (1989) and Tauch et al., FEMS Microbiology Letters 123: 343-347 (1989). 1994). After homologous recombination by means of a "cross-over" event, the resulting strain contains at least two copies of the gene in question.
Mit den oben genannten Methoden lässt sich analog eine Verringerung der enzymatischen Aktivität erzielen. Dies gliedert sich ebenfalls in zwei Strategien, die Reduktion der Expression und/oder die Inhibierung der Enzymaktivität. Zur Reduktion der Expression kann beispielsweise das korrespondierende Gen ganz oder teilweise deletiert werden. Zudem kann die The above-mentioned methods can analogously achieve a reduction in the enzymatic activity. This is also divided into two strategies, the reduction of expression and / or the inhibition of enzyme activity. To reduce the expression, for example, the corresponding gene can be completely or partially deleted. In addition, the
Transkription, beispielsweise durch die Manipulation der Promotorregion oder Verstärkung der Repression (genetisch oder chemisch) bzw. die Herabsetzung der mRNA-Halbwertszeit, inhibiert oder reduziert werden. Ebenfalls kann auf RNA-Ebene die Translation gestört oder reduziert werden. Dem Fachmann sind dazu zahlreiche Techniken bekannt, zum Beispiel die RNAi-Technologie oder die Modifikation der DNA-Sequenz dahingehend, dass es zur Ausbildung von Sekundärstrukturen auf mRNA-Ebene kommt, die die Translation inhibieren oder reduzieren. Die Enzymaktivität kann beispielsweise durch Zugabe von Inhibitoren, durch Einfügen von gerichteten oder ungerichteten Mutationen reduziert werden. Unter der vorstehend und in den nachfolgenden Ausführungen verwendeten Formulierung„eine gegenüber ihrem Wildtyp gesteigerte Aktivität eines Enzyms Ex" ist vorzugsweise stets eine um einen Faktor von mindestens 2, besonders bevorzugt von mindestens 10, darüber hinaus bevorzugt von mindestens 100, darüber hinaus noch mehr bevorzugt von mindestens 1 .000 und am meisten bevorzugt von mindestens 10.000 gesteigerte Aktivität des jeweiligen Enzyms Ex zu verstehen. Weiterhin umfasst die erfindungsgemäße Zelle, welche„eine gegenüber ihrem Wildtyp gesteigerte Aktivität eines Enzyms Ex" aufweist, insbesondere auch eine Zelle, deren Wildtyp keine oder zumindest keine nachweisbare Aktivität dieses Enzyms Ex aufweist und die erst nach Erhöhung der Enzymaktivität, beispielsweise durch Überexpression, eine Transcription, for example, by the manipulation of the promoter region or enhancement of repression (genetically or chemically) or the reduction of the mRNA half-life, be inhibited or reduced. Likewise, at the RNA level, translation can be disturbed or reduced. Numerous techniques are known to the person skilled in the art, for example the RNAi technology or the modification of the DNA sequence in that it is intended for Secondary structure formation occurs at the mRNA level, which inhibit or reduce translation. The enzyme activity can be reduced, for example, by adding inhibitors, by introducing directional or non-directional mutations. The expression "an activity of an enzyme E x which is increased with respect to its wild type" is preferably always a factor greater than or equal to 2, particularly preferably at least 10, more preferably at least 100, and even more preferably of at least 1, 000 and most preferably of at least 10,000 increased activity of the respective enzyme E x Furthermore, the cell according to the invention comprises "an activity of an enzyme E x increased compared to its wild type, in particular also a cell whose Wild type has no or at least no detectable activity of this enzyme E x and only after increasing the enzyme activity, for example by overexpression, a
nachweisbare Aktivität dieses Enzyms Ex zeigt. In diesem Zusammenhang umfasst der Begriff „Überexpression" oder die in den nachfolgenden Ausführungen verwendete Formulierung „Erhöhung der Expression" auch den Fall, dass eine Ausgangszelle, beispielsweise eine Wildtyp-Zelle, keine oder zumindest keine nachweisbare Expression aufweist und erst durch rekombinante Verfahren eine nachweisbare Expression des Enzyms Ex induziert wird. Unter der nachfolgend verwendeten Formulierung„verminderte Aktivität eines Enzyms Ex" wird dementsprechend vorzugsweise eine um einen Faktor von mindestens 0,5, besonders bevorzugt von mindestens 0,1 , darüber hinaus bevorzugt von mindestens 0,01 , darüber hinaus noch mehr bevorzugt von mindestens 0,001 und am meisten bevorzugt von mindestens 0,0001 verminderte Aktivität verstanden. Die Verminderung der Aktivität eines bestimmten Enzyms kann beispielsweise durch gezielte Mutation, genetische Deletion des Gens, durch Zugabe von kompetitiven oder nicht kompetitiven Inhibitoren oder durch andere, weiter oben beschriebene bzw. dem Fachmann bekannte Maßnahmen zur Verminderung der Expression und/oder Funktion eines bestimmten Enzyms erfolgen. Dabei ist es erfindungsgemäß bevorzugt, dass die gentechnisch veränderte Zelle derart gentechnisch verändert ist, dass sie in einem definierten Zeitintervall, vorzugsweise innerhalb von 2 Stunden, noch mehr bevorzugt innerhalb von 8 Stunden und am meisten bevorzugt innerhalb von 24 Stunden, mindestens 2mal, besonders bevorzugt mindestens lOmal, darüber hinaus bevorzugt mindestens lOOmal, darüber hinaus noch mehr bevorzugt mindestens I.OOOmal und am meisten bevorzugt mindestens 10.000 mal mehr C4-Körper bildet als der Wildtyp der Zelle. Die Zunahme der Produktbildung kann dabei beispielsweise dadurch bestimmt werden, dass die erfindungsgemäße Zelle und die Wildtyp-Zelle jeweils getrennt unter gleichen Bedingungen (gleiche Zelldichte, gleiches Nährmedium, gleiche Kulturbedingungen) für ein bestimmtes Zeitintervall in einem geeigneten Nährmedium kultiviert werden und anschließend die Menge an Zielprodukt (C4-Körper) im Nährmedium bestimmt wird. detectable activity of this enzyme E x shows. In this context, the term "overexpression" or the expression "increase in expression" used in the following also encompasses the case that a starting cell, for example a wild-type cell, has no or at least no detectable expression and only by recombinant methods a detectable Expression of the enzyme E x is induced. Accordingly, under the expression "reduced activity of an enzyme E x " used below, it is preferable to use a factor of at least 0.5, more preferably at least 0.1, more preferably at least 0.01, even more preferably at least The reduction of the activity of a particular enzyme may be accomplished, for example, by targeted mutation, genetic deletion of the gene, by the addition of competitive or non-competitive inhibitors, or by others described above It is preferred in accordance with the invention for the genetically modified cell to be genetically modified in such a way that it is metabolized in a defined time interval, preferably within 2 hours, more preferably within a defined time interval 8 hours n and most preferably within 24 hours, at least 2 times, more preferably at least 10 times, more preferably at least 100 times, even more preferably at least I.OOO times, and most preferably at least 10,000 times more C 4 body forms than the wild type of the cell. The increase in product formation can be determined, for example, by culturing the cell according to the invention and the wild-type cell separately under the same conditions (same cell density, same nutrient medium, same culture conditions) for a specific time interval in a suitable nutrient medium and then the amount Target product (C 4 body) is determined in the nutrient medium.
Das Enzym E-ι kann beispielsweise ein Phosphoenolpyruvat (PEP)-abhängiges The enzyme E-1 can, for example, a phosphoenolpyruvate (PEP) -dependent
Phosphotransferase-System (PTS) Enzym I I (Saccharose-spezifisch) (EC 2.7.1 .69; TCDB Klassifikation 4.A.1 .2.1 , 4.A.1 .2.9 oder 4.A.1 .2.-) sein. Phosphotransferase system (PTS) Enzyme II (sucrose-specific) (EC 2.7.1 .69; TCDB classification 4.A.1 .2.1, 4.A.1 .2.9 or 4.A.1 .2.-) ,
Das Enzym E-ι kann beispielsweise durch das scrA Gen kodiert werden. Die kodierende Nukleotidsequenz und die dazugehörige Proteinsequenz können beispielsweise der„Kyoto Encyclopedia of Genes and Genomes" (KEGG-Datenbank) , den Datenbanken des National Center for Biotechnology Information (NCBI) der National Library of Medicine (Bethesda, MD, USA), der Proteindatenbank UniProt (Kooperation des European Bioinformatics Institute (EBI), des Swiss Institute of Bioinformatics (SI B) und des Protein Information Resource (PI R)) oder der Nukleotidsequenz-Datenbank der European Molecular Biologies Laboratories (EMBL, Heidelberg, Deutschland bzw. Cambridge, UK) entnommen werden. In einem besonderen Fall handelt es sich bei Ei um das aus E. coli stammende scrA Gen (SEQ I D NO:41 ) und das daraus resultierende Protein. Darüber hinaus wird das Enzym Ei vorzugsweise von Genen kodiert, die aus der Gruppe derer ausgewählt werden, die Genprodukte kodieren, deren Aminosäuresequenz über einen Bereich von mindestens 100, bevorzugt mindestens 200, insbesondere mindestens 300 Aminosäuren, zu mindestens 60%, vorzugsweise zu mindestens 80%, besonders bevorzugt zu mindestens 95%, ganz besonders bevorzugt zu mindestens 99%, insbesondere zu 100% zu SEQ ID NO: 58 identisch ist. The enzyme E-1 can be coded, for example, by the scrA gene. The coding nucleotide sequence and the associated protein sequence can be found, for example, in the "Kyoto Encyclopedia of Genes and Genomes" (KEGG database), the National Library of Biotechnology Information (NCBI) databases of the National Library of Medicine (Bethesda, MD, USA), the protein database UniProt (cooperation of the European Bioinformatics Institute (EBI), the Swiss Institute of Bioinformatics (SI B) and the Protein Information Resource (PI R)) or the nucleotide sequence database of the European Molecular Biology Laboratories (EMBL, Heidelberg, Germany and Cambridge, In a particular case, egg is the scrA gene (SEQ ID NO: 41) derived from E. coli and the resulting protein, and in addition, the enzyme Ei is preferably encoded by genes derived from the Group of those are selected which encode gene products whose amino acid sequence over a range of at least 100, preferably at least 200, in particular minde at least 300 amino acids, at least 60%, preferably at least 80%, more preferably at least 95%, most preferably at least 99%, in particular 100% to SEQ ID NO: 58 is identical.
In einer Ausführungsform wird das Enzym E-ι von Genen kodiert, die aus der Gruppe derer ausgewählt werden, die Genprodukte kodieren, in deren Aminosäuresequenz bei einer Suche nach in der betreffenden Aminosäuresequenz enthaltenen konservierten Proteindomänen in der NCBI CDD (Version 2.20) mittels RPS-BLAST die Präsenz der konservierten Domäne„PTS-II- BC-sucr" (TIGR01996 oder PssmI D 131051 ) mit einem E-Wert (englisch„e-value") kleiner 1 x 10"5 festgestellt (englisch„domain hit") wird. Zusätzlich zu dem Enzym E-i , das den Transport von Saccharose in die Zelle und die In one embodiment, the enzyme E-1 is encoded by genes which are selected from the group of those encoding gene products in whose amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (Version 2.20) by means of RPS BLAST the presence of the conserved domain "PTS-II BC-sucr" (TIGR01996 or PssmI D 131051) with an E value (English "e-value") less than 1 x 10 "5 detected (English" domain hit ") , In addition to the enzyme egg, which facilitates the transport of sucrose into the cell and the
Umsetzung zu Saccharose-6-Phosphat katalysiert, kann die rekombinante Zelle ferner eine im Vergleich zu ihrem Wildtyp gesteigerte Aktivität mindestens eines Enzyms E2, E3 und E4 oder einer Kombination dieser Enzyme aufweisen. Dabei kann E2 ein Enzym sein, das die Conversion to sucrose-6-phosphate catalyzed, the recombinant cell may further have an increased compared to their wild type activity of at least one enzyme E 2 , E 3 and E 4 or a combination of these enzymes. In this case, E 2 can be an enzyme that the
Umsetzung von Saccharose-6-Phosphat zu -D-Glukose-6-Phosphat und -D-Fruktose katalysiert, E3 kann ein Enzym sein, das die Umsetzung von -D-Fruktose zu -D-Fruktose-6- Phosphat katalysiert, und E4 kann ein Kanal sein, der diffusions-abhängigen Saccharose- Transport in die Zelle erlaubt. In einer Ausführungsform betrifft die Erfindung daher rekombinante Zellen, die im Vergleich zu ihrem Wildtyp eine gesteigerte Aktivität mindestens eines der Enzyme E2, E3 und E4 aufweisen. In einer Ausführungsform ist im Vergleich zum Wildtyp die Aktivität des Enzyms E-i und mindestens eines der Enzyme E2, E3 und E4 gesteigert. In einer anderen Ausführungsform ist im Vergleich zum Wildtyp die Aktivität der Enzyme i) E-i , E2 und E3, ii) E2, E3 und E4, iii) E2 und E3, iv) E3 und E4, v) E2 und E4, oder vi) E-i , E3 und E4 erhöht. In einer weiteren Ausführungsform weist die erfindungsgemäße rekombinante Zelle eine im Vergleich zum Wildtyp erhöhte Aktivität der Enzyme Ei , E2, E3 und E4 auf. Catalysed conversion of sucrose-6-phosphate to -D-glucose-6-phosphate and -D-fructose, E 3 may be an enzyme that catalyzes the conversion of D-fructose to D-fructose-6-phosphate, and E 4 may be a channel that allows diffusion-dependent sucrose transport into the cell. In one embodiment, the invention therefore relates to recombinant cells which have an increased activity of at least one of the enzymes E 2 , E 3 and E 4 in comparison to their wild type. In one embodiment, the activity of the enzyme Ei and at least one of the enzymes E 2 , E 3 and E 4 is increased compared to the wild type. In another embodiment, the activity of the enzymes compared to the wild-type is i) Ei, E 2 and E 3 , ii) E 2 , E 3 and E 4 , iii) E 2 and E 3 , iv) E 3 and E 4 , v) E 2 and E 4 , or vi) Ei, E 3 and E 4 increased. In a further embodiment, the recombinant cell according to the invention has an increased activity of the enzymes Ei, E 2 , E 3 and E 4 in comparison with the wild type.
In weiteren Ausführungsformen kann es sich bei dem Enzym E2 um eine Saccharose-6- Phosphat-Fruktohydrolase (EC 3.2.1 .26), bei dem Enzym E3 um eine Fruktokinase (EC 2.7.1 .4) und/oder bei dem Enzym E4 um ein Saccharose Porin (TCDB Klassifikation 1 .B.3.1 .2) handeln. In further embodiments, the enzyme E 2 may be a sucrose-6-phosphate fructohydrolase (EC 3.2.1 .26), the enzyme E 3 may be a fructokinase (EC 2.7.1 .4) and / or the Enzyme E 4 is a sucrose porin (TCDB Classification 1 .B.3.1 .2).
In einer Ausführungsform der Erfindung wird E2 von dem scrB (£. coli scrB: SEQ I D NO:42), bfrA, sacA, sacB, cscA, fruA oder susH Gen (Streptococcus pneumoniae susH: SEQ I D NO:52) kodiert, E3 wird von dem mac, yajF, mtlZ, rbsK, glcK, pfkB, frcK, frk, sacK, ydhR, kdgK, suk, ydjE, gmuE, ydjE, scrK (£. coli scrK: SEQ ID NO:43) oder cscK Gen (£. coli cscK: SEQ I D NO:46) kodiert und E4 wird von dem scrY Gen (£. coli scrY: SEQ ID NO:44) kodiert. In one embodiment of the invention E 2 is encoded by the scrB (E. coli scrB: SEQ ID NO: 42), bfrA, sacA, sacB, cscA, fruA or susH gene (Streptococcus pneumoniae susH: SEQ ID NO: 52), E 3 is derived from the mac, yajF, mtlZ, rbsK, glcK, pfkB, frcK, frk, sacK, ydhR, kdgK, suk, ydjE, gmuE, ydjE, scrK (E coli scrK: SEQ ID NO: 43) or cscK gene (£ coli ČSČK. SEQ ID NO: 46) encodes and e 4 is of the ScrY gene (£ coli ScrY. 44: SEQ ID NO) coded.
Die Nukleotidsequenzen der vorstehend genannten Gene, deren korrespondierenden The nucleotide sequences of the aforementioned genes, their corresponding
Proteinsequenzen sowie weitere Gene für die Enzyme E2 bis E4 können unter anderem auch der KEGG-, der NCBI-, der UniProt- oder EMBL-Datenbank entnommen werden. In einem besonderen Fall handelt es sich bei E2, E3 und/oder E4 um aus £ coli stammende Gene bzw. die dadurch kodierten Proteine. In einer Ausführungsform wird das Enzym E2 von Genen kodiert, die ausgewählt werden aus der Gruppe derer, die Genprodukte kodieren, deren Aminosäuresequenz über einen Bereich von mindestens 100, bevorzugt mindestens 200, insbesondere mindestens 300 Aminosäuren eine Sequenzidentität von mindestens 60%, vorzugsweise von mindestens 80%, besonders bevorzugt von mindestens 95%, ganz besonders bevorzugt von mindestens 99%, insbesondere von 100% zu SEQ ID NO:59 aufweisen. Protein sequences as well as other genes for the enzymes E 2 to E 4 can also be taken from the KEGG, NCBI, UniProt or EMBL database. In a particular case E 2 , E 3 and / or E 4 are genes derived from E. coli or the proteins encoded thereby. In one embodiment, the enzyme E 2 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, particularly preferably of at least 95%, very particularly preferably of at least 99%, in particular of 100%, to SEQ ID NO: 59.
In verschiedenen Ausführungsformen wird das Enzym E2 von Genen kodiert, die ausgewählt werden aus der Gruppe derer, die Genprodukte kodieren, in deren Aminosäuresequenz bei einer Suche nach in der betreffenden Aminosäuresequenz enthaltenen konservierten In various embodiments, the enzyme E 2 is encoded by genes which are selected from the group of those encoding gene products in their amino acid sequence in a search for conserved contained in the relevant amino acid sequence
Proteindomänen in der NCBI CDD (Version 2.20) mittels RPS-BLAST die Präsenz der konservierten Domäne„scrB_fam" (TIGR01322 oder PssmID 162301 ) mit einem E-Wert (englisch„e-value") kleiner 1 x 10"5 festgestellt wird (englisch„domain hit"). In einer Ausführungsform wird das Enzym E3 von Genen kodiert, die ausgewählt werden aus der Gruppe derer, die Genprodukte kodieren, deren Aminosäuresequenz über einen Bereich von mindestens 100, bevorzugt mindestens 200, insbesondere mindestens 300 Aminosäuren eine Sequenzidentität von mindestens 60%, vorzugsweise von mindestens 80%, besonders bevorzugt von mindestens 95%, ganz besonders bevorzugt von mindestens 99%, insbesondere von 100% zu SEQ ID NO:60 oder SEQ ID NO:63 aufweisen. Protein domains in the NCBI CDD (Version 2.20) by means of RPS-BLAST the presence of the conserved domain "scrB_fam" (TIGR01322 or PssmID 162301) with an E-value (English "e-value") smaller 1 x 10 "5 is found (English "Domain hit"). In one embodiment, the enzyme E 3 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, particularly preferably of at least 95%, very particularly preferably of at least 99%, in particular of 100%, to SEQ ID NO: 60 or SEQ ID NO: 63.
In einer Ausführungsform wird das Enzym E3 von Genen kodiert, die ausgewählt werden aus der Gruppe derer, die Genprodukte kodieren, deren Aminosäuresequenz bei einer Suche nach in der betreffenden Aminosäuresequenz enthaltenen konservierten Proteindomänen in der NCBI CDD (Version 2.20) mittels RPS-BLAST die Präsenz der konservierten DomäneIn one embodiment, the enzyme E 3 is encoded by genes which are selected from the group of those encoding gene products whose amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (Version 2.20) by means of RPS-BLAST Presence of the conserved domain
„bac_FRK" (cd01 167) mit einem E-Wert (englisch„e-value") kleiner 1 x 10"5 festgestellt wird (englisch„domain hit"). "Bac_FRK" (cd01 167) with an E value (English "e-value") smaller 1 x 10 "5 is found (English" domain hit ").
In einer Ausführungsform wird das Enzym E4 von Genen kodiert, die ausgewählt werden aus der Gruppe derer, die Genprodukte kodieren, deren Aminosäuresequenz über einen Bereich von mindestens 100, bevorzugt mindestens 200, insbesondere mindestens 300 Aminosäuren eine Sequenzidentität von mindestens 60%, vorzugsweise von mindestens 80%, besonders bevorzugt von mindestens 95%, ganz besonders bevorzugt von mindestens 99%, insbesondere von 100% zu SEQ ID NO:61 aufweisen. In einer Ausführungsform wird das Enzym E4 von Genen kodiert, die ausgewählt werden aus der Gruppe derer, die Genprodukte kodieren, in deren Aminosäuresequenz bei einer Suche nach in der betreffenden Aminosäuresequenz enthaltenen konservierten Proteindomänen in der NCBI CDD (Version 2.20) mittels RPS-BLAST die Präsenz der konservierten DomäneIn one embodiment, the enzyme E 4 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, more preferably at least 95%, most preferably at least 99%, especially 100% to SEQ ID NO: 61. In one embodiment, the enzyme E 4 is encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST the presence of the conserved domain
„Maltoporin-like" (cd01346 oder PssmID 30073) mit einem E-Wert (englisch„e-value") kleiner 1 x 10"5 festgestellt wird (englisch„domain hit"). "Maltoporin-like" (cd01346 or PssmID 30073) with an E value (English "e-value") smaller 1 x 10 "5 is found (English" domain hit ").
In einer anderen Ausführungsform der Erfindung, zeichnet sich die rekombinante Zelle dadurch aus, dass die im Vergleich zum Wildtyp gesteigerte Saccharoseaufnahme durch die erhöhte Aktivität eines Kanals E5 bewirkt wird, der Saccharose in die Zelle symportiert. Die vorliegende Erfindung erfasst ebenfalls Ausführungsformen, in denen dieser gesteigerte Saccharose- Symport mit den zuvor erwähnten erhöhten Aktivitäten der Enzyme E-i , E2, E3 und/oder E4 kombiniert ist. In another embodiment of the invention, the recombinant cell is characterized by the fact that the increased sucrose uptake compared to the wild type is brought about by the increased activity of a channel E 5 which sorbs sucrose into the cell. The present invention also covers embodiments in which this increased sucrose symport is combined with the aforementioned increased activities of the enzymes Ei, E 2 , E 3 and / or E 4 .
Der Kanal E5 kann beispielsweise eine Saccharose-Permease sein. In einer Ausführungsform wird E5 durch das cscB Gen (beispielsweise £ coli cscB: SEQ ID NO:45) kodiert. The channel E 5 may be, for example, a sucrose permease. In one embodiment, E 5 is encoded by the cscB gene (for example, E. coli cscB: SEQ ID NO: 45).
In weiteren Ausführungsformen kann die rekombinante Zelle zusätzlich zu E5 eine im Vergleich zu ihrem Wildtyp gesteigerte Aktivität mindestens eines der folgenden Enzyme aufweisen: E6, das die Umsetzung von Saccharose zu -D-Glucose und -D-Fruktose katalysiert; und E3, das die Umsetzung von -D-Fruktose zu -D-Fruktose-6-Phosphat katalysiert. In einer In further embodiments, in addition to E 5 , the recombinant cell may have an increased activity compared to its wild type of at least one of the following enzymes: E 6 , which catalyzes the conversion of sucrose to D-glucose and D-fructose; and E 3 , which catalyzes the conversion of D-fructose to D-fructose-6-phosphate. In a
Ausführungsform ist die Aktivität beider Enzyme erhöht.. In einer Ausführungsform der Erfindung, ist das Enzym E5 eine Saccharose-Permease (TCDB Klassifikation 2.A.1 .5.3), das Enzym E3 eine Fruktokinase (EC 2.7.1 .4) und das Enzym E6 eine -D-Fruktofuranosid-Fruktohydrolase (EC 3.2.1 .26). In one embodiment of the invention, the enzyme E 5 is a sucrose permease (TCDB classification 2.A.1 .5.3), the enzyme E 3 is a fructokinase (EC 2.7.1 .4). and the enzyme E 6 is a -D-fructofuranoside fructohydrolase (EC 3.2.1 .26).
In diesem Zusammenhang kann das Enzym E5 durch cscB (beispielsweise £. coli cscB: SEQ ID NO:45), lamB oder scrY (beispielsweise £ coli scrY: SEQ ID NO:44), E3 durch mac, yajF, mtlZ, rbsK, glcK, pfkB, frcK, frk, sacK, ydhR, kdgK, suk, ydjE, gmuE, ydjE, scrK (beispielsweise £ coli scrK: SEQ ID NO:43) oder cscK (beispielsweise £ coli cscK: SEQ ID NO:46) und/oder E6 durch cscA (beispielsweise £ coli cscA: SEQ ID NO:47), susH (beispielsweise Streptococcus pneumoniae susH: SEQ ID NO:52), scrB (beispielsweise £ coli scrB: SEQ ID NO:42), rafD, sacA, fruA oder bfrA kodiert werden. Die Nukleotidsequenzen der vorstehend genannten Gene, deren korrespondierende Proteinsequenz sowie weiterer Gene für die Enzyme E3, E5 und E6 können unter anderem auch der KEGG-, der NCBI-, der UniProt- oder EMBL-Datenbank entnommen werden. In einem besonderen Fall handelt es sich bei E3, E5 und/oder E6 um aus £. coli stammende Gene und die dadurch kodierten Proteine. In this connection, the enzyme E 5 may be replaced by cscB (for example, E. coli cscB: SEQ ID NO: 45), lamB or scrY (for example, E. coli scrY: SEQ ID NO: 44), E 3 by mac, yajF, mtlZ, rbsK , glcK, pfkB, frcK, frk, sacK, ydhR, kdgK, suk, ydjE, gmuE, ydjE, scrK (for example, E. coli scrK: SEQ ID NO: 43) or cscK (for example, E. coli cscK: SEQ ID NO: 46) and / or E 6 by cscA (for example E. coli cscA: SEQ ID NO: 47), susH (for example Streptococcus pneumoniae susH: SEQ ID NO: 52), scrB (for example E. coli scrB: SEQ ID NO: 42), rafD, sacA, fruA or bfrA. The nucleotide sequences of the abovementioned genes, their corresponding protein sequence and other genes for the enzymes E 3 , E 5 and E 6 can be taken from among others the KEGG, the NCBI, the UniProt or EMBL database. In a particular case, E 3 , E 5 and / or E 6 are of £. coli-derived genes and the proteins encoded thereby.
In einer Ausführungsform wird das Enzym E5 von Genen kodiert, die ausgewählt werden aus der Gruppe derer, die Genprodukte kodieren, deren Aminosäuresequenz über einen Bereich von mindestens 100, bevorzugt mindestens 200, insbesondere mindestens 300 Aminosäuren eine Sequenzidentität von mindestens 60%, vorzugsweise von mindestens 80%, besonders bevorzugt von mindestens 95%, ganz besonders bevorzugt von mindestens 99%, insbesondere von 100% zu SEQ ID NO:62 aufweisen. In one embodiment, the enzyme E 5 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, particularly preferably of at least 95%, very particularly preferably of at least 99%, in particular of 100%, to SEQ ID NO: 62.
In einer Ausführungsform wird das Enzym E5 von Genen kodiert, die ausgewählt werden aus der Gruppe derer, die Genprodukte kodieren, in deren Aminosäuresequenz bei einer Suche nach in der betreffenden Aminosäuresequenz enthaltenen konservierten Proteindomänen in der NCBI CDD (Version 2.20) mittels RPS-BLAST die Präsenz der konservierten Domäne „LacY_symp" (PFAM domain 01306 oder PssmID 1 10319) mit einem E-Wert (englisch„e- value") kleiner 1 x 10"5 festgestellt wird (englisch„domain hit"). In one embodiment, the enzyme E 5 is encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) using RPS-BLAST the presence of the conserved domain "LacY_symp" (PFAM domain 01306 or PssmID 1 10319) with an E value (English "e-value") is smaller than 1 x 10 "5 found (English" domain hit ").
In einer Ausführungsform wird das Enzym E6 von Genen kodiert, die ausgewählt werden aus der Gruppe derer, die Genprodukte kodieren, deren Aminosäuresequenz über einen Bereich von mindestens 100, bevorzugt mindestens 200, insbesondere mindestens 300 Aminosäuren eine Sequenzidentität von mindestens 60%, vorzugsweise von mindestens 80%, besonders bevorzugt von mindestens 95%, ganz besonders bevorzugt von mindestens 99%, insbesondere von 100% zu SEQ ID NO:64 oder SEQ ID NO:51 aufweisen. In one embodiment, the enzyme E 6 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, particularly preferably of at least 95%, very particularly preferably of at least 99%, in particular of 100%, to SEQ ID NO: 64 or SEQ ID NO: 51.
In einer Ausführungsform wird das Enzym E6 von Genen kodiert, die ausgewählt werden aus der Gruppe derer, die Genprodukte kodieren, in deren Aminosäuresequenz bei einer Suche nach in der betreffenden Aminosäuresequenz enthaltenen konservierten Proteindomänen in der NCBI CDD (Version 2.20) mittels RPS-BLAST die Präsenz der konservierten Domäne„SacC" (COG1621 oder PssmID 31808) mit einem E-Wert (englisch„e-value") kleiner 1 x 10"5 festgestellt wird(englisch„domain hit"). In einer weiteren Ausführungsform wird die gesteigerte Saccharoseaufnahme der erfindungsgemäßen rekombinanten Zelle dadurch bewirkt, dass sie im Vergleich zu ihrem Wildtyp mindestens eine gesteigerte Aktivität eines Enzymkomplexes aufweist, der Saccharose in die Zelle transportiert. In one embodiment, the enzyme E 6 is encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST the presence of the conserved domain "SacC" (COG1621 or PssmID 31808) with an E value (English "e-value") smaller 1 x 10 "5 is found (English" domain hit "). In a further embodiment, the increased sucrose uptake of the recombinant cell according to the invention is effected by having at least one increased activity of an enzyme complex which transports sucrose into the cell compared to its wild-type.
Dieser Saccharose transportierende Enzymkomplex kann sich beispielsweise aus den This sucrose-transporting enzyme complex can be, for example, from the
Enzymen E7, E8 und E9 zusammensetzen, und umfasst zum Beispiel einen Saccharosespezifischen ABC-Transporter (TCDB Klassifikation 3.A.1 .1.-). In diesem Zusammenhang kann das Enzym E7 durch ein susT1 Gen (beispielsweise Enzymes E 7 , E 8 and E 9 and includes, for example, a sucrose-specific ABC transporter (TCDB classification 3.A.1 .1.-). In this connection, the enzyme E 7 can be replaced by a susT1 gene (for example
Streptococcus pneumoniae susT1 : SEQ ID NO:48), E8 durch ein susT2 Gen (beispielsweise Streptococcus pneumoniae susT2: SEQ ID NO:49) und/oder E9 durch ein susX Gen Streptococcus pneumoniae susT1: SEQ ID NO: 48), E 8 by a susT2 gene (eg Streptococcus pneumoniae susT2: SEQ ID NO: 49) and / or E 9 by a susX gene
(beispielsweise Streptococcus pneumoniae susX: SEQ ID NO:50) kodiert werden. Ferner kann die Zelle mit gesteigerter Saccharose-Transporter-Aktivität eine im Vergleich zu ihrem Wildtyp gesteigerte Aktivität mindestens eines Enzyms E10, E2, E3 oder E6 oder einer beliebige Kombination dieser Enzyme aufweisen. Dabei katalysiert das Enzym E10 die (for example, Streptococcus pneumoniae susX: SEQ ID NO: 50). Further, the cell having increased sucrose transporter activity may have an increased activity of at least one enzyme E 10 , E 2, E 3 or E 6 or any combination of these enzymes as compared to its wild-type. The enzyme E 10 catalyzes the
Umsetzung von Saccharose zu Saccharose-6-Phosphat, das Enzym E2 die Umsetzung von Saccharose-6-Phosphat zu -D-Glukose-6-Phosphat und -D-Fruktose, das Enzym E3 die Umsetzung von -D-Fruktose zu -D-Fruktose-6-Phosphat und das Enzym E6 die Umsetzung von Saccharose zu -D-Glucose und -D-Fruktose. Conversion of sucrose to sucrose-6-phosphate, the enzyme E 2 the conversion of sucrose-6-phosphate to D-glucose-6-phosphate and D-fructose, the enzyme E 3 the conversion of D-fructose to - D-fructose 6-phosphate and the enzyme E 6 the conversion of sucrose to D-glucose and D-fructose.
In einer Ausführungsform weist die rekombinante Zelle eine im Vergleich zu ihrem Wildtyp gesteigerte Aktivität der Enzyme E10, E2, E3 und E6 auf. In one embodiment, the recombinant cell has an increased activity of the enzymes E 10 , E 2 , E 3 and E 6 compared to their wild type.
Enzym E10 kann eine Saccharose-Kinase, Enzym E2 eine Saccharose-6-Phosphat- Fruktohydrolase (EC 3.2.1.26), Enzym E3 eine Fruktokinase (EC 2.7.1 .4) und/oder Enzym E6 eine -D-Fruktofuranosid-Fruktohydrolase (EC 3.2.1 .26) sein. In diesem Zusammenhang kann das Enzym E10 durch ein Saccharose-Kinase Gen, E2 durch ein scrB (beispielsweise £ coli scrB: SEQ ID NO:42), bfrA, sacA, sacB, cscA (beispielsweise £. coli cscA: SEQ ID NO:47), fruA oder susH Gen und/oder E3 durch ein mac, yajF, mtlZ, rbsK, glcK, pfkB, frcK, frk, sacK, ydhR, kdgK, suk, ydjE, gmuE, ydjE, scrK (beispielsweise £ coli scrK: SEQ ID NO:43) oder cscK (beispielsweise £ coli cscK: SEQ ID NO:46) Gen kodiert werden. Die Nukleotidsequenzen der vorstehend genannten Gene, deren korrespondierenden Enzyme E 10 may be a sucrose kinase, enzyme E 2 is a sucrose-6-phosphate fructohydrolase (EC 3.2.1.26), enzyme E 3 is a fructokinase (EC 2.7.1.4) and / or enzyme E 6 is a -D- Fructofuranoside fructohydrolase (EC 3.2.1 .26). In this connection, the enzyme E 10 can be replaced by a sucrose kinase gene, E 2 by a scrB (for example E. coli scrB: SEQ ID NO: 42), bfrA, sacA, sacB, cscA (for example E. coli cscA: SEQ ID NO : 47), fruA or susH gene and / or E 3 by a mac, yajF, mtlZ, rbsK, glcK, pfkB, frcK, frk, sacK, ydhR, kdgK, suk, ydjE, gmuE, ydjE, scrK (for example E. coli scrK: SEQ ID NO: 43) or cscK (for example, E. coli cscK: SEQ ID NO: 46) gene. The nucleotide sequences of the aforementioned genes, their corresponding
Proteinsequenzen sowie weitere alternative Gene für die Enzyme E2, E3, E7, E8, E9 und E10 können unter anderem auch der KEGG-, der NCBI-, der UniProt- oder EMBL-Datenbank entnommen werden. In einem besonderen Fall handelt es sich bei E2, E3, und/oder E10 um aus E. coli und bei E7, E8 und/oder E9 um aus Streptococcus pneumoniae stammende Gene und die dadurch kodierten Proteine. Protein sequences and other alternative genes for the enzymes E 2 , E 3 , E 7 , E 8 , E 9 and E 10 can also be found in the KEGG, NCBI, UniProt or EMBL database. In a particular case, E 2 , E 3 , and / or E 10 are E. coli and E 7 , E 8 and / or E 9 are Streptococcus pneumoniae genes and the proteins encoded thereby.
In einer Ausführungsform wird das Enzym E7 von Genen kodiert, die ausgewählt werden aus der Gruppe derer, die Genprodukte kodieren, deren Aminosäuresequenz über einen Bereich von mindestens 100, bevorzugt mindestens 200, insbesondere mindestens 300 Aminosäuren eine Sequenzidentität von mindestens 60%, vorzugsweise von mindestens 80%, besonders bevorzugt von mindestens 95%, ganz besonders bevorzugt von mindestens 99%, insbesondere von 100% zu SEQ ID NO:65 aufweisen. In one embodiment, the enzyme E 7 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, more preferably at least 95%, most preferably at least 99%, especially 100% to SEQ ID NO: 65.
In einer weiteren Ausführungsform wird das Enzym E7 von Genen kodiert, die ausgewählt werden aus der Gruppe derer, die Genprodukte kodieren, ib deren Aminosäuresequenz bei einer Suche nach in der betreffenden Aminosäuresequenz enthaltenen konservierten In a further embodiment, the enzyme E 7 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has been searched for conserved in the relevant amino acid sequence
Proteindomänen in der NCBI CDD (Version 2.20) mittels RPS-BLAST die Präsenz der konservierten Domäne„LplB" (COG4209 oder PssmID 33938) mit einem E-Wert (englisch„e- value") kleiner 1 x 10"5 festgestellt wird (englisch„domain hit"). Protein domains in the NCBI CDD (Version 2.20) by means of RPS-BLAST the presence of the conserved domain "LplB" (COG4209 or PssmID 33938) with an E-value (English "e-value") smaller 1 x 10 "5 is found (English "Domain hit").
In einer Ausführungsform wird das Enzym E8 von Genen kodiert, die ausgewählt werden aus der Gruppe derer, die Genprodukte kodieren, deren Aminosäuresequenz über einen Bereich von mindestens 100, bevorzugt mindestens 200, insbesondere mindestens 300 Aminosäuren eine Sequenzidentität von mindestens 60%, vorzugsweise von mindestens 80%, besonders bevorzugt von mindestens 95%, ganz besonders bevorzugt von mindestens 99%, insbesondere von 100% zu SEQ ID NO:66 aufweisen. In einer Ausführungsform wird das Enzym E9 von Genen kodiert, die ausgewählt werden aus der Gruppe derer, die Genprodukte kodieren, deren Aminosäuresequenz über einen Bereich von mindestens 100, bevorzugt mindestens 200, insbesondere mindestens 300 Aminosäuren eine Sequenzidentität von mindestens 60%, vorzugsweise von mindestens 80%, besonders bevorzugt von mindestens 95%, ganz besonders bevorzugt von mindestens 99%, insbesondere von 100% zu SEQ ID NO:67 aufweisen. In one embodiment, the enzyme E 8 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, more preferably at least 95%, most preferably at least 99%, especially 100% to SEQ ID NO: 66. In one embodiment, the enzyme E 9 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, especially preferably of at least 95%, very particularly preferably of at least 99%, in particular of 100%, to SEQ ID NO: 67.
In einer Ausführungsform wird das Enzym E9 von Genen kodiert, die ausgewählt werden aus der Gruppe derer, die Genprodukte kodieren, in deren Aminosäuresequenz bei einer Suche nach in der betreffenden Aminosäuresequenz enthaltenen konservierten Proteindomänen in der NCBI CDD (Version 2.20) mittels RPS-BLAST die Präsenz der konservierten Domäne„UgpB" (COG1653 oder PssmID 31839) mit einem E-Wert (englisch„e-value") kleiner 1 x 10"5 festgestellt wird (englisch„domain hit"). In one embodiment, the enzyme E 9 is encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST the presence of the conserved domain "UgpB" (COG1653 or PssmID 31839) with an E value (English "e-value") is less than 1 x 10 "5 is found (English" domain hit ").
In weiteren Ausführungsformen der Erfindung können zwei oder drei der erwähnten In further embodiments of the invention, two or three of the mentioned
Möglichkeiten der Manipulation des Saccharose-Aufnahmewegs miteinander kombiniert werden, so dass die rekombinante Zelle sich durch die gesteigerte Aktivität von zwei oder drei Saccharose-Aufnahmewegen von ihrem Wildtyp unterscheidet. Dies kann jeweils ein, mehrere oder sämtliche Enzyme aus den geschilderten Varianten betreffen. In bestimmten Possibilities of manipulation of the sucrose uptake path are combined so that the recombinant cell differs from the wild type by the increased activity of two or three sucrose uptake pathways. This can in each case relate to one, several or all enzymes from the described variants. In particular
Ausführungsformen ist daher in einer rekombinanten Zelle gemäß der Erfindung die Aktivität der folgenden Enzyme gegenüber dem Wildtyp gesteigert:  Therefore, embodiments in a recombinant cell according to the invention increase the activity of the following enzymes over the wild-type:
E E9; EE 9 ;
Figure imgf000021_0001
Figure imgf000021_0001
E4, E5, E6, E7, E8, E9; E 4 , E 5 , E 6 , E 7 , E 8 , E 9 ;
Ei, E2, E3, E7, E8, Eg! Ε-ιο! Egg, E 2 , E 3 , E 7 , E 8 , Eg! Ε-ιο!
E3, E4, E6, E7, E8, E9; oder E 3 , E 4 , E 6 , E 7 , E 8 , E 9 ; or
E3, E6, E7, E8, E9. E 3 , E 6 , E 7 , E 8 , E 9 .
In anderen Ausführungsformen ist die Aktivität von mindestens einem, vorzugsweise In other embodiments, the activity is at least one, preferably
mindestens 2, 3, 4, 5, 6, 7, 8, 9 oder 10 Enzymen ausgewählt aus der Gruppe der Enzyme E E10 gesteigert. at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 enzymes selected from the group of enzymes EE 10 increased.
In einer Ausführungsform der Erfindung ist die rekombinante Zelle gentechnisch derart verändert, dass sie im Vergleich zu Ihrem Wildtyp eine gesteigerte Aktivität mindestens eines Enzyms aufweist, das die Fixierung von C02 an einen C3-Körper katalysiert. Diese gesteigerte Kohlendioxid-Fixierung kann alternativ oder zusätzlich zu einer Steigerung des Saccharose- Transports in die Zelle vorliegen. Die Steigerung des Saccharosetransports kann mittels der oben dargelegten Techniken erfolgen. Das mindestens eine Enzym, das die Fixierung von C02 an einen C3-Körper katalysiert, kann in bestimmten Ausführungsformen der Erfindung aus der Gruppe ausgewählt werden, die aus folgenden Enzymen besteht: In one embodiment of the invention, the recombinant cell is genetically engineered to have an increased activity of at least one enzyme that catalyzes the fixation of C0 2 to a C 3 body compared to its wild-type. This heightened Carbon dioxide fixation may be alternative or in addition to an increase in sucrose transport into the cell. The increase in sucrose transport can be accomplished by the techniques outlined above. The at least one enzyme that catalyzes the fixation of C0 2 to a C 3 body may, in certain embodiments of the invention, be selected from the group consisting of the following enzymes:
E12, das die Umsetzung von Pyruvat, ATP und C02 zu Oxalacetat, ADP und Phosphat katalysiert; E 12 , which catalyzes the conversion of pyruvate, ATP and C0 2 to oxaloacetate, ADP and phosphate;
Ei3, das die Umsetzung von Phosphoenolpyruvat, H20 und C02 zu Oxalacetat undEi 3 , the reaction of Phosphoenolpyruvat, H 2 0 and C0 2 to oxaloacetate and
Phosphat katalysiert; Catalyzed phosphate;
E14, das die Umsetzung von Phosphoenolpyruvat, ADP/GDP/P, und C02 zu Oxalacetat und ATP/GTP/PPi katalysiert; Which catalyzes the conversion of phosphoenolpyruvate, ADP / GDP / P, and C0 2 to oxaloacetate and ATP / GTP / PPi E 14;
E15, E16, E17 und E18, die die Umsetzung von Pyruvat NAD(P)H+ und C02 zu Malat und NAD(P)+ katalysieren. E 15 , E 16 , E 17 and E 18 , which catalyze the reaction of pyruvate NAD (P) H + and C0 2 to malate and NAD (P) + .
Ebenfalls erfasst durch die vorliegende Erfindung sind beliebige Kombinationen erhöhter Aktivitäten der Enzyme E12-E18 sowie die gesteigerte Aktivität aller Enzyme E12-E18. In einer Ausführungsform ist Also encompassed by the present invention are any combination of increased activities of the enzymes E 12 -E 18 and the increased activity of all enzymes E 12 -E 18 . In one embodiment
E12 eine Pyruvat-Carboxylase (EC 6.4.1 .1 ); E 12 a pyruvate carboxylase (EC 6.4.1.1);
E13 eine Phosphoenolpyruvat-Carboxylase (Phosphat:Oxalacetat-Carboxylase) (EC 4.1 .1 .31 ); E 13 is a phosphoenolpyruvate carboxylase (phosphate: oxaloacetate carboxylase) (EC 4.1 .1 .31);
E14 eine Phosphoenolpyruvat-Carboxykinase (ATP/GTP/PPi:Oxalacetat-Carboxylase) (EC 4.1 .1 .32, EC 4.1 .1 .38 oder EC 4.1 .1 .49); E 14 is a phosphoenolpyruvate carboxykinase (ATP / GTP / PPi: oxaloacetate carboxylase) (EC 4.1 .1 .32, EC 4.1 .1 .38 or EC 4.1 .1 .49);
E15 eine Malatdehydrogenase ((S)-Malat:NAD+ Oxidoreduktase) (EC 1 .1 .1 .38); E 15 a malate dehydrogenase ((S) -malate: NAD + oxidoreductase) (EC 1 .1 .1 .38);
E16 eine Malatdehydrogenase ((S)-Malat:NAD+ Oxidoreduktase) (EC 1 .1 .1 .39); E 16 a malate dehydrogenase ((S) -malate: NAD + oxidoreductase) (EC 1 .1 .1 .39);
E17 eine Malatdehydrogenase ((S)-Malat:NADP+ Oxidoreduktase) (EC 1 .1 .1 .40); E 17 a malate dehydrogenase ((S) -malate: NADP + oxidoreductase) (EC 1 .1 .1 .40);
und/oder  and or
E18 eine D-Malatdehydrogenase ((R)-Malat:NAD+ Oxidoreduktase) (EC 1 .1 .1 .83). E 18 is a D-malate dehydrogenase ((R) -malate: NAD + oxidoreductase) (EC 1 .1 .1 .83).
Das Enzym E12 kann vorzugsweise aus durch ein Gen kodiert werden, das aus der Gruppe umfassend cgl516, aarl62Cp pyrl, pca, cgl0689, pc, pcx, pyc-1 , pyc-2, accC-2, pycA, pycA2, pyc, pycB, pycB1 , pycB2, accC, accA, oadA, pyr, acc und accC1 ausgewählt wird, wobei das pyc-Gen (beispielsweise E. coli pyc: SEQ ID NO:5) besonders bevorzugt ist. The enzyme E 12 may preferably be encoded by a gene selected from the group comprising cgl516, aarl62Cppyrl, pca, cgl0689, pc, pcx, pyc-1, pyc-2, accC-2, pycA, pycA2, pyc, pycB, pycB1, pycB2, accC, accA, oadA, pyr, acc and accC1, with the pyc gene (for example, E. coli pyc: SEQ ID NO: 5) being particularly preferred.
Erfindungsgemäß bevorzugte Pyruvat-Carboxylasen sind insbesondere auch in US 6,455,284, US 6,171 ,833, US 6,884,606, US 6,403,351 , US 6,852,516 und US 6,861 ,246 beschrieben. Eine in diesem Zusammenhang besonders bevorzugte Pyruvat-Carboxylase pyc ist diejenige Mutante, die in„A novel methodology employing Corynebacterium glutamicum genome Pyruvate carboxylases preferred according to the invention are also described in particular in US Pat. Nos. 6,455,284, 6,171,833, 6,884,606, 6,403,351, 6,852,516 and 6,861,246. A pyruvate carboxylase pyc which is particularly preferred in this context is that mutant which is known in the novel methodology employing Corynebacterium glutamicum genome
Information to generate a new L-lysine- producing mutant.", Ohnishi J et al., Applied Information to generate a new L-lysine-producing mutant. ", Ohnishi J et al., Applied
Microbiology and Biotechnology, Vol. 58 (2), Seiten 217-223 (2002) beschrieben ist. Bei dieser Mutante wurde die Aminosäure Prolin an Position 458 durch Serin ersetzt. Der Microbiology and Biotechnology, Vol. 58 (2), pp. 217-223 (2002). In this mutant, the amino acid proline at position 458 was replaced by serine. Of the
Offenbarungsgehalt dieser Veröffentlichung hinsichtlich der Möglichkeiten zur Herstellung von Pyruvat-Carboxylase-Mutanten wird hiermit als Referenz eingeführt und bildet einen Teil der Offenbarung der vorliegenden Erfindung. In bevorzugter Weise stammt pyc aus  The disclosure of this publication regarding the possibilities of producing pyruvate carboxylase mutants is hereby incorporated by reference and forms part of the disclosure of the present invention. Preferably, pyc is derived
Corynebacterium glutamicum. Corynebacterium glutamicum.
Das Enzym E13 kann vorzugsweise durch ein Gen kodiert werden, das ausgewählt wird aus der Gruppe umfassend ppc, capP, pepC und clpA, wobei das ppc-Gen bevorzugt ist. In einer Ausführungsform stammt ppc aus E. coli (SEQ ID NO:1 ). Das Enzym E14 kann vorzugsweise durch ein Gen kodiert werden, das ausgewählt wird aus der Gruppe umfassend pck, pckG, pckA, pckl , pck2 und pck, wobei das pckA-Gen besonders bevorzugt ist. In einer bevorzugten Ausführungsform stammt pckA aus E. coli (SEQ ID NO:2). The enzyme E 13 may preferably be encoded by a gene selected from the group comprising ppc, capP, pepC and clpA, with the ppc gene being preferred. In one embodiment, ppc is from E. coli (SEQ ID NO: 1). The enzyme E 14 may preferably be encoded by a gene selected from the group comprising pck, pckG, pckA, pckI, pck2 and pck, the pckA gene being particularly preferred. In a preferred embodiment pckA is from E. coli (SEQ ID NO: 2).
Erfindungsgemäß bevorzugte Phosphoenolpyruvat-Carboxylasen sind insbesondere auch in US 4,757,009, US 4,980,285, US 5,573,945, US 6,872,553 und US 6,599,732 beschrieben. Der Offenbarungsgehalt dieser Druckschriften im Hinblick auf Phosphoenolpyruvat-Carboxylasen wird hiermit als Referenz eingeführt und bildet einen Teil der Offenbarung der vorliegenden Erfindung. Die Malatdehydrogenasen E15, E16 und E17 können vorzugsweise durch ein Gen kodiert werden, das ausgewählt wird aus der Gruppe umfassend me, me1 , me2, me3, mae, mael , mae2, sfcA, sfcA1 , maeA, maeB, maeB1 , maeB2, tme, yqkJ, ywkA, yqkJ, malS, ytsJ, mleA, mleS, mez, sce59.10c, 2sc7gll.23, malSI, malS2, dme, maeBl, maeB2, mdh, mdh1 , mdh2, dmel cgi 0120, dmel cgi 0120, dme1 -cg5889, fl9kl6.27, f6f22.7, t22p22.60, fl8al7.1 , mod1 , tme, mao, cgl3007, malS und malE, wobei für E15 maeA (beispielsweise £ coli maeA: SEQ ID NO:3), für E16 mmel (beispielsweise Chlamydomonas reinhardti mmel : SEQ ID NO:55) sowie dme und für E17 maeB (beispielsweise £ coli maeB: SEQ ID NO:4) besonders bevorzugt sind. In einer bevorzugten Ausführungsform stammen maeA und maeB aus £ coli. Phosphoenolpyruvate carboxylases preferred according to the invention are also described in particular in US Pat. Nos. 4,757,009, 4,980,285, 5,573,945, 6,872,553 and 6,599,732. The disclosure of these references for phosphoenolpyruvate carboxylases is hereby incorporated by reference and forms part of the disclosure of the present invention. The malate dehydrogenases E 15 , E 16 and E 17 may preferably be encoded by a gene selected from the group comprising me, me1, me2, me3, mae, mael, mae2, sfcA, sfcA1, maeA, maeB, maeB1, maeB2 , tme, yqkJ, ywkA, yqkJ, malS, ytsJ, mleA, mleS, mez, sce59.10c, 2sc7gll.23, malSI, malS2, dme, maeBl, maeB2, mdh, mdh1, mdh2, dmel cgi 0120, dmel cgi 0120 , dme1 -cg5889, fl9kl6.27, f6f22.7, t22p22.60, fl8al7.1, mod1, tme, mao, cgl3007, malS and malE, where for E 15 maeA (for example E. coli maeA: SEQ ID NO: 3), for E 16 mmel (for example Chlamydomonas reinhardti mmel: SEQ ID NO: 55) as well as for dme and for E 17 maeB (for example E. coli maeB : SEQ ID NO: 4) are particularly preferred. In a preferred embodiment, mouse and mouse are from E. coli.
Die Malatdehydrogenase E18 kann vorzugsweise durch ein Gen kodiert werden, das ausgewählt wird aus der Gruppe umfassend yeaU, ycsA, ttuC, ttuC1 , ttuC2, ttuC3, tdh, leuB, leuB1 und dmlA wobei yeaU und dmlA besonders bevorzugt sind. In einer bevorzugten Ausführungsform stammt dmlA aus £. coli (SEQ ID NO:57). The malate dehydrogenase E 18 may preferably be encoded by a gene selected from the group comprising yeaU, ycsA, ttuC, ttuC1, ttuC2, ttuC3, tdh, leuB, leuB1 and dmlA, with yeaU and dmlA being particularly preferred. In a preferred embodiment, dmlA is derived from £. coli (SEQ ID NO: 57).
In einer Ausführungsform wird: In one embodiment:
E12 durch ein pyc Gen; E 12 by a pyc gene;
E13 durch ein ppc Gen; E 13 by a ppc gene;
E14 durch ein pckA Gen; E 14 by a pckA gene;
E15 durch ein maeA Gen; E 15 by a mouse gene;
E16 durch ein mmel Gen; E 16 by a mmel gene;
E17 durch ein maeB Gen; und/oder E 17 by a mouse gene; and or
E18 durch ein dmlA Gen kodiert.. Die vorgenannten Enzyme können einzeln, alle oder in beliebiger Kombination in ihrer Aktivität gesteigert sein. Insbesondere können mindestens eines, mindestens 2, 3, 4, 5, 6, oder 7 Enzyme der Gruppe E12-E18 in ihrer Aktivität gesteigert sein. E 18 encoded by a dmlA gene .. The aforementioned enzymes may be individually, all or in any combination increased in their activity. In particular, at least one, at least 2, 3, 4, 5, 6, or 7 enzymes of the group E 12 -E 18 can be increased in their activity.
Die Nukleotidsequenzen der vorstehend genannten Gene, deren korrespondierende The nucleotide sequences of the above genes, their corresponding
Proteinsequenz sowie weiterer Gene für die Enzyme E12-E18 können unter anderem auch der KEGG-, der NCBI-, der UniProt- oder EMBL-Datenbank entnommen werden. Protein sequence and other genes for the enzymes E 12 -E 18 can also be found in the KEGG, the NCBI, the UniProt or EMBL database.
In einer Ausführungsform wird das Enzym E12 von Genen kodiert, die ausgewählt werden aus der Gruppe derer, die Genprodukte kodieren, deren Aminosäuresequenz über einen Bereich von mindestens 100, bevorzugt mindestens 200, insbesondere mindestens 300 Aminosäuren eine Sequenzidentität von mindestens 60%, vorzugsweise von mindestens 80%, besonders bevorzugt von mindestens 95%, ganz besonders bevorzugt von mindestens 99%, insbesondere von 100% zu SEQ ID NO:68 aufweisen. In einer Ausführungsform wird das Enzym E12 von Genen kodiert, die ausgewählt werden aus der Gruppe derer, die Genprodukte kodieren, in deren Aminosäuresequenz bei einer Suche nach in der betreffenden Aminosäuresequenz enthaltenen konservierten Proteindomänen in der NCBI CDD (Version 2.20) mittels RPS-BLAST die Präsenz der konservierten Domäne „DRE_TIM_PC_TC_5S" (cd07937) oder der konservierten Domäne„pyruvate carboxylase" (PRK12999) mit einem E-Wert (englisch„e-value") kleiner 1 x 10"5 festgestellt wird (englisch „domain hit"). In one embodiment, the enzyme E 12 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, more preferably at least 95%, most preferably at least 99%, especially 100% to SEQ ID NO: 68. In one embodiment, the enzyme E 12 is encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST the presence of the conserved domain "DRE_TIM_PC_TC_5S" (cd07937) or the conserved domain "pyruvate carboxylase" (PRK12999) with an E value (English "e-value") smaller than 1 x 10 "5 is found (English" domain hit ") ,
In einer Ausführungsform wird das Enzym E13 von Genen kodiert, die ausgewählt werden aus der Gruppe derer, die Genprodukte kodieren, deren Aminosäuresequenz über einen Bereich von mindestens 100, bevorzugt mindestens 200, insbesondere mindestens 300 Aminosäuren eine Sequenzidentität von mindestens 60%, vorzugsweise von mindestens 80%, besonders bevorzugt von mindestens 95%, ganz besonders bevorzugt von mindestens 99%, insbesondere von 100% zu SEQ ID NO:69 aufweisen. In one embodiment, the enzyme E 13 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, more preferably at least 95%, most preferably at least 99%, especially 100% to SEQ ID NO: 69.
In einer Ausführungsform wird das Enzym E13 von Genen kodiert, die ausgewählt werden aus der Gruppe derer, die Genprodukte kodieren, inderen Aminosäuresequenz bei einer Suche nach in der betreffenden Aminosäuresequenz enthaltenen konservierten Proteindomänen in der NCBI CDD (Version 2.20) mittels RPS-BLAST die Präsenz der konservierten Domäne „Phosphoenolpyruvate carboxylase" (PssmID 166715 oder COG3252 oder c14574 oder PRK00009) mit einem E-Wert (englisch„e-value") kleiner 1 x 10"5 festgestellt wird (englisch „domain hit"). In one embodiment, the enzyme E 13 is encoded by genes selected from the group of those encoding gene products in an amino acid sequence in a search for conserved protein domains contained in the subject amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST Presence of the conserved domain "phosphoenolpyruvate carboxylase" (PssmID 166715 or COG3252 or c14574 or PRK00009) with an E value (English "e-value") less than 1 x 10 "5 is found (English" domain hit ").
In einer Ausführungsform wird das Enzym E14 von Genen kodiert, die ausgewählt werden aus der Gruppe derer, die Genprodukte kodieren, deren Aminosäuresequenz über einen Bereich von mindestens 100, bevorzugt mindestens 200, insbesondere mindestens 300 Aminosäuren eine Sequenzidentität von mindestens 60%, vorzugsweise von mindestens 80%, besonders bevorzugt von mindestens 95%, ganz besonders bevorzugt von mindestens 99%, insbesondere von 100% zu SEQ ID NO:70 aufweisen. In one embodiment, the enzyme E 14 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, more preferably at least 95%, most preferably at least 99%, especially 100% to SEQ ID NO: 70.
In einer Ausführungsform wird das Enzym E14 von Genen kodiert, die ausgewählt werden aus der Gruppe derer, die Genprodukte kodieren, in deren Aminosäuresequenz bei einer Suche nach in der betreffenden Aminosäuresequenz enthaltenen konservierten Proteindomänen in der NCBI CDD (Version 2.20) mittels RPS-BLAST die Präsenz der konservierten Domäne „PEPCK_ATP" (cd00484) mit einem E-Wert (englisch„e-value") kleiner 1 x 10"5 festgestellt wird (englisch„domain hit"). In one embodiment, the enzyme E 14 is encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST the presence of the conserved domain "PEPCK_ATP" (cd00484) with an E value (English "e-value") smaller 1 x 10 "5 is found (English" domain hit ").
In einer Ausführungsform wird das Enzym E15 von Genen kodiert, die ausgewählt werden aus der Gruppe derer, die Genprodukte kodieren, deren Aminosäuresequenz über einen Bereich von mindestens 100, bevorzugt mindestens 200, insbesondere mindestens 300 Aminosäuren eine Sequenzidentität von mindestens 60%, vorzugsweise von mindestens 80%, besonders bevorzugt von mindestens 95%, ganz besonders bevorzugt von mindestens 99%, insbesondere von 100% zu SEQ ID NO:71 aufweisen. In one embodiment, the enzyme E 15 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, more preferably at least 95%, most preferably at least 99%, especially 100% to SEQ ID NO: 71.
In einer Ausführungsform wird das Enzym E15 von Genen kodiert, die ausgewählt werden aus der Gruppe derer, die Genprodukte kodieren, in deren Aminosäuresequenz bei einer Suche nach in der betreffenden Aminosäuresequenz enthaltenen konservierten Proteindomänen in der NCBI CDD (Version 2.20) mittels RPS-BLAST die Präsenz der konservierten Domäne„NAD(P) binding domain of malic enzyme (ME), subgroup 1 " (cd05312) mit einem E-Wert (englisch„e- value") kleiner 1 x 10"5 festgestellt wird (englisch„domain hit"). In one embodiment, the enzyme E 15 is encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST the presence of the conserved domain "NAD (P) binding domain of malic enzyme (ME), subgroup 1" (cd05312) with an E value (English "e-value") smaller 1 x 10 "5 is found (English" domain hit").
In einer Ausführungsform wird das Enzym E16 von Genen kodiert, die ausgewählt werden aus der Gruppe derer, die Genprodukte kodieren, deren Aminosäuresequenz über einen Bereich von mindestens 100, bevorzugt mindestens 200, insbesondere mindestens 300 Aminosäuren eine Sequenzidentität von mindestens 60%, vorzugsweise von mindestens 80%, besonders bevorzugt von mindestens 95%, ganz besonders bevorzugt von mindestens 99%, insbesondere von 100% zu SEQ ID NO:56 aufweisen. In einer Ausführungsform wird das Enzym E16 von Genen kodiert, die ausgewählt werden aus der Gruppe derer, die Genprodukte kodieren, in deren Aminosäuresequenz bei einer Suche nach in der betreffenden Aminosäuresequenz enthaltenen konservierten Proteindomänen in der NCBI CDD (Version 2.20) mittels RPS-BLAST die Präsenz der konservierten Domäne„malate dehydrogenase" (PRK13529) mit einem E-Wert (englisch„e-value") kleiner 1 x 10"5 festgestellt wird (englisch„domain hit"). In one embodiment, the enzyme E 16 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, more preferably at least 95%, most preferably at least 99%, especially 100% to SEQ ID NO: 56. In one embodiment, the enzyme E 16 is encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST the presence of the conserved domain "malate dehydrogenase" (PRK13529) with an E value (English "e-value") smaller 1 x 10 "5 is found (English" domain hit ").
In einer Ausführungsform wird das Enzym E17 von Genen kodiert, die ausgewählt werden aus der Gruppe derer, die Genprodukte kodieren, deren Aminosäuresequenz über einen Bereich von mindestens 100, bevorzugt mindestens 200, insbesondere mindestens 300 Aminosäuren eine Sequenzidentität von mindestens 60%, vorzugsweise von mindestens 80%, besonders bevorzugt von mindestens 95%, ganz besonders bevorzugt von mindestens 99%, insbesondere von 100% zu SEQ ID NO:54 aufweisen. In einer Ausführungsform wird das Enzym E17 von Genen kodiert, die ausgewählt werden aus der Gruppe derer, die Genprodukte kodieren, in deren Aminosäuresequenz bei einer Suche nach in der betreffenden Aminosäuresequenz enthaltenen konservierten Proteindomänen in der NCBI CDD (Version 2.20) mittels RPS-BLAST die Präsenz der konservierten Domäne„NAD(P) binding domain of malic enzyme (ME), subgroup 2" (cd0531 1 ) mit einem E-Wert (englisch„e- value") kleiner 1 x 10"5 festgestellt wird (englisch„domain hit"). In one embodiment, the enzyme E 17 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a range of at least 100, preferably at least 200, in particular at least 300 amino acids have a sequence identity of at least 60%, preferably of at least 80%, more preferably of at least 95%, most preferably of at least 99%, in particular of 100% to SEQ ID NO: 54. In one embodiment, the enzyme E 17 is encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST the presence of the conserved domain "NAD (P) binding domain of malic enzyme (ME), subgroup 2" (cd0531 1) with an E value (English "e-value") smaller 1 x 10 "5 is found (English" domain hit ").
In einer Ausführungsform wird das Enzym E18 von Genen kodiert, die ausgewählt werden aus der Gruppe derer, die Genprodukte kodieren, deren Aminosäuresequenz über einen Bereich von mindestens 100, bevorzugt mindestens 200, insbesondere mindestens 300 Aminosäuren eine Sequenzidentität von mindestens 60%, vorzugsweise von mindestens 80%, besonders bevorzugt von mindestens 95%, ganz besonders bevorzugt von mindestens 99%, insbesondere von 100% zu SEQ ID NO:53 aufweisen. In one embodiment, the enzyme E 18 is encoded by genes which are selected from the group of those which encode gene products whose amino acid sequence has a sequence identity of at least 60%, preferably of at least 100, preferably at least 200, in particular at least 300 amino acids at least 80%, particularly preferably of at least 95%, very particularly preferably of at least 99%, in particular of 100%, to SEQ ID NO: 53.
In einer Ausführungsform wird das Enzym E18 von Genen kodiert, die ausgewählt werden aus der Gruppe derer, die Genprodukte kodieren, in deren Aminosäuresequenz bei einer Suche nach in der betreffenden Aminosäuresequenz enthaltenen konservierten Proteindomänen in der NCBI CDD (Version 2.20) mittels RPS-BLAST die Präsenz der konservierten Domäne„lso_dh Super-family" (c00445 oder PssmID 174206) mit einem E-Wert (englisch„e-value") kleiner 1 x 10"5 festgestellt wird (englisch„domain hit"). In one embodiment, the enzyme E 18 is encoded by genes selected from the group of those encoding gene products in its amino acid sequence in a search for conserved protein domains contained in the relevant amino acid sequence in the NCBI CDD (version 2.20) by means of RPS-BLAST the presence of the conserved domain "lso_dh super-family" (c00445 or PssmID 174206) with an E-value (English "e-value") smaller 1 x 10 "5 is found (English" domain hit ").
Des Weiteren kann, in einer weiteren Ausführungsform der erfindungsgemäßen Zellen, die rekombinante Zelle zusätzlich derart gentechnisch verändert sein, dass sie im Vergleich zu ihrem Wildtyp einen verminderten Kohlenstofffluss von Saccharose hin zu Gärungsprodukten, die keine C4-Körper sind, aufweist. Furthermore, in a further embodiment of the cells according to the invention, the recombinant cell can additionally be genetically engineered such that it has a reduced carbon flux from sucrose to fermentation products which are not C 4 bodies compared to their wild type.
Diese Ausführungsform umfasst erfindungsgemäße rekombinante Zellen, die zusätzlich gegenüber ihrem Wildtyp dadurch gekennzeichnet sind, dass sie aus Saccharose weniger d-, C2-, und/oder C3-Nebenprodukte erzeugen. Die dazu erforderliche gentechnische Veränderung kann durch eine Manipulation der rekombinanten Zelle geschehen, wodurch der Metabolismus, der die Gärungsprodukte erzeugt, reduziert oder unterbrochen wird. This embodiment encompasses recombinant cells according to the invention which, in addition to their wild type, are characterized by producing less d, C 2 and / or C 3 by- products from sucrose. The requisite genetic modification may be by manipulation of the recombinant cell, thereby reducing or disrupting the metabolism that produces the fermentation products.
In einer Ausführungsform der Erfindung ist die Aktivität von mindestens einem der folgendenIn one embodiment of the invention, the activity is at least one of the following
Enzyme im Vergleich zum Wildtyp verringert: Enzymes reduced compared to wild type:
Ei9, E2o, E21 und E22, die Komponenten eines glukosespezifischen Phosphoenolpyruvat- Phosphotransferasesystems darstellen und als solche den Import von Glukose und in Summe die Umsetzung von Phosphoenolpyruvat und Glukose zu Pyruvat und Glukose- B-Phosphat katalysieren; Ei9, E 2 o, E21 and E22, which are components of a glucose-specific phosphoenolpyruvate phosphotransferase system and, as such, catalyze the import of glucose and in sum the conversion of phosphoenolpyruvate and glucose to pyruvate and glucose B-phosphate;
E23 und E24, die die Umsetzung von Pyruvat und CoA zu Formiat und Acetyl-CoA katalysieren; E 2 3 and E 2 4, which catalyze the reaction of pyruvate and CoA to formate and acetyl-CoA;
E25, das die Umsetzung von Pyruvat, H20 und Ferricytochrom bi zu Acetat, C02 und Ferrocytochrom bi katalysiert; E 2 5, which catalyzes the reaction of pyruvate, H 2 0 and ferricytochrome bi to acetate, C0 2 and ferrocytochrome bi;
E26, das die Umsetzung von S-Methylmalonyl-CoA zu Propanoyl-CoA und C02 katalysiert; E 2 6, which catalyzes the reaction of S-methylmalonyl-CoA to propanoyl-CoA and C0 2 ;
E27 und E28, die die Umsetzung von Acetyl-Phosphat und ADP/P, zu Acetat und ATP/PP, katalysieren; E 2 7 and E 2 8, which catalyze the reaction of acetyl phosphate and ADP / P, to acetate and ATP / PP;
E29, das die Umsetzung von Acetyl-CoA und P, (Orthophosphat) zu Acetyl-Phosphat und CoA katalysiert; E 2 9, which catalyzes the reaction of acetyl CoA and P, (orthophosphate) to acetyl phosphate and CoA;
E30, das die Umsetzung von Pyruvat und NADH zu D-Lactat und NAD+ katalysiert; E 30 , which catalyzes the conversion of pyruvate and NADH to D-lactate and NAD + ;
E31 , das die Umsetzung von Acetyl-CoA zu Acetaldehyd katalysiert;  E31, which catalyzes the conversion of acetyl-CoA to acetaldehyde;
E32, das die Umsetzung von Acetaldehyd zu Ethanol katalysiert; E 32 , which catalyzes the conversion of acetaldehyde to ethanol;
E33, das die Umsetzung von Glyceronphosphat zu Methylglyoxal und Orthophosphat katalysiert; E 33 , which catalyzes the conversion of glycerol phosphate to methylglyoxal and orthophosphate;
E34, E35 und E36, die einen Komplex bilden, der die Umsetzung von Formiat zu C02 katalysiert; E 34 , E 35 and E 36 , which form a complex that catalyzes the conversion of formate to C0 2 ;
E37, das die Umsetzung von Formiat zu C02 katalysiert; E 37 , which catalyzes the conversion of formate to C0 2 ;
E38, E39 und E40, die einen Komplex bilden, der die Umsetzung von Formiat zu C02 katalysiert; E 38 , E 39 and E 40 , which form a complex that catalyzes the conversion of formate to C0 2 ;
E41 , das die Umsetzung von Propionyl-CoA, H20 und Oxalacetat zu 2-Methylcitrat und CoA katalysiert; E41, which catalyzes the reaction of propionyl-CoA, H 2 O and oxalacetate to 2-methyl citrate and CoA;
E42, das die Umsetzung von 2-Methylcitrat zu 2-Methyl-cis-Aconitat oder 2-Methyl-trans- Aconitat und H20 katalysiert; E43, das die Umsetzung von 2-Methyl-trans-Aconitat zu 2-Methyl-cis-Aconitat katalysiert; E44, das die Umsetzung von 2-Methyl-cis-Aconitat zu Succinat und Pyruvat katalysiert; E45, das die Umsetzung von Propionyl-Phosphat und ADP zu Propionat und ATP katalysiert; E 42 which catalyzes the conversion of 2-methyl citrate to 2-methyl cis-aconitate or 2-methyl-trans aconitate and H 2 O; E 43 , which catalyzes the conversion of 2-methyl trans aconitate to 2-methyl cis aconitate; E 44 , which catalyzes the conversion of 2-methyl cis aconitate to succinate and pyruvate; E 45 , which catalyzes the conversion of propionyl phosphate and ADP to propionate and ATP;
E46, welches die Umsetzung von Propionyl-CoA und P, zu Propionyl-Phosphat und CoA katalysiert; E46 , which catalyzes the reaction of propionyl CoA and P, to propionyl phosphate and CoA;
E47, welches die Umsetzung von Pyruvat zu Acetaldehyd und C02 katalysiert; E 47 , which catalyzes the conversion of pyruvate to acetaldehyde and C0 2 ;
E48, welches die Umsetzung von Pyruvat und CoA 2 oxidierten Ferredoxinen zu Acetyl- CoA, C02, und 2 reduzierten Ferredoxinen und 2 H+ katalysiert; E 48 , which catalyzes the reaction of pyruvate and CoA 2 oxidized ferredoxins to acetyl CoA, C0 2 , and 2 reduced ferredoxins and 2 H + ;
E49, welches die Umsetzung von D-Xylulose-5-Phosphat und Phosphat zu E 49 , which involves the reaction of D-xylulose-5-phosphate and phosphate
Acetylphosphat und D-Glyceraldehyd-3-Phosphate und H20 katalysiert; Acetyl phosphate and D-glyceraldehyde-3-phosphate and H 2 O catalyzed;
E50, welches die Umsetzung von (S)-Methylmalonyl-CoA und Pyruvat zu Propionyl-CoA und Oxalacetat katalysiert; E 50 , which catalyzes the reaction of (S) -methylmalonyl-CoA and pyruvate to propionyl-CoA and oxaloacetate;
E51 , welches die Umsetzung von (S)-Methylmalonyl-CoA zu Succinyl-CoA katalysiert; E52, welches die Umsetzung von 2 Pyruvat zu 2-Acetolactat und C02 katalysiert; E51, which catalyzes the reaction of (S) -methylmalonyl-CoA to succinyl-CoA; E 52 , which catalyzes the conversion of 2 pyruvate to 2-acetolactate and C0 2 ;
E53, welches die Umsetzung von 2-Acetolactat zu Acetoin und C02 katalysiert; E 53 , which catalyzes the reaction of 2-acetolactate to acetoin and C0 2 ;
E54, welches die Umsetzung von Acetoin und NAD(P)H+ zu Butan-2,3-diol und NAD(P)+ katalysiert; E 54 , which catalyzes the reaction of acetoin and NAD (P) H + to butane-2,3-diol and NAD (P) + ;
E55, welches die Umsetzung 2 Acetyl-CoA zu Acetoacetyl-CoA katalysiert; E 55 , which catalyzes the reaction of 2 acetyl-CoA to acetoacetyl-CoA;
E56, welches die Umsetzung von Acetoacetyl-CoA und NAD(P)H+ zu 3-Hydroxybutyryl-E 56 , which is the reaction of acetoacetyl-CoA and NAD (P) H + to 3-hydroxybutyryl
CoA und NAD(P)+ katalysiert; CoA and NAD (P) + catalyses;
E57, welches die Umsetzung von Crotonyl-CoA und H20 zu 3-Hydroxybutyryl-CoA katalysiert; E 57 , which catalyzes the reaction of crotonyl-CoA and H 2 O to 3-hydroxybutyryl-CoA;
E58, welches die Umsetzung von Butyraldehyd und NAD(P)H+ zu Butanol und NAD(P)+ katalysiert; E 58 , which catalyzes the reaction of butyraldehyde and NAD (P) H + to butanol and NAD (P) + ;
E59, welches die Umsetzung von Butyryl-CoA und NAD(P)H+ zu Buryraldehyd und NAD(P)+ katalysiert; E 59 , which catalyzes the reaction of butyryl-CoA and NAD (P) H + to buryraldehyde and NAD (P) + ;
E6o, welches die Umsetzung von Acetoacetat und H+ zu Aceton und C02 katalysiert; E6i , welches die Umsetzung von Aceton und NAD(P)H+ zu Propanol und NAD(P)+ katalysiert; E 6 o, which catalyzes the reaction of acetoacetate and H + to acetone and C0 2 ; E 6 i, which catalyzes the reaction of acetone and NAD (P) H + to propanol and NAD (P) + ;
E62, welches die Umsetzung von Acyl-CoA und Carbonsäure zu Acylat und E 62 , which is the reaction of acyl-CoA and carboxylic acid to acylate and
Carbonsäure-CoA katalysiert und Carboxylic CoA catalyzes and
E63, das die Umsetzung von Pyruvat und NADH zu L-Lactat und NAD+ katalysiert. In bestimmten Ausführungsformen können auch mehrere, d.h. mindestens 2, oder alle der vorgenannten Enzyme in ihrer Aktivität verringert sein. E 6 3, which catalyzes the conversion of pyruvate and NADH to L-lactate and NAD + . In certain embodiments, also several, ie at least 2, or all of the aforementioned enzymes may be reduced in their activity.
In bestimmten Ausführungsformen ist/sind: In certain embodiments, / are:
E19 und E2o ein PEP-abhängiges Phosphotransferase-System Enzym I I (EC 2.7.1 .69);E 19 and E 2 o a PEP-dependent phosphotransferase system enzyme II (EC 2.7.1 .69);
E2i ein PEP-abhängiges Phosphotransferase-System Enzym I (EC 2.7.3.9); E 2 i is a PEP-dependent phosphotransferase system Enzyme I (EC 2.7.3.9);
E22 eine Phosphohistidin-Protein (HPr)-Hexose-Phosphotransferase-Komponenente desE 2 2 is a phosphohistidine protein (HPr) -hexose phosphotransferase component of the
PEP-abhängigen Phosphotransferase-Systems; PEP-dependent phosphotransferase system;
E23 und E24 eine Formiat-C-Acetyltransferase (EC 2.3.1 .54); E 2 3 and E 2 4 a formate C-acetyltransferase (EC 2.3.1.54);
E25 eine Pyruvat:Ferricytochrom b-i-Oxidoreduktase (EC 1 .2.2.2); E 25 a pyruvate: ferricytochrome bi-oxidoreductase (EC 1 .2.2.2);
E26 eine Methylmalonyl-CoA-Decarboxylase (EC 4.1 .1 .41 ); E 26 is a methylmalonyl-CoA decarboxylase (EC 4.1 .1 .41);
E27 eine ATP/PPi:Acetat-Phosphotransferase (EC 2.7.2.1 oder EC 2.7.2.1 ); E 27 an ATP / PPi: acetate phosphotransferase (EC 2.7.2.1 or EC 2.7.2.1);
E28 eine Propionat-/Acetat-Kinase (EC 2.7.2.15); E 28 is a propionate / acetate kinase (EC 2.7.2.15);
E29 eine Acetyl-CoA:Phosphat-Acetyltransferase (EC 2.3.1 .8); E 29 an acetyl-CoA: phosphate acetyltransferase (EC 2.3.1 .8);
E30 eine D-Lactatdehydrogenase (EC 1 .1 .1 .28); E 30 is a D-lactate dehydrogenase (EC 1 .1 .1 .28);
E31 eine Acetaldehyd-Dehydrogenase (CoA-acetylierend) (EC 1 .2.1 .10); E 31 an acetaldehyde dehydrogenase (CoA-acetylating) (EC 1 .2.1 .10);
E32 eine NAD-abhängige Alkohol-Dehydrogenase (EC 1 .1 .1 .1 ); E 32 an NAD-dependent alcohol dehydrogenase (EC 1 .1 .1 .1);
E33 eine Glyceronphosphat-Phospholyase (EC 4.2.3.3); E 33 is a glycerone phosphate phospholyase (EC 4.2.3.3);
E34, E35, E36, E37, E38, E39 und E40 Formatdehydrogenasen (EC 1 .2.1 .2); E 34 , E 35 , E 36 , E 37 , E 38 , E 39 and E 40 formate dehydrogenases (EC 1 .2.1 .2);
E41 eine 2-Methylcitrat Synthase (EC 2.3.3.5); E 41 is a 2-methylcitrate synthase (EC 2.3.3.5);
E42 eine 2-Methylcitrat Dehydratase (EC 4.2.1 .79 oder EC 4.2.1 .1 17); E 42 a 2-methylcitrate dehydratase (EC 4.2.1 .79 or EC 4.2.1 .1 17);
E43 eine 2-Methylaconitat Isomerase; E 43 is a 2-methylaconitate isomerase;
E44 eine Methylisocitrat Lyase (EC 4.1 .3.30); E 44 is a methyl isocitrate lyase (EC 4.1 .3.30);
E45 eine Propionatkinase (EC 2.7.2.15); E 45 is a propionate kinase (EC 2.7.2.15);
E46 eine Phosphat-Propionyltransferase (EC 2.3.1 .8); E 46 a phosphate-propionyltransferase (EC 2.3.1 .8);
E47 eine Pyruvatdecarboxylase (EC 4.1 .1 .1 ); E 47 is a pyruvate decarboxylase (EC 4.1 .1 .1);
E48 eine Pyruvat:Ferredoxin Oxidoreduktase (EC 1 .2.7.1 ); E 48 is a pyruvate: ferredoxin oxidoreductase (EC 1 .2.7.1);
E49 eine Phosphoketolase (EC 4.1 .2.9); E 49 is a phosphoketolase (EC 4.1 .2.9);
E50 eine Methylmalonyl-CoA-Carboxytransferase (EC 2.1 .3.1 ); E 50 is a methylmalonyl-CoA-carboxytransferase (EC 2.1 .3.1);
E51 eine Methylmalonyl-CoA-Mutase (EC 5.4.99.2);  E51 a methylmalonyl-CoA mutase (EC 5.4.99.2);
E52 eine Acetolactat-Synthase (EC 2.2.1 .6); E 52 an acetolactate synthase (EC 2.2.1.6);
E53 eine Acetolactat-Decarboxylase (EC 4.1 .1 .5); E 53 an acetolactate decarboxylase (EC 4.1 .1 .5);
E54 eine Butandiol-Dehydrogenase (EC 1 .1 .1 .4 oder EC 1 .1 .1 .76); E 54 a butanediol dehydrogenase (EC 1 .1 .1 .4 or EC 1 .1 .1 .76);
E55 eine Thiolase (EC 2.3.1 .9); E56 eine 3-Hydroxybutyryl-CoA-Dehydrogenase (EC 1 .1 .1.157, EC 1 .1.1.35, EC 1.1 .1 .36 oder EC 1.1 .1.21 1 ); E 55 a thiolase (EC 2.3.1 .9); E 56 is a 3-hydroxybutyryl-CoA dehydrogenase (EC 1 .1 .1.157, EC 1 .1.1.35, EC 1.1 .1 .36 or EC 1.1 .1.21 1);
E57 eine Crotonase (EC 4.2.1 .17); E 57 a crotonase (EC 4.2.1 .17);
E58 eine Butanol-Dehydrogenase (EC 1.1 .1.1 oder EC 1.1.1.2); E 58 is a butanol dehydrogenase (EC 1.1 .1.1 or EC 1.1.1.2);
E59 eine Butyraldehyd-Dehydrogenase (EC 1.2.1.3, EC 1 .2.1.4 oder EC 1.2.1.5); E 59 a butyraldehyde dehydrogenase (EC 1.2.1.3, EC 1 .2.1.4 or EC 1.2.1.5);
E6o eine Acetoacetat-Decarboxylase (EC 4.1 .1.4); E 6 o an acetoacetate decarboxylase (EC 4.1 .1.4);
E6i eine Propanol-Dehydrogenase (EC 1 .1 .1.1 oder EC 1 .1 .1.2); E 6 i is a propanol dehydrogenase (EC 1 .1 .1.1 or EC 1 .1 .1.2);
E62 eine Acyl-CoA:CoA-Transferase (EC 2.8.3.-); und/oder E 6 2 an acyl-CoA: CoA transferase (EC 2.8.3.-); and or
E63 eine L-Lactatdehydrogenase (EC 1.1 .1 .27). E 6 3 an L-lactate dehydrogenase (EC 1.1 .1 .27).
Die erfindungsgemäßen Zellen weisen in einer Ausführungsform eine im Vergleich zum Wildtyp verringerte Aktivität von mindestens einem, vorzugsweise mindestens 2, noch bevorzugter mindestens 3, am bevorzugtesten mindestens 5 der genannten Enzyme auf. In einer Ausführungsform der Erfindung wird die Aktivität von mindestens einem der Enzyme E19-E22 reduziert. Eine solche Verringerung der Enzymaktivität kann vorteilhaft sein, da damit der energieaufwendige Import und die Aktivierung von Glukose unterbunden werden, wodurch der Zelle mehr Energie für den Import und die Aktivierung von Saccharose und für die Fixierung von Kohlendioxid zur Verfügung steht. In one embodiment, the cells according to the invention have a reduced activity compared to the wild type of at least one, preferably at least 2, more preferably at least 3, most preferably at least 5 of said enzymes. In one embodiment of the invention, the activity of at least one of the enzymes E19-E22 is reduced. Such a reduction in enzyme activity may be beneficial as it eliminates the energy-consuming import and activation of glucose, thereby providing the cell with more energy to import and activate sucrose and to fix carbon dioxide.
Das Enzym E26 wird vorzugsweise von einem Gen kodiert, das ausgewählt wird aus der Gruppe umfassend ygfG, mmdA, oadB, oadB2, oadB3, SC1 C2.16, SC1 G7.10, pccBI, mmdB, mmdC und ppcB, wobei das ygfG-Gen besonders bevorzugt ist. In einer Ausführungsform stammt ygfG aus £. coli. The enzyme E 2 6 is preferably encoded by a gene selected from the group comprising ygfG, mmdA, oadB, oadB2, oadB3, SC1C2.16, SC1G7.10, pCCBI, mmdB, mmdC and ppcB, where the ygfG Gene is particularly preferred. In one embodiment, ygfG is from £. coli.
Gemäß verschiedenen Ausführungsformen der Erfindung weist die Zelle eine im Vergleich zu ihrem Wildtyp verminderte Aktivität von mindestens einem Enzym E19-E45 auf, wobei: According to various embodiments of the invention, the cell has a reduced activity of at least one enzyme E 19 -E 45 compared to its wild type, wherein:
E19 durch ein ptsG Gen kodiert wird; E 19 is encoded by a ptsG gene;
E2o durch ein ptsl Gen kodiert wird; E 2 o is encoded by a ptsl gene;
E21 durch ein ptsH Gen kodiert wird;  E21 is encoded by a ptsH gene;
E22 durch ein crr Gen kodiert wird;  E22 is encoded by a crr gene;
E23 durch ein tdcE Gen kodiert wird; E 2 3 is encoded by a tdcE gene;
E24 durch ein pflA oder pflB Gen kodiert wird; E 24 is encoded by a pflA or pflB gene;
E25 durch ein poxB Gen kodiert wird; E26 durch ein ygfG Gen kodiert wird; E 2 5 is encoded by a poxB gene; E 26 is encoded by a ygfG gene;
E27 durch ein ackA Gen kodiert wird; E 27 is encoded by a ackA gene;
E28 durch ein ackA oder tdcD Gen kodiert wird; E 28 is encoded by an ackA or tdcD gene;
E29 durch ein pta Gen kodiert wird; E 2 9 is encoded by a pta gene;
E30 durch ein IdhA Gen kodiert wird; E 30 is encoded by an IdhA gene;
E31 durch ein adhE Gen kodiert wird; E is encoded by a gene adhE 31;
E32 durch ein adhE Gen kodiert wird; E 32 is encoded by an adhE gene;
E33 durch ein mgsA Gen kodiert wird; E 33 is encoded by a mgsA gene;
E34 durch ein fdnG Gen kodiert wird; E 34 is encoded by a fdnG gene;
E35 durch ein fdnH Gen kodiert wird; E is encoded by a gene fdnH 35;
E36 durch ein fdnl Gen kodiert wird; E 36 is encoded by a fdnl gene;
E37 durch ein fdhF Gen kodiert wird; E 37 is encoded by a fdhF gene;
E38 durch ein fdoG Gen kodiert wird; E 38 is encoded by a fdoG gene;
E39 durch ein fdoH Gen kodiert wird; E 39 is encoded by a fdoH gene;
E40 durch ein fdol Gen kodiert wird; E is encoded by a gene fdol 40;
E41 durch ein prpC Gen kodiert wird; E 41 is encoded by a prpC gene;
E42 durch ein prpD oder acnD Gen kodiert wird; E 42 is encoded by a prpD or acnD gene;
E43 durch ein prpF Gen kodiert wird; E 43 is encoded by a prpF gene;
E44 durch ein prpB Gen kodiert wird; E 44 is encoded by a prpB gene;
E45 durch ein tdcD Gen kodiert wird E 45 is encoded by a tdcD gene
E46 durch ein pta Gen kodiert wird; E 46 is encoded by a pta gene;
E47 durch ein pdc Gen kodiert wird; E 47 is encoded by a pdc gene;
E48 durch ein porA, porB, porC oder porD Gen kodiert wird; E 48 is encoded by a porA, porB, porC or porD gene;
E49 durch ein xpkl oder xpk2 Gen kodiert wird E 49 is encoded by an xpkl or xpk2 gene
E50 durch ein Methylmalonyl-CoA-Carboxytransferase Gen kodiert wird;E 50 is encoded by a methylmalonyl CoA carboxytransferase gene;
E51 durch ein sbm oder ein mcmA und ein mcmB Gen kodiert wird;E 51 is encoded by a sbm or a mcmA and a mcmB gene;
E52 durch ein alsS, ilvB, ilvM, ilvN, ilvG, ilvl oder ilvH Gen kodiert wird;E 52 is encoded by a gene as, ilvB, ilvM, ilvN, ilvG, ilvl or ilvH gene;
E53 durch ein alsD Gen kodiert wird; E 53 is encoded by a alsD gene;
E54 durch ein butBGen kodiert wird; E 54 is encoded by a butBGen;
E55 durch ein thl, thIA, thIB oder phaA Gen kodiert wird; E 55 is encoded by a thI, thIA, thIB or phaA gene;
E56 durch ein phaB Gen kodiert wird; E 56 is encoded by a phaB gene;
E57 durch ein crt Gen kodiert wird; E 57 is encoded by a crt gene;
E58 durch ein adhE Gen kodiert wird; E 58 is encoded by an adhE gene;
E59 durch ein adhE, bdhA oder bdhB Gen kodiert wird; E6o durch ein ade Gen kodiert wird; E 59 is encoded by an adhE, bdhA or bdhB gene; E 6 o is encoded by an ade gene;
E6i durch ein adh Gen kodiert wird; E 6 i is encoded by an adh gene;
E62 durch ein ctfA und ein ctfB oder ein atoA und ein atoD Gen kodiert wird und/oder E63 durch ein IdhL Gen kodiert wird. E 6 2 is encoded by a ctfA and a ctfB or an atoA and an atoD gene and / or E 6 3 is encoded by an IdhL gene.
Die Nukleotidsequenzen der vorstehend genannten Gene, deren korrespondierende The nucleotide sequences of the above genes, their corresponding
Proteinsequenz sowie weiterer Gene für die Enzyme E19-E63 können unter anderem auch der KEGG-, der NCBI-, der UniProt- oder EMBL-Datenbank entnommen werden. In einer Ausführungsform der Erfindung, weist die rekombinante Zelle, die ausgehend von Saccharose und C02 als Kohlenstoffquellen mehr C4-Körper als der Wildtyp herstellen kann, eine im Vergleich zu ihrem Wildtyp verminderte Aktivität von mindestens einem, mindestens zwei, mindestens 3, oder mindestens 4 der Enzyme E19, E26, E27, E29, E30, E31, E32 und E46 auf. In einer solchen Ausführungsform sind die Enzyme E19, E26, E27, E29/E46, E30 und E31/E32 IdhA, adhE, ack, pta, ygfG und ptsG. Die erfindungsgemäße Zelle kann auch eine verringerte Aktivität aller 8 Enzyme E19, E26, E27, E29, E30, E31, E32 und E46 bzw. aller 6 Enzyme, die durch die Gene IdhA, adhE, ack, pta, ygfG und ptsG kodiert werden, aufweisen. Protein sequence and other genes for the enzymes E 19 -E 6 3 can be found, inter alia, the KEGG, the NCBI, the UniProt or EMBL database. In one embodiment of the invention, the recombinant cell which, starting from sucrose and C0 2 as carbon sources, can produce more C 4 bodies than the wild type, has a reduced activity of at least one, at least two, at least 3, or their wild-type at least 4 of the enzymes E 19 , E 2 6, E 2 7, E 29 , E 30 , E 31 , E 32 and E 46 on. In such an embodiment, the enzymes E 19 , E 26 , E 27 , E 29 / E 46 , E 30 and E 31 / E 32 are IdhA, adhE, ack, pta, ygfG and ptsG. The cell according to the invention can also have a reduced activity of all the enzymes E 19 , E 26 , E 27 , E 29 , E 30 , E 31 , E 32 and E 46 or of all 6 enzymes which are represented by the genes IdhA, adhE, ack, pta, ygfG and ptsG.
In weiteren Ausführungsformen weist die rekombinante Zelle, die aus Saccharose und C02 als Kohlenstoffquellen mehr C4-Körper als der Wildtyp herstellen kann, eine im Vergleich zu ihrem Wildtyp gesteigerte Aktivität von mindestens einem der Enzyme E E18 und, optional, eine verringerte Aktivität von mindestens einem der Enzyme E19-E63 auf. In further embodiments, the recombinant cell of sucrose and C0 2 can produce 4 -body than wild type as carbon sources more C, increased compared with its wild type activity of at least one of the enzymes EE 18 and, optionally, a reduced activity of at least one of the enzymes E 19 -E 63 .
Bestimmte Ausführungsformen der Erfindung betreffen Zellen, in denen im Vergleich zum Wildtyp die Aktivität: Certain embodiments of the invention relate to cells in which, compared to the wild type, the activity:
(i) der Enzyme E-\ bis E4 und E12 gesteigert und der Enzyme E19, E26, E27, E29 bis E32 und E46 verringert ist; (i) the enzymes E- \ to E 4 and E 12 are increased and the enzymes E 19 , E 26 , E 27 , E 29 to E 32 and E 46 reduced;
(ii) der Enzyme E3, E5, E6 und E12 gesteigert und der Enzyme E19, E26, E27, E29 bis E32 und E45 verringert ist; oder (ii) the enzymes E 3 , E 5 , E 6 and E 12 are increased and the enzymes E 19 , E 26 , E 27 , E 29 to E 32 and E 45 are reduced; or
(iii) der Enzyme E2, E3, E7, E8, E9, E10 und E12 gesteigert und der Enzyme E19, E26, E27, E29 bis E32 und E46 verringert ist. (iii) the enzymes E 2 , E 3 , E 7 , E 8 , E 9 , E 10 and E 12 are increased and the enzymes E 19 , E 26 , E 27 , E 29 to E 32 and E 46 is reduced.
In solchen Ausführungsformen können die Enzyme E-ι bis E10 und E12 bis E18 durch die Gene scrA, scrB, scrK, scrY, cscB, cscA, cscK, susT1 , susT2, susX bzw. pyc, ppc, pckA, maeA, mmel , maeB und dmlA und die Enzyme E19, E20, E2i , E22, E23, E24, E25, E27, E28, E29, E30, E3i , E32, E33, E45, E46, E47, E48, E49, E5o, E55, E56, E57, E58, E59, EQO, Eßi und Εβ3 ptsG, ptsl, ptsH, crr, tdcE, pflA, pflB, poxB, ack, pta, tdcD, IdhA, adhE, mgsA, ygfG, pdc, porA, porB, porC, porD, xpkl , xpk2, thl, thIA, thIB, phaA, phaB, crt, bdhA, bdhB, ade, adh und IdhL kodiert werden. In such embodiments, the enzymes E-1 to E 10 and E 12 to E 18 may be represented by the genes scrA, scrB, scrK, scrY, cscB, cscA, cscK, susT1, susT2, susX or pyc, ppc, pckA, maeA, mm, maeB and dmlA and the enzymes E 19 , E 20 , E 2 i, E 22 , E 23 , E 24 , E 25 , E 27 , E 28 , E 29 , E 30 , E 3 i, E 32 , E 33 , E 45 , E 4 6, E 47, E 4 8, E 49, E 5 O, E 55 , E 5 6, E 57, E 5 8, E 59, EQO, E β i and Εβ 3 ptsG, ptsl, ptsH, crr, tdcE, pflA, pflB, poxB, ack, pta, tdcD, IdhA, adhE, mgsA, ygfG, pdc, porA, porB, porC, porD, xpkl, xpk2, thI, thIA, thIB, phaA, phaB, crt, bdhA, bdhB, ade, adh and IdhL.
In einem weiteren Aspekt betrifft die Erfindung ein Verfahren zur Herstellung einer gentechnisch veränderten Zelle gemäß der Erfindung. In a further aspect, the invention relates to a method for producing a genetically modified cell according to the invention.
In einem solchen Verfahren kann die Erhöhung der Aktivität von mindestens einem der vorstehend beschriebenen Enzyme E E18 und, optional, die Verringerung der Aktivität von mindestens einem der vorstehend beschriebenen Enzyme E19-E63, durch eines der eingangs beschriebenen Verfahren erfolgen. Bevorzugte Kombinationen von Enzymen mit gesteigerter bzw. verringerter Aktivität sind die oben erwähnten Kombinationen. In noch einem weiteren Aspekt betrifft die Erfindung ein Verfahren zur Herstellung mindestens eines C4-Körpers und/oder mindestens einer über diesen C4-Körper hergestellten In such a method, the increase of the activity of at least one of the above-described enzymes EE 18 and, optionally, the reduction of the activity of at least one of the above-described enzymes E 19 -E 63 can be carried out by one of the methods described above. Preferred combinations of enzymes with increased or decreased activity are the above-mentioned combinations. In a still further aspect, the invention relates to a process for producing at least one C 4 body and / or at least one prepared over this C 4 body
Folgeverbindung, wobei das Verfahren die Inkubation einer Zelle gemäß der Erfindung mit einem Nährmedium, das Saccharose enthält, unter Bedingungen, die die Herstellung mindestens eines C4-Körpers und/oder mindestens einer über diesen C4-Körper hergestellten Folgeverbindung aus Saccharose und C02 erlauben, umfasst. Sequential compound, the method comprising incubating a cell according to the invention with a nutrient medium containing sucrose under conditions which include the production of at least one C 4 body and / or at least one sucrose and C0 2 secondary compound produced via this C 4 body allow, includes.
In einer besonderen Ausführungsform dieses Verfahrens enthält das Medium Kohlendioxid. In a particular embodiment of this process, the medium contains carbon dioxide.
Das erfindungsgemäße Verfahren zur Herstellung des mindestens einen C4-Körpers und/oder mindestens einer über diesen C4-Körper hergestellten Folgeverbindung kann zusätzlich auch das Isolieren des mindestens einen C4-Körpers und/oder mindestens einer über diesen C4- Körper hergestellten Folgeverbindung aus dem Nährmedium einschließen. The process according to the invention for producing the at least one C 4 body and / or at least one secondary compound prepared via this C 4 body may additionally also comprise isolating the at least one C 4 body and / or at least one secondary compound produced via this C 4 body from the nutrient medium.
Schließlich richtet sich die Erfindung in noch einem weiteren Aspekt auf die Verwendung der erfindungsgemäßen Zellen zur Herstellung von mindestens einem C4-Körper und/oder mindestens einer über diesen C4-Körper hergestellten Folgeverbindung aus Saccharose und Kohlendioxid als Kohlenstoffquellen. In bestimmten Ausführungsformen des erfindungsgemäßen Verfahrens bzw. der erfindungsgemäßen Verwendung werden die C4-Körper aus folgender Gruppe ausgewählt: Succinat, Malat, Fumarat, Oxalacetat, Aspartat, Asparagin, Threonin, Tetrahydrofuran, Pyrrolidon, Acetoin, 4,4-Bionell, Hydroxysuccinat, Epoxy-v-Butyrolacton, Butensäure, Butyrat, Butandiol, 1 ,2-Butandiol, 1 ,3-Butandiol, 1 ,4-Butandiol, 2,3-Butandiol, Buten, n-Buten, cis-2- Buten, trans-2-Buten, Isobuten, Butadien, 1 ,2-Butadien, 1 ,3-Butadien, 3-Hydroxybutyrolacton, 4-Hydroxybutyrolacton, 1 -Butanol, 2-Butanol, tert-Butanol, Isobutanol, 2-Hydroxybuttersäure, 3- Hydroxybuttersäure, 4-Hydroxybuttersäure, 2-Hydroxyisobuttersäure und 3- Hydroxyisobuttersäure. Finally, in a still further aspect, the invention is directed to the use of the cells according to the invention for the production of at least one C 4 body and / or at least one sucrose and carbon dioxide secondary compound produced via this C 4 body as carbon sources. In certain embodiments of the method according to the invention or the use according to the invention, the C 4 bodies are selected from the following group: succinate, malate, fumarate, oxaloacetate, aspartate, asparagine, threonine, tetrahydrofuran, pyrrolidone, acetoin, 4,4-bionell, hydroxysuccinate, Epoxy-v-butyrolactone, butenoic acid, butyrate, butanediol, 1, 2-butanediol, 1, 3-butanediol, 1, 4-butanediol, 2,3-butanediol, butene, n-butene, cis-2-butene, trans- 2-butene, isobutene, butadiene, 1, 2-butadiene, 1, 3-butadiene, 3-hydroxybutyrolactone, 4-hydroxybutyrolactone, 1-butanol, 2-butanol, tert-butanol, isobutanol, 2-hydroxybutyric acid, 3-hydroxybutyric acid, 4-hydroxybutyric acid, 2-hydroxyisobutyric acid and 3-hydroxyisobutyric acid.
In einer Ausführungsform werden die C4-Körper ausgewählt aus der Gruppe bestehend aus: Malat, Oxalacetat und die über diese C4-Körper hergestellten Folgeverbindungen ausgewählt werden aus der Gruppe bestehend aus durch enzymatische oder chemische Synthese über Malat oder Oxalacetat hergestellte Folgeverbindungen. Die durch enzymatische Synthese über Malat oder Oxalacetat hergestellte Folgeverbindungen können beispielsweise ausgewählt werden aus der Gruppe bestehend aus: Succinat, Aspartat, Asparagin, Threonin, In one embodiment, the C 4 bodies are selected from the group consisting of: malate, oxaloacetate and the derivatives produced via these C 4 bodies are selected from the group consisting of secondary compounds prepared by enzymatic or chemical synthesis via malate or oxalacetate. The secondary compounds produced by enzymatic synthesis via malate or oxalacetate can be selected, for example, from the group consisting of: succinate, aspartate, asparagine, threonine,
Tetrahydrofuran, Butyrat, Butandiol, 1 ,2-Butandiol, 1 ,3-Butandiol, 1 ,4-Butandiol, 2,3-Butandiol, 3- und 4-Hydroxybutyrolacton, 1 -, 2- und tert-Butanol, Isobutanol, 2-, 3- und 4- Hydroxybuttersäure, 2- und 3-Hydroxyisobuttersäure, Methionin und Lysin. Tetrahydrofuran, butyrate, butanediol, 1, 2-butanediol, 1, 3-butanediol, 1, 4-butanediol, 2,3-butanediol, 3- and 4-hydroxybutyrolactone, 1-, 2- and tert-butanol, isobutanol, 2nd , 3- and 4-hydroxybutyric acid, 2- and 3-hydroxyisobutyric acid, methionine and lysine.
Die erfindungsgemäßen, gentechnisch veränderten Zellen können kontinuierlich oder diskontinuierlich im batch-Verfahren (Satzkultivierung) oder im fed-batch-Verfahren The genetically modified cells of the invention can be used continuously or discontinuously in the batch process (batch culturing) or in the fed-batch process
(Zulaufverfahren) oder repeated-fed-batch-Verfahren (repetitives Zulaufverfahren) zum Zwecke der Produktion von C4-Körpern, beispielsweise C4-Carbonsäuren wie Succinat, mit dem (Feed process) or repeated-fed-batch process (repetitive feed process) for the purpose of producing C 4 bodies, for example C 4 carboxylic acids such as succinate, with the
Nährmedium in Kontakt gebracht und kultiviert werden. Ebenfalls praktikabel ist ein Bring nutrient medium into contact and cultivated. Also practicable is a
semikontinuierliches Verfahren, wie es in der GB-A-1009370 beschrieben wird. Eine semi-continuous process as described in GB-A-1009370. A
Zusammenfassung der bekannten Kultivierungsmethoden ist im Lehrbuch von Chmiel Summary of the known cultivation methods is in the textbook of Chmiel
(„Bioprozesstechnik 1 . Einführung in die Bioverfahrenstechnik" (Gustav Fischer Verlag, Stuttgart, 1991 )) oder im Lehrbuch von Storhas („Bioreaktoren und periphere Einrichtungen", Vieweg Verlag, Braunschweig/Wiesbaden, 1994) zu finden. Das zu verwendende Kulturmedium muss in geeigneter Weise den Ansprüchen des jeweiligen Wirtszellstammes genügen. ("Bioprocess Technique 1: Introduction to Bioprocess Engineering" (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas ("Bioreactors and Peripheral Facilities", Vieweg Verlag, Braunschweig / Wiesbaden, 1994). The culture medium to be used must suitably satisfy the requirements of the respective host cell strain.
Beschreibungen von Kulturmedien für verschiedene Mikroorganismen sind im Handbuch "Manual of Methods for General Bacteriology" der American Society for Bacteriology Descriptions of culture media for various microorganisms are given in the Manual of Methods for General Bacteriology of the American Society for Bacteriology
(Washington D. C, USA, 1981 ) enthalten. In den verwendeten Nährmedien dienen insbesondere Saccharose und/oder Kohlendioxid als Kohlenstoffquellen. Als Stickstoffquellen können organische stickstoffhaltige Verbindungen wie Peptone, (Washington D.C, USA, 1981). In particular, sucrose and / or carbon dioxide serve as carbon sources in the nutrient media used. As nitrogen sources, organic nitrogen-containing compounds such as peptones,
Hefeextrakt, Fleischextrakt, Malzextrakt, Maisquellwasser, Sojabohnenmehl und Harnstoff oder anorganische Verbindungen wie Ammoniumsulfat, Ammoniumchlorid, Ammoniumphosphat, Ammoniumcarbonat und Ammoniumnitrat verwendet werden. Die Stickstoffquellen können einzeln oder als Mischung verwendet werden.  Yeast extract, meat extract, malt extract, corn steep liquor, soybean meal and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources can be used singly or as a mixture.
Als Phosphorquelle können Phosphorsäure, Kaliumdihydrogenphosphat oder As a phosphorus source can phosphoric acid, potassium dihydrogen phosphate or
Dikaliumhydrogenphosphat oder die entsprechenden natriumhaltigen Salze verwendet werden. Dipotassium hydrogen phosphate or the corresponding sodium-containing salts are used.
Das Kulturmedium kann weiterhin Metallsalze enthalten, wie z. B. Magnesiumsulfat oder Eisensulfat, die für das Wachstum der Zellen notwendig sind. The culture medium may further contain metal salts, such as. As magnesium sulfate or iron sulfate, which are necessary for the growth of the cells.
Schließlich können dem Medium noch weitere Stoffe, wie z.B. Aminosäuren und/oder Vitamine zugesetzt werden. Dem Kulturmedium können überdies geeignete Vorstufen zugesetzt werden. Die genannten Einsatzstoffe können zur Kultur in Form eines einmaligen Ansatzes Finally, the medium may contain other substances, e.g. Amino acids and / or vitamins are added. In addition, suitable precursors can be added to the culture medium. The stated feedstocks can be used for culture in the form of a unique approach
hinzugegeben oder in geeigneter Weise während der Kultivierung zugefüttert werden. be added or fed in a suitable manner during the cultivation.
Zur pH-Kontrolle der Kultur werden basische Verbindungen wie Natriumhydroxid, For pH control of the culture basic compounds such as sodium hydroxide,
Kaliumhydroxid, Ammoniak bzw. Ammoniakwasser oder saure Verbindungen wie Potassium hydroxide, ammonia or ammonia water or acidic compounds such as
Phosphorsäure oder Schwefelsäure in geeigneter Weise eingesetzt. Zur Kontrolle der Phosphoric acid or sulfuric acid used in a suitable manner. To control the
Schaumentwicklung können Antischaummittel wie z. B. Fettsäurepolyglykolester eingesetzt werden. Zur Aufrechterhaltung der Stabilität von Plasmiden können dem Medium geeignete selektiv wirkende Stoffe wie z. B. Antibiotika hinzugefügt werden. Foaming can anti-foaming agents such. B. fatty acid polyglycol esters are used. To maintain the stability of plasmids, the medium suitable selective substances such. B. antibiotics are added.
Die Temperatur der Kultur liegt normalerweise bei 20°C bis 45°C und vorzugsweise bei 25°C bis 40°C. The temperature of the culture is usually 20 ° C to 45 ° C, and preferably 25 ° C to 40 ° C.
Die Aufreinigung des/der C4-Körper(s) oder Folgeprodukten aus der Nährlösung erfolgt vorzugsweise kontinuierlich, wobei es in diesem Zusammenhang weiterhin bevorzugt ist, auch die Herstellung des C4-Körpers durch Fermentation kontinuierlich durchzuführen, so dass der gesamte Prozess von der Herstellung des C4-Körpers bis zur dessen Aufreinigung aus der Fermentationsbrühe kontinuierlich durchgeführt werden kann. Zur kontinuierlichen Aufreinigung des C4-Körpers aus der Fermentationsbrühe wird diese kontinuierlich über eine Vorrichtung zur Abtrennung der bei der Fermentation eingesetzten Mikroorganismen, vorzugsweise über einen Filter mit einer Ausschlussgröße in einem Bereich von 20 bis 200 kDa geführt, in dem eine Fest/Flüssig-T rennung stattfindet. Denkbar ist auch der Einsatz einer Zentrifuge, einer geeigneten Sedimentationsvorrichtung oder eine Kombination dieser Vorrichtungen, wobei es besonders bevorzugt ist, zumindest einen Teil der Mikroorganismen zunächst durch The purification of the C 4 body (s) or secondary products from the nutrient solution is preferably carried out continuously, it being further preferred in this context to carry out the production of the C 4 body by fermentation continuously, so that the entire process from the production of the C 4 body to its purification from the fermentation broth can be carried out continuously. For the continuous purification of the C 4 body from the fermentation broth, this is continuously passed through a device for separating the microorganisms used in the fermentation, preferably via a filter with an exclusion size in a range of 20 to 200 kDa, in which a solid / liquid Separation takes place. It is also conceivable to use a centrifuge, a suitable sedimentation device or a combination of these devices, it being particularly preferred that at least some of the microorganisms pass through first
Sedimentation abzutrennen und anschließend die von den Mikroorganismen teilweise befreite Fermentationsbrühe einer Ultrafiltration oder Zentrifugationsvorrichtung zuzuführen. Separate sedimentation and then supply the partially freed from the microorganisms fermentation broth an ultrafiltration or centrifugation device.
Das hinsichtlich seines C4-Körper-Anteils angereicherte Fermentationserzeugnis wird nach der Abtrennung der Mikroorganismen einer vorzugsweise mehrstufigen Trennanlage zugeführt. In dieser Trennanlage sind mehrere hintereinander geschaltete Trennstufen vorgesehen, aus denen jeweils Rückführleitungen ausmünden, die zum Fermentationstank zurückgeführt sind. Weiterhin führen aus den jeweiligen Trennstufen Ableitungen heraus. Die einzelnen The enriched in terms of its C 4 body portion fermentation product is fed after separation of the microorganisms of a preferably multi-stage separation plant. In this separation plant a plurality of series-connected separation stages are provided, from which each return lines lead, which are returned to the fermentation tank. Furthermore lead out of the respective separation stages derivatives. The single ones
Trennstufen können nach dem Prinzip der Elektrodialyse, der Umkehrosmose, der Ultrafiltration oder der Nanofiltration arbeiten. In der Regel handelt es sich um Membran-Trenneinrichtungen in den einzelnen Trennstufen. Die Auswahl der einzelnen Trennstufen ergibt sich aus Art und Umfang der Gärungsnebenprodukte und Substratreste. Separation stages can operate on the principle of electrodialysis, reverse osmosis, ultrafiltration or nanofiltration. As a rule, these are membrane separation devices in the individual separation stages. The selection of the individual separation stages results from the nature and extent of the fermentation by-products and substrate residues.
Neben der Abtrennung des C4-Körpers mittels Elektrodialyse, Umkehrosmose, Ultrafiltration oder Nanofiltration, in deren Verlauf als Endprodukt eine wässrige C4-Körper-Lösung erhalten wird, kann der C4-Körper auch durch Extraktionsverfahren aus der von Mikroorganismen befreiten Fermentationslösung abgetrennt werden, wobei in diesem Fall letztendlich der reine C4-Körper erhalten werden kann. Zur Abtrennung einer C4-Carbonsäure durch Extraktion können der Fermentationslösung beispielsweise Ammoniumverbindungen oder Amine zugesetzt werden, um ein Ammoniumsalz der C4-Carbonsäure zu bilden. Dieses In addition to the removal of the C 4 body by means of electrodialysis, reverse osmosis, ultrafiltration or nanofiltration, in the course of which an aqueous C 4 -body solution is obtained, the C 4 body can also be separated by extraction from the freed from microorganisms fermentation solution in which case, ultimately, the pure C 4 body can be obtained. For separation of a C 4 carboxylic acid by extraction, for example, ammonium compounds or amines may be added to the fermentation solution to form an ammonium salt of C 4 carboxylic acid. This
Ammoniumsalz kann dann aus der Fermentationslösung abgetrennt werden, in dem ein organisches Extraktionsmittel zugesetzt und die so erhaltene Mischung anschließend erhitzt wird, wodurch das Ammoniumsalz sich in der organischen Phase anreichert. Aus dieser Phase kann die C4-Carbonsäure dann unter Erhalt der reinen C4-Carbonsäure beispielsweise durch weitere Extraktionsschritte isoliert werden. Genauere Einzelheiten bezüglich dieses Ammonium salt can then be separated from the fermentation solution by adding an organic extractant and then heating the mixture thus obtained, whereby the ammonium salt accumulates in the organic phase. From this phase, the C 4 carboxylic acid can then be isolated to obtain the pure C 4 carboxylic acid, for example by further extraction steps. More details regarding this
Trennverfahrens sind der WO- A-02/090312 zu entnehmen, deren Offenbarungsgehalt hiermit als Referenz eingeführt wird und einen Teil der Offenbarung der vorliegenden Anmeldung bildet. Separation process can be found in WO-A-02/090312, the disclosure of which hereby is incorporated by reference and forms part of the disclosure of the present application.
Je nach Art und Weise der Abtrennung des C4-Körpers aus der Fermentationslösung wird entweder eine wässrige C4-Körper-Lösung, beinhaltend 2 bis 90 Gew.-%, vorzugsweise 7,5 bis 50 Gew.-% und besonders bevorzugt 10 bis 25 Gew.-% an C4-Körper, oder aber reiner C4- Körper erhalten. Depending on the manner in which the C 4 body is separated from the fermentation solution, either an aqueous C 4 -body solution containing from 2 to 90% by weight, preferably from 7.5 to 50% by weight and more preferably from 10 to 25 wt .-% of C 4 body, or pure C 4 - body obtained.
Des Weiteren können die durch das erfindungsgemäße Verfahren hergestellte C4- Carbonsäuren vor, während oder nach der Aufreinigung noch neutralisiert werden, wobei hierzu Basen wie etwa Kalziumhydroxid oder Natriumhydroxid eingesetzt werden können. Furthermore, the C 4 -carboxylic acids prepared by the process according to the invention can still be neutralized before, during or after the purification, it being possible to use bases such as calcium hydroxide or sodium hydroxide for this purpose.
Weitere Ausführungsformen der Erfindung sind in den Ansprüchen und den Beispielen enthalten. Die folgenden Beispiele dienen der Veranschaulichung der Erfindung, wobei die Erfindung nicht auf diese speziellen Ausführungsformen beschränkt ist. Further embodiments of the invention are contained in the claims and the examples. The following examples serve to illustrate the invention, but the invention is not limited to these specific embodiments.
Beispiel 1 example 1
Herstellung von E. co//'-Expressionsvektoren für die Gene ppc, pck, maeA und maeB aus E. coli sowie pyc aus C. glutamicum Preparation of E. co // 'expression vectors for the genes ppc, pck, maeA and MAEB from E. coli and C. glutamicum pyc from
Zur Herstellung von £ co//-Expressionsvektoren für die Gene ppc (SEQ ID NO: 1 ), pck (SEQ ID NO: 2), maeA (SEQ ID NO: 3) und maeB (SEQ ID NO: 4) aus £ coli sowie pyc aus C. For the production of £ co // expression vectors for the genes ppc (SEQ ID NO: 1), pck (SEQ ID NO: 2), mAca (SEQ ID NO: 3) and mAb (SEQ ID NO: 4) from E. coli as well as pyc from C.
glutamicum (SEQ ID NO: 5) werden diese Gene aus chromosomaler DNA von £. coli MG1655 bzw. C. glutamicum ATCC 13032 per PCR amplifiziert und gleichzeitig über die verwendeten Oligonukleotide eine Schnittstelle stromaufwärts der jeweiligen Ribosomenbindungstelle und eine Schnittstelle stromabwärts des Stopcodons eingeführt. Die Präparation der glutamicum (SEQ ID NO: 5), these genes are made from chromosomal DNA of £. coli MG1655 or C. glutamicum ATCC 13032 were amplified by PCR and introduced simultaneously via the oligonucleotides used an interface upstream of the respective ribosome binding site and an interface downstream of the stop codon. The preparation of
chromosomalen DNA von £ coli MG 1655 bzw. C. glutamicum ATCC 13032 erfolgt mittels DNeasy Blood & Tissue Kit (Qiagen, Hilden) gemäß den Angaben des Herstellers. Bei der Amplifikation der Gene ppc, pck, maeA und maeB aus £ coli sowie pyc aus C. glutamicum mit chromosomaler DNA von £ coli MG 1655 bzw. C. glutamicum ATCC 13032 als Matrize kommen folgende Oligonukleotide zum Einsatz: 5'-ATA GAG CTC AGG GCT ATC AAA CGA TAA GAT GGG GTG-3' (SEQ ID NO: 6) Chromosomal DNA of E. coli MG 1655 or C. glutamicum ATCC 13032 is carried out using DNeasy Blood & Tissue Kit (Qiagen, Hilden) according to the manufacturer's instructions. The following oligonucleotides are used in the amplification of the genes ppc, pck, maeA and maeB from E. coli and pyc from C. glutamicum with chromosomal DNA of E. coli MG 1655 or C. glutamicum ATCC 13032 as template. 5'-ATA GAG CTC AGG GCT ATC AAA CGA TAA GAT GGG GTG-3 '(SEQ ID NO: 6)
5'-ATA CCT GCA GGT TAG CCG GTA TTA CGC ATA CCT GCC G-3' (SEQ ID NO: 7) 5'-ATA CCT GCA GGT TAG CCG GTA TTA CGC ATA CCT GCC G-3 '(SEQ ID NO: 7)
E. coli pck: pck-fw: 5'-ATA GGA TCC TTA CTA TTC AGG CAA TAC ATA TTG GCT AAG E. coli pck: pck-fw: 5'-ATA GGA TCC TTA CTA TTC AGG CAA TAC ATA TTG GCT AAG
GA-3' (SEQ ID NO: 8) pck-rv: 5'-ATA CCT GCA GGT CAT TAC AGT TTC GGA CCA GCC GCT AC-3'  GA-3 '(SEQ ID NO: 8) pck-rv: 5'-ATA CCT GCA GGT CAT TAC AGT TTC GGA CCA GCC GCT AC-3'
(SEQ ID NO: 9)  (SEQ ID NO: 9)
5'-ATA GAG CTC GTG GAT ATT CAA AAA AGA GTG AGT GAC ATG GAA C-3' (SEQ ID NO: 10) 5'-ATA GAG CTC GTG GAT ATT CAA AAA AGA GTG AGT GAC ATG GAA C-3 '(SEQ ID NO: 10)
5'-ATA CCT GCA GGT TAG ATG GAG GTA CGG CGG TAG TCG-3' (SEQ ID NO: 1 1 ) 5'-ATA CCT GCA GGT TAG ATG GAG GTA CGG CGG TAG TCG-3 '(SEQ ID NO: 1 1)
5'-ATA GAG CTC TAC GTG AAA GGA ACA ACC AAA TGG ATG ACC-3' (SEQ ID NO: 12) 5'-ATA GAG CTC TAC GTG AAA GGA ACA ACC AAA TGG ATG ACC-3 '(SEQ ID NO: 12)
5'-ATA CCT GCA GGT TAC AGC GGT TGG GTT TGC GCT TC-3' (SEQ ID NO: 13) C. glutamicum pyc: pyc-fw: 5'-ATA GAG CTC TGA AAG GAA TAA TTA CTC TAG TGT CGA CTC-3' 5'-ATA CCT GCA GGT TAC AGC GGT TGG GTT TGC GCT TC-3 '(SEQ ID NO: 13) C. glutamicum pyc: pyc-fw: 5'-ATA GAG CTC TGA AAG GAA TAA TTA CTC TAG TGT CGA CTC-3 '
(SEQ ID NO: 14) pyc-rv: 5'-ATA CCT GCA GGG GTT TAG GAA ACG ACG ACG ATC AAG-3'  (SEQ ID NO: 14) pyc-rv: 5'-ATA CCT GCA GGG GTT TAG GAA ACG ACG ACG ATC AAG-3 '
(SEQ ID NO: 15)  (SEQ ID NO: 15)
Folgende Parameter werden für die PCR eingesetzt: 1 x: initiale Denaturierung, 98 °C, 0:30 min; 30 x: Denaturierung, 98 °C, 0:10 min, Annealing, 60 °C, 0:30 min; Elongation, 72 °C, 3 min; 1 x: terminale Elongation, 72 °C, 10 min. Für die Amplifikation wird der Phusion™ High-Fidelity Master Mix von New England Biolabs (Frankfurt) entsprechend den Empfehlungen des The following parameters are used for the PCR: 1 x: initial denaturation, 98 ° C, 0:30 min; 30x: denaturation, 98 ° C, 0:10 min, annealing, 60 ° C, 0:30 min; Elongation, 72 ° C, 3 min; 1 x: terminal elongation, 72 ° C, 10 min. For the amplification, the Phusion ™ High-Fidelity Master Mix from New England Biolabs (Frankfurt) is used according to the recommendations of the
Herstellers verwendet. Jeweils 10 μΙ der PCR-Reaktionen werden anschließend auf einem 0,8 %igen Agarosegel aufgetrennt. Die Durchführung der PCR, der Agarosegel-Elektrophorese, Ethidiumbromidfärbung der DNA und Bestimmung der PCR-Fragmentgrößen erfolgt in dem Fachmann bekannter Art und Weise. In allen Fällen können PCR-Fragmente der erwarteten Größe amplifiziert werden. Diese beträgt für ppc 2710 bp, für pck 1686 bp, für maeA 1745 bp, für maeB 3220 bp und für pyc 3466 bp. Die PCR-Produkte werden mit Sac\ und Sbfi {ppc, maeA, maeB und pyc) bzw. ßamHI und Sbfi {pck) entsprechend den Empfehlungen des Herstellers der Restriktiosendonukleasen (New England Biolabs; Bad Schwalbach) verdaut und in den mit Sac\ und Sbfi {ppc, maeA, maeB und pyc) bzw. ßamHI und Sbfi {pck) geschnittenen Vektor pEC-XC99E (SEQ ID NO: 16) ligiert. pEC-XC99E ist ein £ coli-C. g/yfam/ci/m-Shuttle-Vektor, der in beiden Organismen  Manufacturer used. Each 10 μΙ of the PCR reactions are then separated on a 0.8% agarose gel. The carrying out of the PCR, the agarose gel electrophoresis, ethidium bromide staining of the DNA and determination of the PCR fragment sizes are carried out in a manner known to the person skilled in the art. In all cases, PCR fragments of the expected size can be amplified. For ppc this is 2710 bp, for pck 1686 bp, for maeA 1745 bp, for maeB 3220 bp and for pyc 3466 bp. The PCR products are digested with Sac \ and Sbfi {ppc, maeA, maeB and pyc) and / or bamHI and Sbfi {pck) according to the recommendations of the manufacturer of the Restriktiosendonukleasen (New England Biolabs, bath Schwalbach) and in those with Sac \ and Sbfi {ppc, maeA, maeB and pyc) or ßamHI and Sbfi {pck) cut vector pEC-XC99E (SEQ ID NO: 16) ligated. pEC-XC99E is a coli coli C. g / yfam / ci / m shuttle vector present in both organisms
Chloramphenicol-Resistenz vermittelt sowie einen ColE1 -Replikationsursprung (hohe Chloramphenicol resistance and a ColE1 origin of replication (high
Kopiezahl: > 500 Kopien pro £ co//-Zelle) und einen synthetischen irc-Promotor ohne Copy number:> 500 copies per £ co // cell) and a synthetic irc promoter without
Ribosomenbindungstelle trägt. Ligation sowie Transformation chemisch kompetenter £. coli DH5 -Zellen (Gibco-BRL, Karlsruhe) erfolgen in dem Fachmann bekannter Art und Weise. Carries ribosome binding site. Ligation and transformation of chemically competent £. coli DH5 cells (Gibco-BRL, Karlsruhe) are carried out in a manner known to the person skilled in the art.
Die korrekte Insertion der ppc-, pck-, maeA-, maeB- bzw. pyc-Fragmente wird durch eine Restriktion mit Sac\ und Sbfi {ppc, maeA, maeB und pyc) bzw. ßamHI und Sbfi {pck) überprüft. Die Authentizität der inserierten Fragmente wird durch DNA-Sequenzierung überprüft. Die fertiggestellten £ co//-Expressionsvektoren werden als pEC-XC99E-ppc (SEQ ID NO:17), pEC- XC99E-pck (SEQ ID NO:18), pEC-XC99E-maeA (SEQ ID NO:19), pEC-XC99E-maeB (SEQ ID NO:20) und pEC-XC99E-pyc (SEQ ID NO:21 ) bezeichnet. Beispiel 2 The correct insertion of the ppc, pck, maeA, maeB or pyc fragments is checked by a restriction with Sac \ and Sbfi {ppc, maeA, maeB and pyc) or ßamHI and Sbfi {pck). The authenticity of the inserted fragments is checked by DNA sequencing. The completed £ co // expression vectors are identified as pEC-XC99E-ppc (SEQ ID NO: 17), pEC-XC99E-pck (SEQ ID NO: 18), pEC-XC99E-maeA (SEQ ID NO: 19), pEC XC99E-maeB (SEQ ID NO: 20) and pEC-XC99E-pyc (SEQ ID NO: 21). Example 2
Herstellung von E. co//'-Expressionsvektoren für die Loci cscA-cscKB aus E. coli sowie scrK-scrYAB aus pUR400 Construction of E. co // 'expression vectors for the loci CSCA-cscKB from E. coli and SCRK-scrYAB from pUR400
Zur Herstellung von £ co//-Expressionsvektoren für die Loci cscA-cscKB aus £. coli DSM 1 1 16 (SEQ ID NO: 22) sowie scrK-scrYAB aus pUR400 (SEQ ID NO: 23) werden diese aus chromosomaler DNA von £ coli DSM 1 1 16 bzw. Gesamt-DNA von £ co//'-K12 (pUR400) per PCR amplifiziert und gleichzeitig über die verwendeten Oligonukleotide je eine For the production of £ co // expression vectors for the loci cscA-cscKB from £. coli DSM 1 1 16 (SEQ ID NO: 22) and SCRk-scrYAB from pUR400 (SEQ ID NO: 23), these are from chromosomal DNA of £ coli DSM 1 1 16 and total DNA of £ co // '-K12 (pUR400) amplified by PCR and simultaneously via the oligonucleotides used one
Restriktionsschnittstelle am 5'- bzw. 3'-Ende eingeführt. Die Präparation der chromosomalen DNA aus £ coli DSM 1 1 16 bzw. Gesamt-DNA aus £ co//'-K12 (pUR400) erfolgt mittels DNeasy Blood & Tissue Kit (Qiagen, Hilden) gemäß den Angaben des Herstellers Bei der Amplifikation der Loci cscA-cscKB aus chromosomaler DNA von £ coli DSM 1 1 16 bzw. von scrK-scrYAB aus Gesamt-DNA von £ co//-K12 (pUR400) als Matrize kommen folgende Oligonukleotide zum Einsatz: Restriction interface introduced at the 5 'or 3' end. The preparation of the chromosomal DNA from E. coli DSM 1 1 16 or total DNA from £ co // ' -K12 (pUR400) is carried out using the DNeasy Blood & Tissue Kit (Qiagen, Hilden) according to the manufacturer's instructions in the amplification of the loci cscA-cscKB from chromosomal DNA of E. coli DSM 1 1 16 or scrK-scrYAB from total DNA of coco // - K12 (pUR400) as template, the following oligonucleotides are used:
£ coli cscA-cscKB: csc-fw: 5'-ATA CAT ATG TTA TTA ACC CAG TAG CCA GAG TGC TCC AT GT-Coli cscA-cscKB: csc-fw: 5'-ATA CAT ATG TTA TTA ACC CAG TAG CCA GAG TGC TCC AT GT-
3' (SEQ ID NO: 24) csc-rv: 5'-ATA CTC GAG CTA CTA TAT TGC TGA AGG TAC AGG CGT TTC C-3 '(SEQ ID NO: 24) csc-rv: 5'-ATA CTC GAG CTA CTA TAT TGC TGA AGG TAC AGG CGT TTC C-
3' (SEQ ID NO: 25) 3 '(SEQ ID NO: 25)
£ coli scrK-scrYAB scr-fw: 5'-ATA CCA TGG TCC GCC AGT TCA TCC GGG AAC GG-3' (SEQ ID Coli coli scrK-scrYAB scr-fw: 5'-ATA CCA TGG TCC GCC AGT TCA TCC GGG AAC GG-3 '(SEQ ID
NO: 26) scr-rv: 5'-ATA GCG GCC GCT TAT TCT ACC ATG CAA GTT CGC AGC-3'  NO: 26) scr-rv: 5'-ATA GCG GCC GCT TAT TCT ACC ATG CAA GTT CGC AGC-3 '
(SEQ ID NO: 27) Folgende Parameter werden für die PCR eingesetzt: 1 x: initiale Denaturierung, 98 °C, 0:30 min; 30 x: Denaturierung, 98 °C, 0:10 min, Annealing, 60 °C, 0:30 min; Elongation, 72 °C, 5 min; 1 x: terminale Elongation, 72 °C, 10 min. Für die Amplifikation wird der Phusion™ High-Fidelity Master Mix von New England Biolabs (Frankfurt) entsprechend den Empfehlungen des (SEQ ID NO: 27) The following parameters are used for the PCR: 1 x: initial denaturation, 98 ° C, 0:30 min; 30x: denaturation, 98 ° C, 0:10 min, annealing, 60 ° C, 0:30 min; Elongation, 72 ° C, 5 min; 1 x: terminal elongation, 72 ° C, 10 min. For the amplification, the Phusion ™ High-Fidelity Master Mix from New England Biolabs (Frankfurt) is used according to the recommendations of the
Herstellers verwendet. Jeweils 10 μΙ der PCR-Reaktionen werden anschließend auf einem 0,8 %igen Agarosegel aufgtrennt. Die Durchführung der PCR, der Agarosegel-Elektrophorese, Ethidiumbromidfärbung der DNA und Bestimmung der PCR-Fragmentgrößen erfolgen in dem Fachmann bekannter Art und Weise. In beiden Fällen können PCR-Fragmente der erwarteten Größe amplifiziert werden. Diese beträgt für cscA-cscKB 3907 bp und für scrK-scrYAB 5800 bp. Die PCR-Produkte werden mit Λ/col- und Not\ (scrK-scrYAB) bzw. Nde\ und Xho\ (cscA-cscKB) entsprechend den  Manufacturer used. Each 10 μΙ of the PCR reactions are then aufgtrennt on a 0.8% agarose gel. The carrying out of the PCR, agarose gel electrophoresis, ethidium bromide staining of the DNA and determination of the PCR fragment sizes are carried out in a manner known to the person skilled in the art. In both cases, PCR fragments of the expected size can be amplified. This is 3907 bp for cscA-cscKB and 5800 bp for scrK-scrYAB. The PCR products are labeled with Λ / col and Not \ (scrK-scrYAB) or Nde \ and Xho \ (cscA-cscKB) according to the
Empfehlungen des Herstellers der Restriktiosendonukleasen (New England Biolabs; Bad Schwalbach) verdaut und in den Λ/col- und Not\ (scrK-scrYAB) bzw. Nde\ und Xho\ (cscA- cscKB) geschnittenen Vektor pCOLADuet-1 (SEQ ID NO: 28; Merck Biosciences; Nottigham, UK) ligiert. pCOLADuet-1 ist ein £. co//-Vektor mit niedriger Kopiezahl (20 - 40 Kopien pro Zelle), der Kanamycin-Resistenz vermittelt sowie einen ColA-Replikationsursprung trägt. Recommendations of the manufacturer of Restriktiosendonukleasen (New England Biolabs, bath Schwalbach) digested and in the Λ / col and Not \ (scrK-scrYAB) and / or Nde \ and Xho \ (cscA cscKB) cut vector pCOLADuet-1 (SEQ ID NO : 28; Merck Biosciences; Nottigham, UK). pCOLADuet-1 is a £. co // vector of low copy number (20-40 copies per cell) that mediates kanamycin resistance and carries a ColA origin of replication.
Ligation sowie Transformation chemisch kompetenter E. coli DH5 -Zellen (Gibco-BRL, Ligation and transformation of chemically competent E. coli DH5 cells (Gibco-BRL,
Karlsruhe) erfolgen in dem Fachmann bekannter Art und Weise. Die korrekte Insertion der cscA-cscKB- und scr -scrY^ß-Fragmente wird durch eine Restriktion mit Λ/col- und Not\ (scrK-scrYAB) bzw. Nde\ und Xho\ (cscA-cscKB) überprüft. Die Authentizität der inserierten Fragmente wird durch DNA-Sequenzierung überprüft. Die fertiggestellten E. coli- Expressionsvektoren werden als pCOLA-csc (SEQ ID NO:29) bzw. pCOLA-scr (SEQ ID NO:30) bezeichnet. Karlsruhe) take place in a manner known to the person skilled in the art. The correct insertion of the cscA-cscKB and scr -scrYββ fragments is checked by restriction with Λ / col and Not \ (scrK-scrYAB) or Nde \ and Xho \ (cscA-cscKB). The authenticity of the inserted fragments is checked by DNA sequencing. The completed E. coli expression vectors are designated pCOLA-csc (SEQ ID NO: 29) and pCOLA-scr (SEQ ID NO: 30), respectively.
Beispiel 3 Example 3
Chromatographische Quantifizierung von Edukten und Produkten Chromatographic quantification of educts and products
Die chromatographische Quantifizierung von Saccharose erfolgt auf folgende Art und Weise: Die Quantifizierung von Saccharose wird mittels Hochleistungsflüssigkeitschromatografie durchgeführt. Für die Trennung von Zuckern wird eine Anionenaustauscher-Säule mit The chromatographic quantification of sucrose is carried out in the following way: The quantification of sucrose is carried out by means of high-performance liquid chromatography carried out. For the separation of sugars is an anion exchange column with
Aminophase, 5 μηι Partikelgröße und den Maßen 250 x 4,6 mm (Waters Spherisorb NH2; Waters, Eschborn) verwendet. Die mobile Phase besteht aus einem Gemisch aus Acetonitril (56% v/v), Aceton (26% v/v) und Wasser (16% v/v). Die Proben werden sterilfiltriert und unverdünnt vermessen. Das Injektionsvolumen beträgt 10 μΙ_, der Durchfluss der mobilen Phase 2 mL/min und die Säulentemperatur 30°C. Saccharose wird mittels Brechungsindex- Detektor (Agilent 1200 Series RID; Agilent Technologies, Böblingen) quantifiziert. Die Aminophase, 5 μηι particle size and the dimensions 250 x 4.6 mm (Waters Spherisorb NH2, Waters, Eschborn) used. The mobile phase consists of a mixture of acetonitrile (56% v / v), acetone (26% v / v) and water (16% v / v). The samples are sterile filtered and measured undiluted. The injection volume is 10 μΙ_, the mobile phase flow 2 mL / min and the column temperature 30 ° C. Sucrose is quantified by refractive index detector (Agilent 1200 Series RID, Agilent Technologies, Böblingen). The
Referenzsubstanz (Sigma-Aldrich, Steinheim) wird in Wasser gelöst vermessen. Dabei liegt bis zu einer Konzentration von 100 g/L eine lineare Abhängigkeit zwischen der Peakfläche und der Substanzkonzentration vor. Die Bestimmungsgrenze liegt für Saccharose bei 1 g/L. Reference substance (Sigma-Aldrich, Steinheim) is measured dissolved in water. There is a linear dependence between the peak area and the substance concentration up to a concentration of 100 g / L. The limit of quantification for sucrose is 1 g / L.
Die chromatographische Quantifizierung von Succinat, Acetat, Lactat, Formiat und Ethanol erfolgte auf folgende Art und Weise: Die Trennung der organischen Säuren und Ethanol wird mit Hilfe der Hochleistungsflüssigkeitschromatografie durchgeführt. Dabei findet die The chromatographic quantification of succinate, acetate, lactate, formate and ethanol was carried out in the following manner: The separation of the organic acids and ethanol is carried out by means of high-performance liquid chromatography. It finds the
lonenausschluss-Säule Aminex® HPX 87H mit den Maßen 300 mm x 7,8 mm (Bio Rad Laboratories, München) als stationäre Phase Verwendung. Als mobile Phase wird 10 mMIon exclusion column Aminex® HPX 87H with the dimensions 300 mm x 7.8 mm (Bio Rad Laboratories, Munich) as a stationary phase use. As the mobile phase becomes 10 mM
Schwefelsäure eingesetzt. Die Säulentemperatur beträgt 40°C, der Durchfluss 0,6 ml/min. Die Probe wird mit 0,5 M Schwefelsäure auf einen pH-Wert von 4 bis 5 angesäuert und mit einem Volumen von 20 μί in die Säule injiziert. Die Detektion erfolgt mittels Diodenarraydetektor (Agilent 1200 Series DAD, Agilent Technologies, Böblingen) bei einer Wellenlänge von 190 bis 400 nm sowie Brechungsindex-Detektor (Agilent 1200 Series RID, Agilent Technologies, Böblingen). Die Referenzsubstanzen werden in Konzentrationen von 0,1 g/L bis 20 g/L in Wasser gelöst vermessen. Die Bestimmungsgrenzen liegen für Lactat, Acetat, Succinat und Formiat bei 0,8 g/L, Ethanol kann bis zu einer Konzentration von 1 g/L bestimmt werden. Im Bereich von 0,8 g/L bis 20 g/L liegt eine lineare Abhängigkeit der Peakflächen von der Sulfuric acid used. The column temperature is 40 ° C, the flow rate 0.6 ml / min. The sample is acidified with 0.5 M sulfuric acid to a pH of 4 to 5 and injected into the column with a volume of 20 μί. Detection is performed using a diode array detector (Agilent 1200 Series DAD, Agilent Technologies, Böblingen) at a wavelength of 190 to 400 nm and a refractive index detector (Agilent 1200 Series RID, Agilent Technologies, Böblingen). The reference substances are measured in concentrations of 0.1 g / L to 20 g / L dissolved in water. The limits of determination for lactate, acetate, succinate and formate are 0.8 g / L, ethanol can be determined up to a concentration of 1 g / L. In the range of 0.8 g / L to 20 g / L there is a linear dependence of the peak areas of the
Substanzkonzentration vor. Substance concentration.
Beispiel 4 Example 4
Produktion von Succinat durch E. co//'-Stämme mit Expressionsvektoren für die Gene ppc, pck, maeA und maeB aus E. coli sowie pyc aus C. glutamicum in Kombination mit Expressionsvektoren für die Loci cscA-cscKB aus E. coli sowie scrK-scrYAB aus pUR400 Zur Erzeugung von £ co//-Stämmen mit Expressionsvektoren für die Gene ppc, pck, maeA und maeB aus £ coli sowie pyc aus C. glutamicum in Kombination mit Expressionsvektoren für die Loci cscA-cscKB aus £. co// sowie scrK-scrYAB aus pUR400 werden kompetente Zellen von £ co// MG1655 hergestellt. Dazu wird der Stamm in Luria-Bertani Bouillon nach Miller (Merck, Darmstadt) als 10 mL-Vorkultur angezogen. Eine 100 mL LB-Bouillon wird mit 2 mL der Vorkultur inokuliert und bei 37°C und 200 U/min im Inkubationsschüttler bis zu einer optischen Dichte (600 nm) von 0,5 kultiviert. Die elektrokompetenten Zellen werden durch Waschen mit einer sterilen 10% (w/v) Glycerinlösung wie folgt hergestellt: Die 100 mL Kultur wird durch 10minütige Zentrifugation bei 4°C und 5500 x g geerntet und durch Resuspendieren in 10% Glycerinlösung gewaschen. Nach einem weiteren Zentrifugations- und Waschschritt werden die pellettierten E.coli MG1655 Zellen in 0,5 mL 10% Glycerinlösung aufgenommen und in Aliquots von 50 L bei -80°C bis zur Elektroporation gelagert. Ppc production of succinate by E. co // 'strains with expression vectors for the genes, pck, maeA and MAEB from E. coli and C. glutamicum pyc out in combination with expression vectors for the loci csca-cscKB from E. coli and SCRk -scrYAB from pUR400 For the generation of £ co // strains with expression vectors for the genes ppc, pck, maeA and maeB from E. coli as well as pyc from C. glutamicum in combination with expression vectors for the loci cscA-cscKB from £. co // and scrK-scrYAB from pUR400 are used to prepare competent cells of £ co // MG1655. For this purpose, the strain is grown in Luria-Bertani Bouillon Miller (Merck, Darmstadt) as a 10 mL pre-culture. A 100 mL LB broth is inoculated with 2 mL of preculture and cultured at 37 ° C and 200 rpm in the incubator shaker to an optical density (600 nm) of 0.5. The electrocompetent cells are prepared by washing with a sterile 10% (w / v) glycerol solution as follows: The 100 mL culture is harvested by centrifugation at 4 ° C and 5500 xg for 10 minutes and washed by resuspension in 10% glycerol solution. After another centrifugation and washing step, the pelleted E. coli MG1655 cells are taken up in 0.5 mL 10% glycerol solution and stored in aliquots of 50 L at -80 ° C until electroporation.
Diese werden dann sequentiell oder in Kombination mit den Plasmiden pEC-XC99E, pEC- XC99E-ppc, pEC-XC99E-pck, pEC-XC99E-maeA, pEC-XC99E-maeB, pEC-XC99E-pyc, pCOLADuet-1 , pCOLA-csc und pCOLA-scr transformiert und auf LB-Platten mit These are then used sequentially or in combination with the plasmids pEC-XC99E, pEC-XC99E-ppc, pEC-XC99E-pck, pEC-XC99E-maeA, pEC-XC99E-maeB, pEC-XC99E-pyc, pCOLADuet-1, pCOLA- csc and pCOLA-scr transformed and on LB plates with
Chloramphenicol (50 Mg/ml) und/oder Kanamycin (50 Mg/ml) ausplattiert. Transformanten werden durch Plasmidpräparation und analytische Restriktionsanalyse bezüglich Präsenz der authentischen Plasmide überprüft. Chloramphenicol (50 μg / ml) and / or kanamycin (50 μg / ml). Transformants are checked by plasmid preparation and analytical restriction analysis for the presence of the authentic plasmids.
So wurden folgende £ So the following were £
• £ coli MG 1655 (pCOLADuet-1 , pEC-XC99E) Coli coli 1655 (pCOLADuet-1, pEC-XC99E)
• £ coli MG 1655 (pCOLA-csc, pEC-XC99E)  Coli coli 1655 (pCOLA-csc, pEC-XC99E)
• £ coli MG 1655 (pCOLA-csc, pEC-XC99E-ppc)  Coli coli 1655 (pCOLA-csc, pEC-XC99E-ppc)
• £ coli MG 1655 (pCOLA-csc, pEC-XC99E-pck)  Coli coli 1655 (pCOLA-csc, pEC-XC99E-pck)
• £ coli MG 1655 (pCOLA-csc, pEC-XC99E-maeA)  Coli coli 1655 (pCOLA-csc, pEC-XC99E-maeA)
• £ coli MG 1655 (pCOLA-csc, pEC-XC99E-maeB)  Coli coli 1655 (pCOLA-csc, pEC-XC99E-maeB)
• £ coli MG 1655 (pCOLA-csc, pEC-XC99E-pyc)  Coli coli 1655 (pCOLA-csc, pEC-XC99E-pyc)
• £ coli MG 1655 (pCOLA-scr, pEC-XC99E)  Coli coli 1655 (pCOLA-scr, pEC-XC99E)
• £ coli MG 1655 (pCOLA-scr, pEC-XC99E-ppc)  Coli coli 1655 (pCOLA-scr, pEC-XC99E-ppc)
• £ coli MG 1655 (pCOLA-scr, pEC-XC99E-pck)  • coli coli 1655 (pCOLA-scr, pEC-XC99E-pck)
• £ coli MG 1655 (pCOLA-scr, pEC-XC99E-maeA)  Coli coli 1655 (pCOLA-scr, pEC-XC99E-maeA)
• £ coli MG 1655 (pCOLA-scr, pEC-XC99E-maeB) • E. coli MG1655 (pCOLA-scr, pEC-XC99E-pyc) Coli coli 1655 (pCOLA-scr, pEC-XC99E-maeB) E. coli MG1655 (pCOLA-scr, pEC-XC99E-pyc)
Diese Stämme werden im Anschluss verwendet, um ihre Fähigkeit zur Produktion von Succinat unter anaeroben Bedingungen zu analysieren. Dabei wird wie folgt vorgegangen: Die Stämme werden einem mehrstufigen Kultivierungsprozess unterzogen. Die Vorkultur zur Produktion von Biomasse wird aerob durchgeführt in einem modifizierten M9-Medium, das zusätzlich Hefeextrakt als Komplexbestandteil enthält. Das Medium, bestehend aus 38 mM Dinatriumhydrogenphosphat-Dihydrat (Merck, Darmstadt), 22 mM Kaliumdihydrogenphosphat (Merck, Darmstadt), 8,6 mM Natriumchlorid (Merck, Darmstadt), 18,7 mM Ammoniumchlorid (Merck, Darmstadt), 1 % (w/v) Hefeextrakt (Merck, Darmstadt), 2% (w/v) Saccharose (Sigma- Aldrich, Steinheim), 0,1 mM Calciumchlorid-Dihydrat (Sigma-Aldrich, Steinheim), 1 mM These strains are then used to analyze their ability to produce succinate under anaerobic conditions. The procedure is as follows: The strains are subjected to a multi-stage cultivation process. The preculture for the production of biomass is carried out aerobically in a modified M9 medium which additionally contains yeast extract as complex component. The medium consisting of 38 mM disodium hydrogen phosphate dihydrate (Merck, Darmstadt), 22 mM potassium dihydrogen phosphate (Merck, Darmstadt), 8.6 mM sodium chloride (Merck, Darmstadt), 18.7 mM ammonium chloride (Merck, Darmstadt), 1% ( w / v) yeast extract (Merck, Darmstadt), 2% (w / v) sucrose (Sigma-Aldrich, Steinheim), 0.1mM calcium chloride dihydrate (Sigma-Aldrich, Steinheim), 1mM
Magnesiumsulfat-Heptahydrat (Merck, Darmstadt), 200 mM MOPS (3-(N-Morpholino)- Propansulfonsäure, Sigma-Aldrich, Steinheim) und 1 % (v/v) MEM Vitaminlösung (Sigma- Aldrich, Steinheim), wird mit Natriumhydroxid auf einen pH-Wert von 7,0 eingestellt und zu 10 ml_ mit Chloramphenicol (50 g/mL) und/oder Kanamycin (50 g/mL) in einen 100 mL Magnesium sulfate heptahydrate (Merck, Darmstadt), 200 mM MOPS (3- (N-morpholino) -propanesulfonic acid, Sigma-Aldrich, Steinheim), and 1% (v / v) MEM vitamin solution (Sigma-Aldrich, Steinheim) is treated with sodium hydroxide adjusted to a pH of 7.0 and to 10 ml with chloramphenicol (50 g / ml) and / or kanamycin (50 g / ml) in a 100 ml
Erlenmeyerkolben mit Schikane vorgelegt und mit einer Einzelkolonie des zu untersuchenden Stammes inokuliert. Die Kultivierung erfolgt bei 37°C und 250 U/min in einem  Erlenmeyer flask presented with chicane and inoculated with a single colony of the strain to be examined. The cultivation takes place at 37 ° C. and 250 rpm in one
Inkubationsschüttler. Nach einer Kultivierungsdauer von 8 Stunden werden 200 mL Incubator. After a culture period of 8 hours, 200 mL
modifiziertes M9-Medium mit 0,2 mg/L Biotin und 0,5 mM IPTG in einem 1000 ml_- Erlenmeyerkolben mit Schikane mit der Vorkultur inokultiert, so dass eine optische Dichte (600 nm) von 0,2 erreicht wird. Nach einer Kultivierungsdauer von mindestens 12 Stunden bei 37°C und 250 U/min wird die Kulturbrühe durch 10minütige Zentrifugation bei 3300 g und 4°C geerntet. Die sedimentierten Bakterienzellen werden mit steriler 0,9% Natriumchloridlösung gewaschen und anschließend in Medium für die anaerobe Kultivierung aufgenommen. Die anaerobe Hauptkultur erfolgt in modifiziertem M9 Medium ohne Komplexbestandteile (38 mM Dinatriumhydrogenphosphat-Dihydrat, 22 mM Kaliumdihydrogen-phosphat, 8,6 mM modified M9 medium with 0.2 mg / L biotin and 0.5 mM IPTG in a 1000 ml Erlenmeyer flask with chicane inocultivated with the preculture so that an optical density (600 nm) of 0.2 is achieved. After a culture period of at least 12 hours at 37 ° C and 250 rpm, the culture broth is harvested by centrifugation at 3300 g and 4 ° C for 10 minutes. The sedimented bacterial cells are washed with sterile 0.9% sodium chloride solution and then taken up in anaerobic culture medium. The main anaerobic culture is carried out in modified M9 medium without complex components (38 mM disodium hydrogen phosphate dihydrate, 22 mM potassium dihydrogen phosphate, 8.6 mM
Natriumchlorid, 18,7 mM Ammoniumchlorid, 2% (w/v) Saccharose, 0,1 mM Calciumchlorid- Dihydrat, 1 mM Magnesiumsulfat-Heptahydrat, 200 mM MOPS, 1 % (v/v) MEM Vitaminlösung, 0,1 % (w/v) Natriumhydrogencarbonat, pH 7,0 mit Natriumhydroxid) unter Zusatz von 0,2 mg/L Biotin, 50 g/L Magnesiumcarbonat und 0,5 mM IPTG, sowie 1 mg/L Resazurin als Sodium chloride, 18.7 mM ammonium chloride, 2% (w / v) sucrose, 0.1 mM calcium chloride dihydrate, 1 mM magnesium sulfate heptahydrate, 200 mM MOPS, 1% (v / v) MEM vitamin solution, 0.1% ( w / v) sodium bicarbonate, pH 7.0 with sodium hydroxide) with the addition of 0.2 mg / L biotin, 50 g / L magnesium carbonate and 0.5 mM IPTG, and 1 mg / L resazurin as
Sauerstoffindikator. 50 mL Medium werden in 100 mL Laborglasflaschen (Schott, Mainz) vorgelegt und mit der Zellsuspension inokuliert, so dass eine optische Dichte (600 nm) von 20 erreicht wird. Die Flaschen werden mit einer Verschlusskappe mit Stopfen gasdicht verschlossen, die Kultivierung erfolgt bei 37°C und 250 U/min im Inkubationsschüttler für eine Dauer von 24 Stunden. Die Anaerobiose stellt sich innerhalb der ersten Stunde der Kultivierung ein und wird anhand der Entfärbung des Resazurin festgestellt. Während der Kultivierung werden Proben von 1 ml_ steril mit einer Kanüle durch den Stopfen entnommen, die nach Sterilfiltration mittels in Beispiel 3 beschriebener chromatografischer Methoden hinsichtlich ihres Saccharosegehaltes und des Gehaltes organischer Säuren analysiert werden. Oxygen indicator. 50 mL of medium are placed in 100 mL laboratory glass bottles (Schott, Mainz) and inoculated with the cell suspension so that an optical density (600 nm) of 20 is achieved. The bottles are gas-tight with a cap with a stopper closed, the cultivation is carried out at 37 ° C and 250 rev / min in incubation shaker for a period of 24 hours. Anaerobiosis occurs within the first hour of cultivation and is detected by decolorization of resazurin. During culture, samples of 1 ml_ sterile are withdrawn through the stopper with a cannula which, after sterile filtration, is analyzed for its sucrose content and organic acid content by means of chromatographic methods described in Example 3.
Beispiel 5 Produktion von Succinat durch E. co//'-Stämme mit Deletionen in den Genen ack-pta, adhE, IdhA und ygfG, in welchen das Gen ptsG durch den scrK-scrYAB-Locus aus dem Plasmid pUR400 ersetzt wurde und welcher das Gen pyc, kodierend für eine Example 5 was the production of succinate by E. co // 'strains with deletions in the genes ack-pta, adhE, IDHA and ygfG, in which the gene is replaced by the ptsG SCRk-scrYAB locus from the plasmid pUR400 and which the Gene pyc, coding for a
Pyruvatcarboxylase aus Corynebacterium glutamicum, überexprimiert. Pyruvate carboxylase from Corynebacterium glutamicum, overexpressed.
Zunächst wurde ein £. co//-Stamm mit Deletionen in den Genen ack-pta (SEQ ID NO:31 ), adhE (SEQ ID NO:32), IdhA (SEQ ID NO:33) und ygfG (SEQ ID NO:34) und einem Austausch des pisG-Gens (SEQ ID NO:35) durch den scrK-scrYAB-Locus konstruiert. Es wurde der E. coli- Stamm MG1655 ack-pta adhE IdhA ygfG ptsG:: scrK-scrYAB mittels Methoden generiert, die dem Fachmann bekannt sind (z.B. siehe Datsenko KA, Wanner BL. Proc Natl Acad Sei U S A. 2000. 97(12):6640-5.). Die DNA-Sequenz der einzelnen Loci nach Deletion bzw. Insertion ist in SEQ ID NO:36 ( ack-pta), SEQ ID NO:37 ( adhE), SEQ ID NO:38 ( IdhA), SEQ ID NO:39 ( ygfG) und SEQ ID NO:40 {ptsGwscrK-scrYAB) wiedergegeben. First, a pound. co // strain with deletions in the genes ack-pta (SEQ ID NO: 31), adhE (SEQ ID NO: 32), IdhA (SEQ ID NO: 33) and ygfG (SEQ ID NO: 34) and an exchange of the pisG gene (SEQ ID NO: 35) is constructed by the scrK scrYAB locus. The E. coli strain MG1655 ack-pta adhE IdhAggfG ptsG :: scrK-scrYAB was generated by methods known to the person skilled in the art (eg see Datsenko KA, Wanner BL Proc Natl Acad Sei USA 2000. 97 ( 12): 6640-5).. The DNA sequence of the individual loci after deletion or insertion is shown in SEQ ID NO: 36 (ack-pta), SEQ ID NO: 37 (adhE), SEQ ID NO: 38 (IdhA), SEQ ID NO: 39 (ygfG ) and SEQ ID NO: 40 {ptsGwscrK-scrYAB).
£. co// MG1655 ack-pta adhE IdhA ygfG ptsG:: scrK-scrYAB wurde im Anschluss analog zu Beispiel 4 mit den Plasmiden pEC-XC99E und pEC-XC99E-pyc transformiert. Die £. co//'-Stämme MG1655 ack-pta adhE IdhA ygfG ptsG:: scrK-scrYAB (pEC-XC99E) und ack-pta adhE IdhA ygfG ptsG:: scrK-scrYAB (pEC-XC99E-pyc) wurden daraufhin analog zu Beispiel 4 kultiviert und die Konzentration von Saccharose, Succinat und £. co // MG1655 ack-pta adhE IdhA ygfG ptsG :: scrK-scrYAB was subsequently transformed analogously to Example 4 with the plasmids pEC-XC99E and pEC-XC99E-pyc. The £. co // ' strains MG1655 ack-pta adhE IdhA ygfG ptsG :: scrK-scrYAB (pEC-XC99E) and ack-pta adhE IdhA ygfG ptsG :: scrK-scrYAB (pEC-XC99E-pyc) were then analogous to Example 4 cultured and the concentration of sucrose, succinate and
Nebenprodukten analog zu Beispiel 3 quantifiziert. Die Ergebnisse sind in Tab. 1 und 2 dargestellt. Tab. 1 . Produktion von Succinat mit E. coli MG1655 ack-pta adhE IdhA ygfG ptsGv. scrK-scrYAB (pEC-XC99E). Angegeben sind die Konzentration von Succinat und By-products analogously to Example 3 quantified. The results are shown in Tab. 1 and 2. Tab. 1. Production of Succinate with E. coli MG1655 ack-pta adhE IdhA ygfG ptsGv. scrK-scrYAB (pEC-XC99E). Indicated are the concentration of succinate and
Nebenprodukten sowie die Produktausbeute (Yp/s) und die durchschnittliche Raum-Zeit- Ausbeute (RZA) in den ersten 24 Stunden. Ethanol, Lactat, Pyruvat und Formiat konnten nicht nachgewiesen werden. By-products and the product yield (Yp / s) and the average space-time yield (RZA) in the first 24 hours. Ethanol, lactate, pyruvate and formate could not be detected.
Figure imgf000047_0001
Tab. 2. Produktion von Succinat mit £. coli MG1655 ack-pta adhE IdhA ygfG ptsGv. scrK-scrYAB (pEC-XC99E-pyc). Angegeben sind die Konzentration von Succinat und den Nebenprodukten Acetat, Ethanol, Lactat und Formiat sowie die Produktausbeute (Yp/s) und die durchschnittliche Raum-Zeit-Ausbeute (RZA) in den ersten 24 Stunden. Ethanol, Lactat, Pyruvat und Formiat konnten nicht nachgewiesen werden.
Figure imgf000047_0001
Tab. 2. Production of succinate with £. coli MG1655 ack-pta adhE IdhA ygfG ptsGv. scrK-scrYAB (pEC-XC99E-pyc). Indicated are the concentration of succinate and the by-products acetate, ethanol, lactate and formate as well as the product yield (Yp / s) and the average space-time yield (RZA) in the first 24 hours. Ethanol, lactate, pyruvate and formate could not be detected.
Figure imgf000047_0002
Figure imgf000047_0002
Der Offenbarungsgehalt aller hierin zitierter Dokumente ist in seiner Gesamtheit durch Bezugnahme eingeschlossen und bildet einen Teil der Offenbarung der vorliegenden The disclosure of all documents cited herein is incorporated by reference in its entirety and forms a part of the disclosure of the present application
Anmeldung. Registration.
Die Erfindung wird hierin durch Bezugnahme auf bestimmte Ausführungsformen beschrieben, ist aber nicht auf diese beschränkt. Insbesondere ist für den Fachmann ohne Weiteres ersichtlich, dass verschiedene Änderungen an der beschriebenen Erfindung vorgenommen werden können, ohne vom Sinn und Umfang der Erfindung, wie er durch die angefügten Patentansprüche bestimmt wird, abzuweichen. Der Umfang der Erfindung wird somit durch die Patentansprüche bestimmt und es ist beabsichtigt, dass die Erfindung alle Modifikationen und Änderungen, die in den Deutungs- und Äquivalenzbereich der Ansprüche fallen, umfasst. The invention is described herein by reference to certain embodiments, but is not limited thereto. In particular, it will be readily apparent to those skilled in the art that various changes are made to the invention described without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is thus defined by the claims, and it is intended that the invention encompass all modifications and changes that fall within the scope of the claims and equivalents of the claims.

Claims

Patentansprüche claims
1 . Rekombinante Zelle, welche gegenüber ihrem Wildtyp derart gentechnisch verändert wurde, dass sie aus Saccharose und Kohlendioxid als Kohlenstoffquellen im Vergleich zu ihrem Wildtyp mehr C4-Körper und/oder mehr über diese C4-Körper hergestellte Folgeverbindungen zu bilden vermag. 1 . Recombinant cell which has been altered in such genetically compared to its wild type that it is able to form a result Compounds prepared via this C 4 -body of sucrose and carbon dioxide as the carbon sources as compared to its wild type more C 4 -body and / or more.
2. Die rekombinante Zelle nach Anspruch 1 , wobei die C4-Körper ausgewählt werden aus der Gruppe bestehend aus: Succinat, Malat, Fumarat, Oxalacetat, Aspartat, Asparagin, Threonin, Tetrahydrofuran, Pyrrolidon, Acetoin, 4,4-Bionell, 2. The recombinant cell of claim 1, wherein the C 4 bodies are selected from the group consisting of: succinate, malate, fumarate, oxaloacetate, aspartate, asparagine, threonine, tetrahydrofuran, pyrrolidone, acetoin, 4,4-bionell,
Hydroxysuccinat, Epoxy-v-Butyrolacton, Butensäure, Butyrat, Butandiol, 1 ,2- Butandiol, 1 ,3-Butandiol, 1 ,4-Butandiol, 2,3-Butandiol, Buten, n-Buten, cis-2-Buten, trans-2-Buten, Isobuten, Butadien, 1 ,2-Butadien, 1 ,3-Butadien, 3- Hydroxybutyrolacton, 4-Hydroxybutyrolacton, 1 -Butanol, 2-Butanol, tert-Butanol, Isobutanol, 2-Hydroxybuttersäure, 3-Hydroxybuttersäure, 4-Hydroxybuttersäure, 2- Hydroxyisobuttersäure und 3-Hydroxyisobuttersäure.  Hydroxysuccinate, epoxy-v-butyrolactone, butenoic acid, butyrate, butanediol, 1, 2-butanediol, 1, 3-butanediol, 1, 4-butanediol, 2,3-butanediol, butene, n-butene, cis-2-butene, trans-2-butene, isobutene, butadiene, 1, 2-butadiene, 1, 3-butadiene, 3-hydroxybutyrolactone, 4-hydroxybutyrolactone, 1-butanol, 2-butanol, tert-butanol, isobutanol, 2-hydroxybutyric acid, 3- Hydroxybutyric acid, 4-hydroxybutyric acid, 2-hydroxyisobutyric acid and 3-hydroxyisobutyric acid.
3. Die rekombinante Zelle nach Anspruch 1 oder 2, wobei die C4-Körper ausgewählt werden aus der Gruppe bestehend aus: Malat, Oxalacetat und die über diese C4- Körper hergestellte Folgeverbindungen ausgewählt werden aus der Gruppe bestehend aus durch enzymatische oder chemische Synthese über Malat oder Oxalacetat hergestellte Folgeverbindungen. 3. The recombinant cell of claim 1 or 2, wherein the C 4 bodies are selected from the group consisting of: malate, oxaloacetate and the follow-up compounds prepared via these C 4 - are selected from the group consisting of by enzymatic or chemical synthesis Follow-up compounds prepared via malate or oxaloacetate.
4. Die rekombinante Zelle nach Anspruch 3, wobei die durch enzymatische Synthese über Malat oder Oxalacetat hergestellte Folgeverbindungen ausgewählt werden aus der Gruppe bestehend aus: Aspartat, Asparagin, Threonin, Tetrahydrofuran, Butyrat, Butandiol, 1 ,2-Butandiol, 1 ,3-Butandiol, 1 ,4-Butandiol, 2,3-Butandiol, 3- und 4- Hydroxybutyrolacton, 1 -, 2- und tert-Butanol, Isobutanol, 2-, 3- und 4- Hydroxybuttersäure, 2- und 3-Hydroxyisobuttersäure, Methionin und Lysin. 4. The recombinant cell according to claim 3, wherein the secondary compounds produced by enzymatic synthesis via malate or oxaloacetate are selected from the group consisting of: aspartate, asparagine, threonine, tetrahydrofuran, butyrate, butanediol, 1, 2-butanediol, 1, 3 Butanediol, 1,4-butanediol, 2,3-butanediol, 3- and 4-hydroxybutyrolactone, 1-, 2- and tert-butanol, isobutanol, 2-, 3- and 4-hydroxybutyric acid, 2- and 3-hydroxyisobutyric acid, Methionine and lysine.
5. Die rekombinante Zelle nach mindestens einem der Ansprüche 1 bis 4, wobei es sich bei der Zelle um eine mikrobielle Zelle handelt. The recombinant cell of any one of claims 1 to 4, wherein the cell is a microbial cell.
6. Die rekombinante Zelle nach mindestens einem der Ansprüche 1 bis 5, wobei es sich bei der mikrobiellen Zelle um eine Escherichia coli, Alcaligenes latus, Bacillus megaterium, Bacillus subtilis, Brevibacterium flavum, Brevibacterium lactofermentum, Escherichia coli, Basfia succiniciproducens, Wollinella succinogenes, Fibrobacter succinogenes, Ruminococcus flavefaciens, Anaerobiospirillum succiniciproducens,6. The recombinant cell according to at least one of claims 1 to 5, wherein the microbial cell is an Escherichia coli, Alcaligenes latus, Bacillus megaterium, Bacillus subtilis, Brevibacterium flavum, Brevibacterium lactofermentum, Escherichia coli, Basfia succiniciproducens, Wollinella succinogenes, Fibrobacter succinogenes, Ruminococcus flavefaciens, Anaerobiospirillum succiniciproducens,
Mannheimia succiniciproducens, Actinobacillus succinogenes, Corynebacterium glutamicum, Corynebacterium efficiens, Zymonomas mobilis, Methylobacterium extorquens, Ralstonia eutropha, Saccharomyces cerevisiae, Rhodobacter sphaeroides, Paracoccus versutus, Pseudomonas aeruginosa, Acinetobacter calcoaceticus, Clostridium acetobutylicum, Clostridium saccharoperbutylacetonicum,Saccharomyces cerevisiae, Rhodobacter sphaeroides, Paracoccus versutus, Pseudomonas aeruginosa, Acinetobacter calcoaceticus, Clostridium acetobutylicum, Clostridium saccharoperbutylacetonicum,
Clostridium beijerinckii, Rhodospirillum rubrum, Burkholderia thailandensis oder Pseudomonas putida Zelle handelt. Clostridium beijerinckii, Rhodospirillum rubrum, Burkholderia thailandensis or Pseudomonas putida cell.
7. Die rekombinante Zelle nach mindestens einem der Ansprüche 1 bis 6, wobei die Zelle im Vergleich zu ihrem Wildtyp mehr Saccharose aufnehmen kann. 7. The recombinant cell according to any one of claims 1 to 6, wherein the cell can receive more sucrose compared to its wild type.
8. Die rekombinante Zelle nach mindestens einem der Ansprüche 1 bis 7, wobei die Zelle im Vergleich zu ihrem Wildtyp mehr Kohlendioxid fixieren kann. 9. Die rekombinante Zelle nach mindestens einem der Ansprüche 1 bis 8, wobei die8. The recombinant cell according to any one of claims 1 to 7, wherein the cell can fix more carbon dioxide compared to their wild type. 9. The recombinant cell according to any one of claims 1 to 8, wherein the
Zelle eine im Vergleich zu Ihrem Wildtyp gesteigerte Aktivität mindestens eines Enzyms aufweist, das den Transport von Saccharose in die Zelle katalysiert. Cell has an increased compared to their wild type activity of at least one enzyme that catalyzes the transport of sucrose into the cell.
10. Die rekombinante Zelle nach Anspruch 9, wobei das mindestens eine Enzym ein Enzym E1 umfasst, das den Transport von Saccharose in die Zelle und die 10. The recombinant cell of claim 9, wherein the at least one enzyme comprises an enzyme E1 which facilitates the transport of sucrose into the cell and the
Umsetzung zu Saccharose-6-Phosphat katalysiert.  Conversion to sucrose-6-phosphate catalyzed.
1 1 . Die rekombinante Zelle nach Anspruch 10, wobei das Enzym E1 ein 1 1. The recombinant cell of claim 10, wherein the enzyme is E1
Phosphoenolpyruvat (PEP)-abhängiges Phosphotransferase-System (PTS) Enzym II (Saccharose-spezifisch) (EC2.7.1 .69; TCDB Klassifikation 4.A.1.2.1 , 4.A.1 .2.9 oder Phosphoenol pyruvate (PEP) -dependent phosphotransferase system (PTS) Enzyme II (sucrose-specific) (EC2.7.1.69; TCDB classification 4.A.1.2.1, 4.A.1 .2.9 or
4.A.1 .2.-) ist. 4.A.1 .2.-).
12. Die rekombinante Zelle nach Anspruch 10 oder 1 1 , wobei das Enzym E1 von Genen kodiert wird, die aus der Gruppe derer ausgewählt werden, die Genprodukte kodieren, deren Aminosäuresequenz über einen Bereich von mindestens 100 Aminosäuren, zu mindestens 60% zu SEQ ID NO: 58 identisch ist. The recombinant cell of claim 10 or 11, wherein the enzyme E1 is encoded by genes selected from the group consisting of gene products whose amino acid sequence is identical over a range of at least 100 amino acids, at least 60% to SEQ ID NO: 58.
13. Die rekombinante Zelle nach mindestens einem der Ansprüche 1 bis 12, wobei die Zelle ferner eine im Vergleich zu ihrem Wildtyp gesteigerte Aktivität mindestens eines Enzyms E2, das die Umsetzung von Saccharose-6-Phosphat zu -D-Glukose-6- Phosphat und -D-Fruktose katalysiert; und/oder mindestens eines Enzyms E3, welches die Umsetzung von -D-Fruktose zu -D-Fruktose-6-Phosphat katalysiert; und/oder mindestens eines Kanals E4, der diffusions-abhängigen Saccharose- Transport in die Zelle erlaubt, aufweist. 13. The recombinant cell according to any one of claims 1 to 12, wherein the cell further comprises an increased compared to their wild-type activity of at least one enzyme E 2 , which is the conversion of sucrose-6-phosphate to -D-glucose-6-phosphate and D-fructose catalyzes; and / or at least one enzyme E 3 , which catalyzes the conversion of D-fructose to D-fructose-6-phosphate; and / or at least one channel E 4 permitting diffusion-dependent sucrose transport into the cell.
14. Die rekombinante Zelle nach Anspruch 13, wobei 14. The recombinant cell of claim 13, wherein
das Enzym E2 eine Saccharose-6-Phosphat-Fruktohydrolase (EC 3.2.1 .26) ist; und/oder the enzyme E 2 is a sucrose-6-phosphate fructohydrolase (EC 3.2.1 .26); and or
das Enzym E3 eine Fruktokinase (EC 2.7.1 .4) ist; und/oder the enzyme E 3 is a fructokinase (EC 2.7.1.4); and or
das Enzym E4 ein Saccharose Porin (TCDB Klassifikation 1 .B.3.1 .2) ist. the enzyme E 4 is a sucrose porin (TCDB classification 1 .B.3.1 .2).
15. Die rekombinante Zelle nach Anspruch 14, wobei 15. The recombinant cell of claim 14, wherein
E2 durch ein scrB oder susH Gen kodiert wird; und/oder E 2 is encoded by a scrB or susH gene; and or
E3 durch ein scrK oder cscK Gen kodiert wird; und/oder E 3 is encoded by a scrK or cscK gene; and or
E4 durch ein scrY Gen kodiert wird. E 4 is encoded by a scrY gene.
16. Die rekombinante Zelle nach Anspruch 9, wobei das mindestens eine Enzym einen Kanal E5 umfasst, der Saccharose in die Zelle symportiert. The recombinant cell of claim 9, wherein the at least one enzyme comprises a channel E5 which sorbs sucrose into the cell.
17. Die rekombinante Zelle nach Anspruch 16, wobei der Kanal E5 eine Saccharose- Permease (TCDB Klassifikation 2.A.1 .5.3) ist. 17. The recombinant cell according to claim 16, wherein the channel E5 is a sucrose permease (TCDB classification 2.A.1 .5.3).
18. Die rekombinante Zelle nach Anspruch 17, wobei der Kanal E5 durch ein cscB Gen kodiert wird. The recombinant cell of claim 17, wherein the E5 channel is encoded by a cscB gene.
19. Die rekombinante Zelle nach mindestens einem der Ansprüche 16 bis 18, wobei die Zelle ferner eine im Vergleich zu ihrem Wildtyp gesteigerte Aktivität mindestens eines Enzyms E6, das die Umsetzung von Saccharose zu -D-Glucose und -D-Fruktose katalysiert; und/oder mindestens eines Enzyms E3, welches die Umsetzung von -D- Fruktose zu -D-Fruktose-6-Phosphat katalysiert, aufweist. The recombinant cell of any one of claims 16 to 18, wherein the cell further comprises an increased activity of at least one enzyme E6, which is the conversion of sucrose to D-glucose and D-fructose, compared to its wild-type catalyzes; and / or at least one enzyme E3 which catalyzes the conversion of D-fructose to D-fructose-6-phosphate.
20. Die rekombinante Zelle nach Anspruch 19, wobei The recombinant cell of claim 19, wherein
das Enzym E3 eine Fruktokinase (EC 2.7.1.4) ist; und/oder the enzyme E 3 is a fructokinase (EC 2.7.1.4); and or
das Enzym E6 eine -D-Fruktofuranosid-Fruktohydrolase (EC 3.2.1 .26) ist. the enzyme E 6 is a -D-fructofuranoside fructohydrolase (EC 3.2.1 .26).
21 . Die rekombinante Zelle nach Anspruch 20, wobei 21. The recombinant cell of claim 20, wherein
E3 durch ein scrK oder cscK Gen kodiert wird; und/oder E 3 is encoded by a scrK or cscK gene; and or
E6 durch ein cscA Gen kodiert wird. E 6 is encoded by a cscA gene.
22. Die rekombinante Zelle nach Anspruch 9, wobei das mindestens eine Enzym einen Enzymkomplex aus E7, E8 und E9 umfasst, der Saccharose in die Zelle transportiert. 22. The recombinant cell of claim 9, wherein the at least one enzyme comprises an enzyme complex of E7, E8 and E9 which transports sucrose into the cell.
23. Die rekombinante Zelle nach Anspruch 22, wobei der Enzymkomplex aus E7, E8 und E9 ein Saccharose-spezifischer ABC-Transporter (TCDB Klassifikation 3.A.1.1 .-) ist. 23. The recombinant cell of claim 22, wherein the enzyme complex of E7, E8 and E9 is a sucrose-specific ABC transporter (TCDB classification 3.A.1.1 .-).
24. Die rekombinante Zelle nach Anspruch 22 oder 23, wobei 24. The recombinant cell of claim 22 or 23, wherein
E7 durch ein susT1 Gen; und/oder E 7 by a susT1 gene; and or
E8 durch ein susT2 Gen; und/oder E 8 by a susT2 gene; and or
E9 durch ein susX Gen kodiert wird. E 9 is encoded by a susX gene.
25. Die rekombinante Zelle nach mindestens einem der Ansprüche 22 bis 24, wobei die Zelle ferner eine im Vergleich zu ihrem Wildtyp gesteigerte Aktivität mindestens eines Enzyms E10, das die Umsetzung von Saccharose zu Saccharose-6-Phosphat katalysiert; und/oder mindestens eines Enzyms E2, welches die Umsetzung von Saccharose-6-Phosphat zu -D-Glukose-6-Phosphat und -D-Fruktose katalysiert; und/oder mindestens eines Enzyms E3, welches die Umsetzung von -D-Fruktose zuThe recombinant cell of any one of claims 22 to 24, wherein the cell further comprises an increased activity of at least one enzyme E10, which catalyzes the conversion of sucrose to sucrose-6-phosphate, compared to its wild-type; and / or at least one enzyme E2 which catalyzes the conversion of sucrose-6-phosphate to D-glucose-6-phosphate and D-fructose; and / or at least one enzyme E3 which promotes the conversion of D-fructose
-D-Fruktose-6-Phosphat katalysiert; aufweist. D-fructose-6-phosphate catalyzed; having.
26. Die rekombinante Zelle nach Anspruch 25 wobei die Zelle eine im Vergleich zu ihrem Wildtyp gesteigerte Aktivität der Enzyme E10, E2, E3 und E6 aufweist. 26. The recombinant cell according to claim 25, wherein the cell has an increased activity of the enzymes E10, E2, E3 and E6 compared to their wild type.
27. Die rekombinante Zelle nach Anspruch 26, wobei das Enzym E10 eine Saccharose-Kinase ist; und/oder 27. The recombinant cell of claim 26, wherein the enzyme E 10 is a sucrose kinase; and or
das Enzym E2 eine Saccharose-6-Phosphat-Fruktohydrolase (EC 3.2.1 .26) ist; das Enzym E3 eine Fruktokinase (EC 2.7.1 .4) ist und/oder the enzyme E 2 is a sucrose-6-phosphate fructohydrolase (EC 3.2.1 .26); the enzyme E 3 is a fructokinase (EC 2.7.1 .4) and / or
das Enzym E6 -D-Fruktofuranosid-Fruktohydrolase (EC 3.2.1 .26) ist. the enzyme E 6 -D-fructofuranoside fructohydrolase (EC 3.2.1 .26).
28. Die rekombinante Zelle nach Anspruch 26 oder 27, wobei The recombinant cell of claim 26 or 27, wherein
Eio durch ein Saccharose-Kinase Gen; und/oder  Eio by a sucrose kinase gene; and or
E2 durch ein scrB oder susH Gen; E 2 by a scrB or susH gene;
E3 durch ein scrK oder cscK Gen und/oder E 3 by a scrK or cscK gene and / or
E6 durch ein cscA Gen kodiert wird E 6 is encoded by a cscA gene
29. Die rekombinante Zelle nach einem der Ansprüche 1 bis 28, wobei die Zelle eine im Vergleich zu Ihrem Wildtyp gesteigerte Aktivität mindestens eines Enzyms aufweist, das die Fixierung von C02 an einen C3-Körper katalysiert. 29. The recombinant cell according to any one of claims 1 to 28, wherein the cell has an increased activity of at least one enzyme, which catalyzes the fixation of C0 2 to a C 3 body compared to its wild type.
30. Die rekombinante Zelle nach Anspruch 29, wobei das mindestens eine Enzym, das die Fixierung von C02 an einen C3-Körper katalysiert, aus der Gruppe ausgewählt wird, die aus folgenden Enzymen besteht: The recombinant cell of claim 29, wherein the at least one enzyme that catalyzes the fixation of C0 2 to a C 3 body is selected from the group consisting of the following enzymes:
E12, das die Umsetzung von Pyruvat, ATP und C02 zu Oxalacetat, ADP und Phosphat katalysiert; E 12 , which catalyzes the conversion of pyruvate, ATP and C0 2 to oxaloacetate, ADP and phosphate;
E13, das die Umsetzung von Phosphoenolpyruvat, H20 und C02 zu Oxalacetat und Phosphat katalysiert; E 13 , which catalyzes the reaction of phosphoenolpyruvate, H 2 O and C0 2 to oxaloacetate and phosphate;
E14, das die Umsetzung von Phosphoenolpyruvat, ADP/GDP/P, und C02 zu Oxalacetat und ATP/GTP/PPi katalysiert; Which catalyzes the conversion of phosphoenolpyruvate, ADP / GDP / P, and C0 2 to oxaloacetate and ATP / GTP / PPi E 14;
Eis, Ei6, Ei7 und E18, die die Umsetzung von Pyruvat NAD(P)H+ und C02 zu Malat und NAD(P)+ katalysieren. Ice, Ei 6 , Ei 7 and E 18 , which catalyze the conversion of pyruvate NAD (P) H + and C0 2 to malate and NAD (P) + .
31 . Die rekombinante Zelle nach Anspruch 30, wobei: 31. The recombinant cell of claim 30, wherein:
E12 eine Pyruvat-Carboxylase (EC 6.4.1 .1 ) ist; E 12 is a pyruvate carboxylase (EC 6.4.1.1);
Ei3 eine Phosphoenolpyruvat-Carboxylase (Phosphat:Oxalacetat-Carboxylase) (EC 4.1 .1 .31 ) ist; Ei 3 is a phosphoenolpyruvate carboxylase (phosphate: oxaloacetate carboxylase) (EC 4.1 .1 .31);
E14 eine Phosphoenolpyruvat-Carboxykinase (ATP/GTP/PPi:Oxalacetat-E 14 is a phosphoenolpyruvate carboxykinase (ATP / GTP / PPi: oxaloacetate
Carboxylase) (EC 4.1 .1 .32, EC 4.1 .1 .38 oder EC 4.1 .1 .49) ist; Carboxylase) (EC 4.1 .1 .32, EC 4.1 .1 .38 or EC 4.1 .1 .49);
Eis eine Malatdehydrogenase ((S)-Malat:NAD+ Oxidoreduktase) (EC 1 .1 .1 .38) ist; Ei6 eine Malatdehydrogenase ((S)-Malat:NAD+ Oxidoreduktase) (EC 1.1.1.39) ist; Ei7 eine Malatdehydrogenase ((S)-Malat:NADP+ Oxidoreduktase) (EC 1.1 .1 .40) ist; und/oder Ice is a malate dehydrogenase ((S) -malate: NAD + oxidoreductase) (EC 1 .1 .1 .38); Egg 6 is a malate dehydrogenase ((S) -malate: NAD + oxidoreductase) (EC 1.1.1.39); Egg 7 is a malate dehydrogenase ((S) -malate: NADP + oxidoreductase) (EC 1.1 .1 .40); and or
Ei8 eine D-Malatdehydrogenase ((R)-Malat:NAD+ Oxidoreduktase) (EC 1.1 .1 .83) ist. Egg 8 is a D-malate dehydrogenase ((R) -malate: NAD + oxidoreductase) (EC 1.1 .1 .83).
32. Die rekombinante Zelle nach Anspruch 31 , wobei: 32. The recombinant cell of claim 31, wherein:
Ei2 durch ein pyc Gen;  Ei2 by a pyc gene;
Ei3 durch ein ppc Gen; Egg 3 by a ppc gene;
EM durch ein pckA Gen;  EM by a pckA gene;
Eis durch ein maeA Gen;  Ice through a mouse gene;
Ei6 durch ein mme1 Gen; Egg 6 by a mme1 gene;
Ei7 durch ein maeB Gen; und/oder Egg 7 by a mouse gene; and or
E18 durch ein dmlA Gen kodiert wird. E 18 is encoded by a dmlA gene.
33. Die rekombinante Zelle nach mindestens einem der Ansprüche 13 bis 15, wobei die Zelle ferner eine im Vergleich zu ihrem Wildtyp erhöhte Aktivität eines Enzym E12, das die Umsetzung von Pyruvat, ATP und C02 zu Oxalacetat, ADP und Phosphat katalysiert, aufweist. The recombinant cell of any one of claims 13 to 15, wherein the cell further has an increased activity of an enzyme E12, which catalyzes the conversion of pyruvate, ATP and C0 2 to oxaloacetate, ADP and phosphate, compared to its wild-type.
34. Die rekombinante Zelle nach Anspruch 33, wobei E12 durch ein pyc Gen kodiert wird. 34. The recombinant cell of claim 33, wherein E12 is encoded by a pyc gene.
35. Die rekombinante Zelle nach mindestens einem der Ansprüche 1 bis 34, wobei die Zelle einen im Vergleich zu ihrem Wildtyp verminderten Kohlenstofffluss von 35. The recombinant cell according to at least one of claims 1 to 34, wherein the cell has a decreased carbon flux compared to its wild-type
Saccharose hin zu Gärungsprodukten, die keine C4-Körper sind, aufweist. Sucrose towards fermentation products which are not C 4 bodies.
36. Die rekombinante Zelle nach Anspruch 35, wobei in der Zelle im Vergleich zu ihrem Wildtyp aus Saccharose weniger d-, C2- und/oder C3-Nebenprodukte erzeugt werden. 36. The recombinant cell of claim 35, wherein less d, C 2 and / or C 3 by- products are produced in the cell from sucrose compared to its wild type.
37. Die rekombinante Zelle nach Anspruch 35 oder 36, wobei der Metabolismus, der Gärungsprodukte erzeugt, reduziert oder unterbrochen ist. Die rekombinante Zelle nach mindestens einem der Ansprüche 35 bis 37, wobei die Zelle eine im Vergleich zu ihrem Wildtyp verminderte Aktivität von mindestens einem Enzym aufweist, das aus der Gruppe, die aus den folgenden Enzymen besteht, ausgewählt wird: 37. The recombinant cell of claim 35 or 36, wherein the metabolism that produces fermentation products is reduced or disrupted. The recombinant cell according to any one of claims 35 to 37, wherein the cell has a reduced activity of at least one enzyme selected from the group consisting of the following enzymes compared to its wild-type:
Ei9, E2o, E2i und E22, die Komponenten eines glukosespezifischen Ei9, E 2 o, E 2 i and E 2 2, the components of a glucose-specific
Phosphoenolpyruvat-Phosphotransferasesystems darstellen und als solche den Represent phosphoenolpyruvate Phosphotransferasesystems and as such the
Import von Glukose und in Summe die Umsetzung von Phosphoenolpyruvat undImport of glucose and in sum the implementation of phosphoenolpyruvate and
Glukose zu Pyruvat und Glukose-6-Phosphat katalysieren; Catalyze glucose to pyruvate and glucose-6-phosphate;
E23 und E24, die die Umsetzung von Pyruvat und CoA zu Formiat und Acetyl-CoA katalysieren; E 2 3 and E 2 4, which catalyze the reaction of pyruvate and CoA to formate and acetyl-CoA;
E25, das die Umsetzung von Pyruvat, H20 und Ferricytochrom bi zu Acetat, C02 und Ferrocytochrom bi katalysiert; E 25 , which catalyzes the reaction of pyruvate, H 2 O and ferricytochrome bi to acetate, C0 2 and ferrocytochrome bi;
E26, das die Umsetzung von S-Methylmalonyl-CoA zu Propanoyl-CoA und C02 katalysiert; E 26 , which catalyzes the conversion of S-methylmalonyl-CoA to propanoyl-CoA and C0 2 ;
E27 und E28, die die Umsetzung von Acetyl-Phosphat und ADP/P, zu Acetat und ATP/PPi katalysieren; E 27 and E 28 , which catalyze the reaction of acetyl phosphate and ADP / P, to acetate and ATP / PPi;
E29, das die Umsetzung von Acetyl-CoA und P, (Orthophosphat) zu Acetyl- Phosphat und CoA katalysiert; E 29 , which catalyzes the conversion of acetyl-CoA and P, (orthophosphate) to acetyl phosphate and CoA;
E30, das die Umsetzung von Pyruvat und NADH zu D-Lactat und NAD+ katalysiert; E 30 , which catalyzes the conversion of pyruvate and NADH to D-lactate and NAD + ;
E31, das die Umsetzung von Acetyl-CoA zu Acetaldehyd katalysiert; E 31 , which catalyzes the conversion of acetyl-CoA to acetaldehyde;
E32, das die Umsetzung von Acetaldehyd zu Ethanol katalysiert; E 32 , which catalyzes the conversion of acetaldehyde to ethanol;
E33, das die Umsetzung von Glyceronphosphat zu Methylglyoxal und E 33 , which involves the conversion of glycerol phosphate to methylglyoxal and
Orthophosphat katalysiert;  Orthophosphate catalyzes;
E34, E35 und E36, die einen Komplex bilden, der die Umsetzung von Formiat zu C02 katalysiert; E 34 , E 35 and E 36 , which form a complex that catalyzes the conversion of formate to C0 2 ;
E37, das die Umsetzung von Formiat zu C02 katalysiert; E 37 , which catalyzes the conversion of formate to C0 2 ;
E38, E39 und E40, die einen Komplex bilden, der die Umsetzung von Formiat zuE 38 , E 39 and E 40 , which form a complex that promotes the conversion of formate
C02 katalysiert; C0 2 catalyzes;
E41, das die Umsetzung von Propionyl-CoA, H20 und Oxalacetat zu 2- Methylcitrat und CoA katalysiert; E 41 , which catalyzes the reaction of propionyl CoA, H 2 O and oxalacetate to 2-methyl citrate and CoA;
E42, das die Umsetzung von 2-Methylcitrat zu 2-Methyl-cis-Aconitat oder 2- Methyl-trans-Aconitat und H20 katalysiert; E43, das die Umsetzung von 2-Methyl-trans-Aconitat zu 2-Methyl-cis-Aconitat katalysiert; E 42 which catalyzes the conversion of 2-methyl citrate to 2-methyl cis-aconitate or 2-methyl-trans aconitate and H 2 O; E 43 , which catalyzes the conversion of 2-methyl trans aconitate to 2-methyl cis aconitate;
E44, das die Umsetzung von 2-Methyl-cis-Aconitat zu Succinat und Pyruvat katalysiert; E 44 , which catalyzes the conversion of 2-methyl cis aconitate to succinate and pyruvate;
E45, das die Umsetzung von Propionyl-Phosphat und ADP zu Propionat und ATP katalysiert; E 45 , which catalyzes the conversion of propionyl phosphate and ADP to propionate and ATP;
E46, welches die Umsetzung von Propionyl-CoA und P, zu Propionyl-Phosphat und CoA katalysiert; E46 , which catalyzes the reaction of propionyl CoA and P, to propionyl phosphate and CoA;
E47, welches die Umsetzung von Pyruvat zu Acetaldehyd und C02 katalysiert; E48, welches die Umsetzung von Pyruvat und CoA 2 oxidierten Ferredoxinen zu Acetyl-CoA, C02, und 2 reduzierten Ferredoxinen und 2 H+ katalysiert; E 47 , which catalyzes the conversion of pyruvate to acetaldehyde and C0 2 ; E 48 , which catalyzes the reaction of pyruvate and CoA 2 oxidized ferredoxins to acetyl-CoA, C0 2 , and 2 reduced ferredoxins and 2 H + ;
E49, welches die Umsetzung von D-Xylulose-5-Phosphat und Phosphat zu Acetylphosphat und D-Glyceraldehyd-3-Phosphate und H20 katalysiert; E 49 , which catalyzes the reaction of D-xylulose-5-phosphate and phosphate to acetyl phosphate and D-glyceraldehyde-3-phosphates and H 2 O;
E50, welches die Umsetzung von (S)-methylmalonyl-CoA und Pyruvat zu Propionyl-CoA und Oxalacetat katalysiert; E 50 , which catalyzes the reaction of (S) -methylmalonyl-CoA and pyruvate to propionyl-CoA and oxaloacetate;
E51 , welches die Umsetzung von (S)-Methylmalonyl-CoA zu Succinyl-CoA katalysiert;  E51, which catalyzes the reaction of (S) -methylmalonyl-CoA to succinyl-CoA;
E52, welches die Umsetzung von 2 Pyruvat zu 2-Acetolactat und C02 katalysiert; E53, welches die Umsetzung von 2-Acetolactat zu Acetoin und C02 katalysiert; E54, welches die Umsetzung von Acetoin und NAD(P)H+ zu Butan-2,3-diol und NAD(P)+ katalysiert; E 52 , which catalyzes the conversion of 2 pyruvate to 2-acetolactate and C0 2 ; E 53 , which catalyzes the reaction of 2-acetolactate to acetoin and C0 2 ; E 54 , which catalyzes the reaction of acetoin and NAD (P) H + to butane-2,3-diol and NAD (P) + ;
E55, welches die Umsetzung 2 Acetyl-CoA zu Acetoacetyl-CoA katalysiert; E 55 , which catalyzes the reaction of 2 acetyl-CoA to acetoacetyl-CoA;
E56, welches die Umsetzung von Acetoacetyl-CoA und NAD(P)H+ zu 3- Hydroxybutyryl-CoA und NAD(P)+ katalysiert; E 56 , which catalyzes the reaction of acetoacetyl-CoA and NAD (P) H + to 3-hydroxybutyryl-CoA and NAD (P) + ;
E57, welches die Umsetzung von Crotonyl-CoA und H20 zu 3-Hydroxybutyryl- CoA katalysiert; E 57 , which catalyzes the reaction of crotonyl CoA and H 2 O to 3-hydroxybutyryl CoA;
E58, welches die Umsetzung von Butyraldehyd und NAD(P)H+ zu Butanol und NAD(P)+ katalysiert; E 58 , which catalyzes the reaction of butyraldehyde and NAD (P) H + to butanol and NAD (P) + ;
E59, welches die Umsetzung von Butyryl-CoA und NAD(P)H+ zu Buryraldehyd und NAD(P)+ katalysiert; E 59 , which catalyzes the reaction of butyryl-CoA and NAD (P) H + to buryraldehyde and NAD (P) + ;
E6o, welches die Umsetzung von Acetoacetat und H+ zu Aceton und C02 katalysiert; E 6 o, which catalyzes the reaction of acetoacetate and H + to acetone and C0 2 ;
E6i , welches die Umsetzung von Aceton und NAD(P)H+ zu Propanol und NAD(P)+ katalysiert; E62, welches die Umsetzung von Acyl-CoA und Carbonsäure zu Acylat und Carbonsäure-CoA katalysiert; und E 6 i, which catalyzes the reaction of acetone and NAD (P) H + to propanol and NAD (P) + ; E 62 , which catalyzes the reaction of acyl-CoA and carboxylic acid to acylate and carboxylic acid CoA; and
E63, das die Umsetzung von Pyruvat und NADH zu L-Lactat und NAD+ katalysiert. E 6 3, which catalyzes the conversion of pyruvate and NADH to L-lactate and NAD + .
39. Die rekombinante Zelle nach Anspruch 38, wobei: 39. The recombinant cell of claim 38, wherein:
E19 und E2o ein PEP-abhängiges Phosphotransferase-System Enzym I I (EC 2.7.1 .69); E 19 and E 2 o a PEP-dependent phosphotransferase system enzyme II (EC 2.7.1 .69);
E2i ein PEP-abhängiges Phosphotransferase-System Enzym I (EC 2.7.3.9);E 2 i is a PEP-dependent phosphotransferase system Enzyme I (EC 2.7.3.9);
E22 eine Phosphohistidin-Protein (HPr)-Hexose-Phosphotransferase-E 2 2 is a phosphohistidine protein (HPr) -hexose phosphotransferase
Komponenente des PEP-abhängigen Phosphotransferase-Systems; Components of the PEP-dependent phosphotransferase system;
E23 und E24 eine Formiat-C-Acetyltransferase (EC 2.3.1 .54); E 2 3 and E 2 4 a formate C-acetyltransferase (EC 2.3.1.54);
E25 eine Pyruvat: Ferricytochrom b-i-Oxidoreduktase (EC 1 .2.2.2); E 25 a pyruvate: ferricytochrome bi-oxidoreductase (EC 1 .2.2.2);
E26 eine Methylmalonyl-CoA-Decarboxylase (EC 4.1 .1 .41 ); E 26 is a methylmalonyl-CoA decarboxylase (EC 4.1 .1 .41);
E27 eine ATP/PPi:Acetat-Phosphotransferase (EC 2.7.2.1 oder EC 2.7.2.1 );E 27 an ATP / PPi: acetate phosphotransferase (EC 2.7.2.1 or EC 2.7.2.1);
E28 eine Propionat-/Acetat-Kinase (EC 2.7.2.15); E 28 is a propionate / acetate kinase (EC 2.7.2.15);
E29 eine Acetyl-CoA:Phosphat-Acetyltransferase (EC 2.3.1 .8); E 29 an acetyl-CoA: phosphate acetyltransferase (EC 2.3.1 .8);
E30 eine D-Lactatdehydrogenase (EC 1 .1 .1 .28); E 30 is a D-lactate dehydrogenase (EC 1 .1 .1 .28);
E31 eine Acetaldehyd-Dehydrogenase (CoA-acetylierend) (EC 1 .2.1 .10);E 31 an acetaldehyde dehydrogenase (CoA-acetylating) (EC 1 .2.1 .10);
E32 eine NAD-abhängige Alkohol-Dehydrogenase (EC 1 .1 .1 .1 ); E 32 an NAD-dependent alcohol dehydrogenase (EC 1 .1 .1 .1);
E33 eine Glyceronphosphat-Phospholyase (EC 4.2.3.3); E 33 is a glycerone phosphate phospholyase (EC 4.2.3.3);
E34, E35, E36, E37, E38, E39 und E40 Formatdehydrogenasen (EC 1 .2.1 .2);E 34 , E 35 , E 36 , E 37 , E 38 , E 39 and E 40 formate dehydrogenases (EC 1 .2.1 .2);
E41 eine 2-Methylcitrat Synthase (EC 2.3.3.5); E 41 is a 2-methylcitrate synthase (EC 2.3.3.5);
E42 eine 2-Methylcitrat Dehydratase (EC 4.2.1 .79 oder EC 4.2.1 .1 17); E 42 a 2-methylcitrate dehydratase (EC 4.2.1 .79 or EC 4.2.1 .1 17);
E43 eine 2-Methylaconitat Isomerase; E 43 is a 2-methylaconitate isomerase;
E44 eine Methylisocitrat Lyase (EC 4.1 .3.30); E 44 is a methyl isocitrate lyase (EC 4.1 .3.30);
E45 eine Propionatkinase (EC 2.7.2.15); E 45 is a propionate kinase (EC 2.7.2.15);
E46 eine Phosphat-Propionyltransferase (EC 2.3.1 .8); E 46 a phosphate-propionyltransferase (EC 2.3.1 .8);
E47 eine Pyruvatdecarboxylase (EC 4.1 .1 .1 ); E 47 is a pyruvate decarboxylase (EC 4.1 .1 .1);
E48 eine Pyruvat:Ferredoxin Oxidoreduktase (EC 1 .2.7.1 ); E 48 is a pyruvate: ferredoxin oxidoreductase (EC 1 .2.7.1);
E49 eine Phosphoketolase (EC 4.1 .2.9); E 49 is a phosphoketolase (EC 4.1 .2.9);
E50 eine Methylmalonyl-CoA-Carboxytransferase (EC 2.1 .3.1 ); E 50 is a methylmalonyl-CoA-carboxytransferase (EC 2.1 .3.1);
E51 eine Methylmalonyl-CoA-Mutase (EC 5.4.99.2);  E51 a methylmalonyl-CoA mutase (EC 5.4.99.2);
E52 eine Acetolactat-Synthase (EC 2.2.1 .6); E53 eine Acetolactat-Decarboxylase (EC 4.1 .1.5); E 52 an acetolactate synthase (EC 2.2.1.6); E 53 an acetolactate decarboxylase (EC 4.1 .1.5);
E54 eine Butandiol-Dehydrogenase (EC 1.1 .1 .4 oder EC 1 .1 .1 .76); E 54 a butanediol dehydrogenase (EC 1.1 .1 .4 or EC 1 .1 .1 .76);
E55 eine Thiolase (EC 2.3.1.9); E 55 a thiolase (EC 2.3.1.9);
E56 eine 3-Hydroxybutyryl-CoA-Dehydrogenase (EC 1 .1 .1.157, EC 1.1 .1.35, ECE 56 a 3-hydroxybutyryl-CoA dehydrogenase (EC 1 .1 .1.157, EC 1.1 .1.35, EC
1 .1 .1.36 oder EC 1 .1.1 .21 1 ); 1 .1 .1.36 or EC 1 .1.1 .21 1);
E57 eine Crotonase (EC 4.2.1 .17); E 57 a crotonase (EC 4.2.1 .17);
E58 eine Butanol-Dehydrogenase (EC 1.1 .1.1 oder EC 1.1.1.2); E 58 is a butanol dehydrogenase (EC 1.1 .1.1 or EC 1.1.1.2);
E59 eine Butyraldehyd-Dehydrogenase (EC 1.2.1.3, EC 1 .2.1.4 oder EC 1.2.1.5)E 59 a butyraldehyde dehydrogenase (EC 1.2.1.3, EC 1 .2.1.4 or EC 1.2.1.5)
E6o eine Acetoacetat-Decarboxylase (EC 4.1 .1.4); E 6 o an acetoacetate decarboxylase (EC 4.1 .1.4);
E6i eine Propanol-Dehydrogenase (EC 1 .1 .1.1 oder EC 1 .1 .1.2); E 6 i is a propanol dehydrogenase (EC 1 .1 .1.1 or EC 1 .1 .1.2);
E62 eine Acyl-CoA:CoA-Transferase (EC 2.8.3.-); und/oder E 6 2 an acyl-CoA: CoA transferase (EC 2.8.3.-); and or
E63 eine L-Lactatdehydrogenase (EC 1 .1.1.27) ist. E 6 3 is an L-lactate dehydrogenase (EC 1 .1.1.27).
40. Die rekombinante Zelle nach Anspruch 40, wobei: 40. The recombinant cell of claim 40, wherein:
E19 durch ein ptsG Gen kodiert wird; E 19 is encoded by a ptsG gene;
E2o durch ein ptsl Gen kodiert wird; E 2 o is encoded by a ptsl gene;
E21 durch ein ptsH Gen kodiert wird;  E21 is encoded by a ptsH gene;
E22 durch ein crr Gen kodiert wird;  E22 is encoded by a crr gene;
E23 durch ein tdcE Gen kodiert wird; E 2 3 is encoded by a tdcE gene;
E24 durch ein pflA oder pflB Gen kodiert wird; E 2 4 is encoded by a pflA or pflB gene;
E25 durch ein poxB Gen kodiert wird; E 2 5 is encoded by a poxB gene;
E26 durch ein ygfG Gen kodiert wird; E 2 6 is encoded by a ygfG gene;
E27 durch ein ackA Gen kodiert wird; E 2 7 is encoded by a ackA gene;
E28 durch ein ackA oder tdcD Gen kodiert wird; E 2 8 is encoded by an ackA or tdcD gene;
E29 durch ein pta Gen kodiert wird; E 2 9 is encoded by a pta gene;
E30 durch ein IdhA Gen kodiert wird; E 30 is encoded by an IdhA gene;
E31 durch ein adhE Gen kodiert wird;  E31 is encoded by an adhE gene;
E32 durch ein adhE Gen kodiert wird; E 32 is encoded by an adhE gene;
E33 durch ein mgsA Gen kodiert wird; E 33 is encoded by a mgsA gene;
E34 durch ein fdnG Gen kodiert wird; E 34 is encoded by a fdnG gene;
E35 durch ein fdnH Gen kodiert wird; E is encoded by a gene fdnH 35;
E36 durch ein fdnl Gen kodiert wird; E 36 is encoded by a fdnl gene;
E37 durch ein fdhF Gen kodiert wird; E 37 is encoded by a fdhF gene;
E38 durch ein fdoG Gen kodiert wird; E39 durch ein fdoH Gen kodiert wird; E 38 is encoded by a fdoG gene; E39 is encoded by a fdoH gene;
E40 durch ein fdol Gen kodiert wird;  E40 is encoded by a fdol gene;
E41 durch ein prpC Gen kodiert wird;  E41 is encoded by a prpC gene;
E42 durch ein prpD oder acnD Gen kodiert wird;  E42 is encoded by a prpD or acnD gene;
E43 durch ein prpF Gen kodiert wird;  E43 is encoded by a prpF gene;
E44 durch ein prpB Gen kodiert wird;  E44 is encoded by a prpB gene;
E45 durch ein tdcD Gen kodiert wird  E45 is encoded by a tdcD gene
E46 durch ein pta Gen kodiert wird;  E46 is encoded by a pta gene;
E47 durch ein pdc Gen kodiert wird;  E47 is encoded by a pdc gene;
E48 durch ein porA, porB, porC oder porD Gen kodiert wird;  E48 is encoded by a porA, porB, porC or porD gene;
E49 durch ein xpkl oder xpk2 Gen kodiert wird  E49 is encoded by an xpkl or xpk2 gene
E50 durch ein Methylmalonyl-CoA-Carboxytransferase Gen kodiert wird;  E50 is encoded by a methylmalonyl CoA carboxytransferase gene;
Ε5ι durch ein sbm oder ein mcmA und ein mcmB Gen kodiert wird; 5 ι is encoded by a sbm or a mcmA and a mcmB gene;
E52 durch ein alsS, ilvB, ilvM, ilvN, ilvG, ilvl oder ilvH Gen kodiert wird; E 52 is encoded by a gene as, ilvB, ilvM, ilvN, ilvG, ilvl or ilvH gene;
E53 durch ein alsD Gen kodiert wird; E 53 is encoded by a alsD gene;
E54 durch ein butB Gen kodiert wird;  E54 is encoded by a butB gene;
E55 durch ein thl, thIA, thIB oder phaA Gen kodiert wird;  E55 is encoded by a thI, thIA, thIB or phaA gene;
E56 durch ein phaB Gen kodiert wird; E 56 is encoded by a phaB gene;
E57 durch ein crt Gen kodiert wird; E 57 is encoded by a crt gene;
E58 durch ein adhE Gen kodiert wird; E 58 is encoded by an adhE gene;
E59 durch ein adhE, bdhA oder bdhB Gen kodiert wird;  E59 is encoded by an adhE, bdhA or bdhB gene;
Εβο durch ein ade Gen kodiert wird;  It is encoded by an ade gene;
E6i durch ein adh Gen kodiert wird; E 6 i is encoded by an adh gene;
Εβ2 durch ein ctfA und ein ctfB oder ein atoA und ein atoD Gen kodiert wird; und/oder  Εβ2 is encoded by a ctfA and a ctfB or an atoA and an atoD gene; and or
Εβ3 durch ein IdhL Gen kodiert wird.  Εβ3 is encoded by an IdhL gene.
Die rekombinante Zelle nach mindestens einem der Ansprüche 13 bis 15 oder 33 oder 34, wobei die Zelle eine im Vergleich zu ihrem Wildtyp verminderte Aktivität mindestens eines der Enzyme E19, E20, E21 , E22, E23, E24, E25, E26, E27, E28, E29, E30, E31 , E32, E33, E45, E46, E47, E48, E49, E50, E55, E56, E57, E58, E59, E60, E61 und E63 aufweist. The recombinant cell according to at least one of claims 13 to 15 or 33 or 34, wherein the cell has a reduced activity compared to its wild type of at least one of the enzymes E19, E20, E21, E22, E23, E24, E25, E26, E27, E28 , E29, E30, E31, E32, E33, E45, E46, E47, E48, E49, E50, E55, E56, E57, E58, E59, E60, E61 and E63.
42. Die rekombinante Zelle nach Anspruch 42, wobei die Enzyme E19, E20, E21 , E22, E23, E24, E25, E27, E28, E29, E30, E31 , E32, E33, E45, E46, E47, E48, E49, E50, E55, E56, E57, E58, E59, E60, E61 und E63 ptsG, ptsl, ptsH, crr, tdcE, pflA, pflB, poxB, ack, pta, tdcD, IdhA, adhE, mgsA, ygfG, pdc, porA, porB, porC, porD, xpkl , xpk2, thl, thIA, thIB, phaA, phaB, crt, bdhA, bdhB, ade, adh und IdhL sind. 42. The recombinant cell of claim 42, wherein the enzymes E19, E20, E21, E22, E23, E24, E25, E27, E28, E29, E30, E31, E32, E33, E45, E46, E47, E48, E49, E50, E55, E56, E57, E58, E59, E60, E61 and E63 ptsG, ptsl, ptsH, crr, tdcE, pflA, pflB, poxB, ack, pta, tdcD, IdhA, adhE, mgsA, ygfG, pdc, porA , porB, porC, porD, xpkl, xpk2, thL, thIA, thIB, phaA, phaB, crt, bdhA, bdhB, ade, adh, and idhL.
43. Verfahren zur Herstellung einer gentechnisch veränderten Zelle nach mindestens einem der Ansprüche 1 bis 42, umfassend die Erhöhung im Vergleich zum Wildtyp der Aktivität mindestens eines der in den Ansprüchen 9 bis 34 genannten Enzyme in der Zelle. 43. A method for producing a genetically modified cell according to any one of claims 1 to 42, comprising increasing compared to the wild type of activity of at least one of the enzymes mentioned in claims 9 to 34 in the cell.
44. Das Verfahren nach Anspruch 43, ferner umfassend die Verminderung der Aktivität im Vergleich zum Wildtyp mindestens eines der in den Ansprüchen 33 bis 40 genannten Enzyme in der Zelle. 44. The method of claim 43, further comprising reducing the activity compared to the wild-type at least one of the enzymes in the cell mentioned in claims 33 to 40.
45. Verfahren zur Herstellung mindestens eines C4-Körpers und/oder mindestens einer über diesen C4-Körper hergestellte Folgeverbindung, wobei das Verfahren die Inkubation einer Zelle nach einem der Ansprüche 1 bis 42 mit einem Nährmedium, das Saccharose enthält, unter Bedingungen, die die Herstellung mindestens eines C4-Körpers aus Saccharose und C02 erlauben, umfasst. 45. A method for producing at least one C 4 -Körpers and / or at least one C 4 -body on this sequence produced compound, which method comprises incubating a cell of any one of claims 1 to 42 with a nutrient medium containing sucrose, under conditions which permit the production of at least one C 4 body from sucrose and C0 2 .
46. Das Verfahren nach Anspruch 45, wobei das Medium ferner Kohlendioxid enthält. 46. The method of claim 45, wherein the medium further contains carbon dioxide.
47. Das Verfahren nach Anspruch 45 oder 46, ferner umfassend Isolieren des 47. The method of claim 45 or 46, further comprising isolating the
mindestens einen C4-Körpers und/oder der mindestens einen über diesen C4-Körper hergestellten Folgeverbindung aus dem Nährmedium. at least one C 4 body and / or the at least one follow-up compound prepared from this C 4 body from the nutrient medium.
48. Das Verfahren nach mindestens einem der Ansprüche 45 bis 47, wobei der 48. The method according to at least one of claims 45 to 47, wherein the
mindestens eine C4-Körper ausgewählt wird aus der Gruppe bestehend aus Succinat, Malat, Fumarat, Oxalacetat, Aspartat, Asparagin, Threonin, Tetrahydrofuran, at least one C 4 body is selected from the group consisting of succinate, malate, fumarate, oxaloacetate, aspartate, asparagine, threonine, tetrahydrofuran,
Pyrrolidon, Acetoin, 4,4-Bionell, Hydroxysuccinat, Epoxy-v-Butyrolacton, Butensäure, Butyrat, Butandiol, 1 ,2-Butandiol, 1 ,3-Butandiol, 1 ,4-Butandiol, 2,3-Butandiol, Buten, n-Buten, cis-2-Buten, trans-2-Buten, Isobuten, Butadien, 1 ,2-Butadien, 1 ,3-Butadien, 3-Hydroxybutyrolacton, 4-Hydroxybutyrolacton, 1 -Butanol, 2-Butanol, tert-Butanol, Isobutanol, 2-Hydroxybuttersäure, 3-Hydroxybuttersäure, 4-Hydroxybuttersäure, 2- Hydroxyisobuttersäure und 3-Hydroxyisobuttersäure. Pyrrolidone, acetoin, 4,4-bionell, hydroxysuccinate, epoxy-v-butyrolactone, butenoic acid, butyrate, butanediol, 1, 2-butanediol, 1, 3-butanediol, 1, 4-butanediol, 2,3-butanediol, butene, n-butene, cis-2-butene, trans-2-butene, isobutene, butadiene, 1, 2-butadiene, 1, 3-butadiene, 3-hydroxybutyrolactone, 4-hydroxybutyrolactone, 1-butanol, 2-butanol, tert-butyl butanol, Isobutanol, 2-hydroxybutyric acid, 3-hydroxybutyric acid, 4-hydroxybutyric acid, 2-hydroxyisobutyric acid and 3-hydroxyisobutyric acid.
49. Das Verfahren nach mindestens einem der Ansprüche 45 bis 48, wobei die C4-Körper ausgewählt werden aus der Gruppe bestehend aus: Malat, Oxalacetat und die über diese C4-Körper hergestellten Folgeverbindungen ausgewählt werden aus der Gruppe bestehend aus durch enzymatische oder chemische Synthese über Malat oder Oxalacetat hergestellte Folgeverbindungen. 49. The method according to at least one of claims 45 to 48, wherein the C 4 bodies are selected from the group consisting of: malate, oxaloacetate and the followers prepared via these C 4 bodies are selected from the group consisting of by enzymatic or chemical synthesis via malate or oxaloacetate prepared follow-up compounds.
50. Das Verfahren nach Anspruch 49, wobei die durch enzymatische Synthese über Malat oder Oxalacetat hergestellten Folgeverbindungen ausgewählt werden aus der Gruppe bestehend aus: Aspartat, Asparagin, Threonin, Tetra hydrofu ran, Butyrat, Butandiol, 1 ,2-Butandiol, 1 ,3-Butandiol, 1 ,4-Butandiol, 2,3-Butandiol, 3- und 4- Hydroxybutyrolacton, 1 -, 2- und tert-Butanol, Isobutanol, 2-, 3- und 4- Hydroxybuttersäure, 2- und 3-Hydroxyisobuttersäure, Methionin und Lysin. 50. The process according to claim 49, wherein the secondary compounds produced by enzymatic synthesis via malate or oxaloacetate are selected from the group consisting of: aspartate, asparagine, threonine, tetrahydrofuran, butyrate, butanediol, 1,2-butanediol, 1,3 Butanediol, 1,4-butanediol, 2,3-butanediol, 3- and 4-hydroxybutyrolactone, 1-, 2- and tert-butanol, isobutanol, 2-, 3- and 4-hydroxybutyric acid, 2- and 3-hydroxyisobutyric acid , Methionine and lysine.
51 . Verwendung einer Zelle nach einem der Ansprüche 1 bis 42 zur mikrobiologischen Herstellung von mindestens einem C4-Körper und/oder mindestens einer über diesen C4-Körper hergestellten Folgeverbindung aus Saccharose und Kohlendioxid. 51. Use of a cell according to one of claims 1 to 42 for the microbiological production of at least one C 4 body and / or at least one sucrose and carbon dioxide secondary compound produced via this C 4 body.
PCT/EP2011/059619 2010-06-11 2011-06-09 Microbiological production of c4 bodies from saccharose and carbon dioxide WO2011154503A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102010029973.1 2010-06-11
DE102010029973A DE102010029973A1 (en) 2010-06-11 2010-06-11 Microbiological production of C4 bodies from sucrose and carbon dioxide

Publications (1)

Publication Number Publication Date
WO2011154503A1 true WO2011154503A1 (en) 2011-12-15

Family

ID=44356275

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2011/059619 WO2011154503A1 (en) 2010-06-11 2011-06-09 Microbiological production of c4 bodies from saccharose and carbon dioxide

Country Status (2)

Country Link
DE (1) DE102010029973A1 (en)
WO (1) WO2011154503A1 (en)

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102876760A (en) * 2012-10-22 2013-01-16 南京工业大学 Application of Bacillus subtilis in high-yield adenosine and acetoin co-production
DE102012201360A1 (en) 2012-01-31 2013-08-01 Evonik Industries Ag New cell capable of forming rhamnolipid compound, which is useful for the production of cosmetic, dermatological or pharmaceutical formulations, of plant protection formulations and of care and cleaning agents and surfactant concentrates
WO2013134167A1 (en) * 2012-03-05 2013-09-12 E. I. Du Pont De Nemours And Company Variant sucrose transporter polypeptides
WO2013142033A1 (en) * 2012-03-20 2013-09-26 Metabolix, Inc. Genetically engineered microorganisms for the production of poly-4-hydroxybutyrate
FR3002774A1 (en) * 2013-03-04 2014-09-05 Agronomique Inst Nat Rech MUTANT YEAS HAVING INCREASED PRODUCTION OF LIPIDS AND CITRIC ACID
US8871488B2 (en) 2010-06-18 2014-10-28 Butamax Advanced Biofuels Llc Recombinant host cells comprising phosphoketolases
WO2015158716A1 (en) * 2014-04-16 2015-10-22 Novamont S.P.A. Process for the production of 1,4-butanediol
US9725746B2 (en) 2012-12-21 2017-08-08 Evonik Degussa Gmbh Producing amines and diamines from a carboxylic acid or dicarboxylic acid or a monoester thereof
US9765370B2 (en) 2012-04-02 2017-09-19 Evonik Degussa Gmbh Method for aerobically producing alanine or a compound produced using alanine
US9765366B2 (en) 2012-02-22 2017-09-19 Evonik Degussa Gmbh Biotechnological method for producing butanol and butyric acid
US9850192B2 (en) 2012-06-08 2017-12-26 Cj Cheiljedang Corporation Renewable acrylic acid production and products made therefrom
US9914941B2 (en) 2011-11-09 2018-03-13 Amyris, Inc. Production of acetyl-coenzyme a derived isoprenoids
WO2018091525A1 (en) 2016-11-15 2018-05-24 Danmarks Tekniske Universitet Bacterial cells with improved tolerance to diacids
US10174353B2 (en) 2014-05-26 2019-01-08 Evonik Degussa Gmbh Methods of producing rhamnolipids
CN110651037A (en) * 2017-05-19 2020-01-03 巴斯夫欧洲公司 Method for producing organic compounds
CN111139193A (en) * 2019-12-05 2020-05-12 天津科技大学 Grape juice yeast strain with low yield of higher alcohol and strong degradation malic acid and application thereof
US10786064B2 (en) 2010-02-11 2020-09-29 Cj Cheiljedang Corporation Process for producing a monomer component from a genetically modified polyhydroxyalkanoate biomass

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102012101153A1 (en) * 2012-02-14 2013-08-14 Thyssenkrupp Uhde Gmbh PROCESS FOR THE BIOTECHNOLOGICAL MANUFACTURE OF ORGANIC ACIDS BY AT LEAST ONE HEFESTAMM
CN103103147B (en) * 2012-07-18 2014-11-12 大连理工大学 Mercurial paracoccus strain simultaneously having denitrification and iron reduction functions as well as cultivation method and application of mercurial paracoccus strain

Citations (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1009370A (en) 1962-12-18 1965-11-10 Ajinomoto I I A process for the production of 1-glutamic acid by fermentation
US4489160A (en) 1981-08-26 1984-12-18 Kyowa Hakko Kogyo Co., Ltd. Plasmid pCG2
US4601893A (en) 1984-02-08 1986-07-22 Pfizer Inc. Laminate device for controlled and prolonged release of substances to an ambient environment and method of use
US4757009A (en) 1983-08-29 1988-07-12 Ajinomoto Co., Inc. Recombinant DNA having a phosphoenol pyruvate carboxylase gene inserted therein, bacteria carrying said recombinant DNA and a process for producing amino acids using said bacteria
US4980285A (en) 1984-10-04 1990-12-25 Ajinomoto Co., Inc. Method for producing L-amino acids
EP0472869A2 (en) 1990-08-30 1992-03-04 Degussa Ag Plasmids of corynebacterium glutamicum and derived plasmid vectors
US5158891A (en) 1984-08-21 1992-10-27 Asahi Kasei Kogyo Kabushiki Kaisha Plasmid containing a gene for tetracycline resistance and DNA fragments derived therefrom
WO1996015246A1 (en) 1994-11-11 1996-05-23 Forschungszentrum Jülich GmbH Dna which regulates gene expression in coryne-form bacteria
US5573945A (en) 1994-01-10 1996-11-12 Ajinomoto Co., Inc. Mutant and method for producing L-glutamic acid by fermentation
JPH10229891A (en) 1997-02-20 1998-09-02 Mitsubishi Rayon Co Ltd Production of malonic acid derivative
US6171833B1 (en) 1998-12-23 2001-01-09 Massachusetts Institute Of Technology Pyruvate carboxylase from corynebacterium glutamicum
DE10031999A1 (en) 1999-09-09 2001-04-19 Degussa Process for the fermentative production of D-pantothenic acid using coryneform bacteria
US6455284B1 (en) 1998-04-13 2002-09-24 The University Of Georgia Research Foundation, Inc. Metabolically engineered E. coli for enhanced production of oxaloacetate-derived biochemicals
WO2002090312A1 (en) 2001-05-07 2002-11-14 Cargill, Incorporated Process for preparing carboxylic acids and derivatives thereof
US6599732B1 (en) 1999-06-29 2003-07-29 Archer-Daniels-Midland Company Regulation of carbon assimilation
US6852516B2 (en) 1999-01-13 2005-02-08 Ajinomoto Co., Inc. Method for producing L-glutamic acid by fermentation
US6861246B2 (en) 1999-07-07 2005-03-01 Degussa Ag L-lysine-producing corynebacteria and process for the preparation of lysine
US6872553B2 (en) 1999-10-20 2005-03-29 Degussa Ag Nucleotide sequences which code for the pck gene
WO2008137402A1 (en) * 2007-05-02 2008-11-13 E. I. Du Pont De Nemours And Company Method for the production of 1-butanol
WO2009078687A2 (en) * 2007-12-18 2009-06-25 Korea Advanced Institute Of Science And Technology Recombinant microorganism having an ability of using sucrose as a carbon source

Patent Citations (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1009370A (en) 1962-12-18 1965-11-10 Ajinomoto I I A process for the production of 1-glutamic acid by fermentation
US4489160A (en) 1981-08-26 1984-12-18 Kyowa Hakko Kogyo Co., Ltd. Plasmid pCG2
US4757009A (en) 1983-08-29 1988-07-12 Ajinomoto Co., Inc. Recombinant DNA having a phosphoenol pyruvate carboxylase gene inserted therein, bacteria carrying said recombinant DNA and a process for producing amino acids using said bacteria
US4601893A (en) 1984-02-08 1986-07-22 Pfizer Inc. Laminate device for controlled and prolonged release of substances to an ambient environment and method of use
US5158891A (en) 1984-08-21 1992-10-27 Asahi Kasei Kogyo Kabushiki Kaisha Plasmid containing a gene for tetracycline resistance and DNA fragments derived therefrom
US4980285A (en) 1984-10-04 1990-12-25 Ajinomoto Co., Inc. Method for producing L-amino acids
EP0472869A2 (en) 1990-08-30 1992-03-04 Degussa Ag Plasmids of corynebacterium glutamicum and derived plasmid vectors
US5573945A (en) 1994-01-10 1996-11-12 Ajinomoto Co., Inc. Mutant and method for producing L-glutamic acid by fermentation
WO1996015246A1 (en) 1994-11-11 1996-05-23 Forschungszentrum Jülich GmbH Dna which regulates gene expression in coryne-form bacteria
JPH10229891A (en) 1997-02-20 1998-09-02 Mitsubishi Rayon Co Ltd Production of malonic acid derivative
US6455284B1 (en) 1998-04-13 2002-09-24 The University Of Georgia Research Foundation, Inc. Metabolically engineered E. coli for enhanced production of oxaloacetate-derived biochemicals
US6171833B1 (en) 1998-12-23 2001-01-09 Massachusetts Institute Of Technology Pyruvate carboxylase from corynebacterium glutamicum
US6403351B1 (en) 1998-12-23 2002-06-11 Archer Daniels Midland Company Pyruvate carboxylase polypeptide from Corynebacterium glutamicum
US6884606B2 (en) 1998-12-23 2005-04-26 Massachusetts Institute Of Technology Pyruvate carboxylase from Corynebacterium glutamicum
US6852516B2 (en) 1999-01-13 2005-02-08 Ajinomoto Co., Inc. Method for producing L-glutamic acid by fermentation
US6599732B1 (en) 1999-06-29 2003-07-29 Archer-Daniels-Midland Company Regulation of carbon assimilation
US6861246B2 (en) 1999-07-07 2005-03-01 Degussa Ag L-lysine-producing corynebacteria and process for the preparation of lysine
DE10031999A1 (en) 1999-09-09 2001-04-19 Degussa Process for the fermentative production of D-pantothenic acid using coryneform bacteria
US6872553B2 (en) 1999-10-20 2005-03-29 Degussa Ag Nucleotide sequences which code for the pck gene
WO2002090312A1 (en) 2001-05-07 2002-11-14 Cargill, Incorporated Process for preparing carboxylic acids and derivatives thereof
WO2008137402A1 (en) * 2007-05-02 2008-11-13 E. I. Du Pont De Nemours And Company Method for the production of 1-butanol
WO2009078687A2 (en) * 2007-12-18 2009-06-25 Korea Advanced Institute Of Science And Technology Recombinant microorganism having an ability of using sucrose as a carbon source

Non-Patent Citations (46)

* Cited by examiner, † Cited by third party
Title
"E. coli Plasmid Vectors", 2003, HUMANA PRESS
"Manual of Methods for General Bacteriology", 1981, AMERICAN SOCIETY FOR BACTERIOLOGY
"Methods Enzymol.", vol. 185, 1988, article "Vectors : a survey of molecular cloning vectors and their uses, 179-204, Butterworth, Stoneham; Goeddel, D. V. (1990), Systems for heterologous gene expression", pages: 3 - 7
CHMIEL: "Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik", 1991, GUSTAV FISCHER VERLAG
DONAHUE ET AL., JOURNAL OF BACTERIOLOGY, vol. 182, no. 19, 2000, pages 5624 - 5627
DUNICAN, SHIVNAN, BIO/TECHNOLOGY, vol. 7, 1989, pages 1067 - 1070
EIKMANNS ET AL., GENE, vol. 102, 1991, pages 93 - 98
EIKMANNS ET AL., GENE, vol. 107, 1991, pages 69 - 74
FREEDBERG ET AL., JOURNAL OF BACTERIOLOGY, vol. 115, no. 3, 1973, pages 816 - 823
GLOVER, D. M.: "DNA cloning: a practical approach", vol. 1-111, 1985, IRL PRESS LTD.
GUERRERO ET AL., GENE, vol. 138, 1994, pages 35 - 41
H. LIN ET AL: "Increasing the Acetyl-CoA Pool in the Presence of Overexpressed Phosphoenolpyruvate Carboxylase or Pyruvate Carboxylase Enhances Succinate Production in Escherichia coli", BIOTECHNOLOGY PROGRESS, vol. 20, no. 5, 1 October 2004 (2004-10-01), pages 1599 - 1604, XP055006788, ISSN: 8756-7938, DOI: 10.1021/bp049843a *
HERMANN ET AL., ELECTOPHORESIS, vol. 22, 2001, pages 1712 - 23
HERMANN ET AL., ELECTROPHORESIS, vol. 22, 2001, pages 1712.23
JAHREIS K ET AL: "Adaptation of sucrose metabolism in the Escherichia coli wild-type strain EC3132", JOURNAL OF BACTERIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, WASHINGTON, DC; US, vol. 184, no. 19, 1 October 2002 (2002-10-01), pages 5307 - 5316, XP002984526, ISSN: 0021-9193, DOI: 10.1128/JB.184.19.5307-5316.2002 *
JENSEN, HAMMER, BIOTECHNOLOGY AND BIOENGINEERING, vol. 58, 1998, pages 191 - 195
JIAN WANG ET AL: "Succinate production from different carbon sources under anaerobic conditions by metabolic engineered Escherichia coli strains", METABOLIC ENGINEERING, vol. 13, no. 3, 1 May 2011 (2011-05-01), pages 328 - 335, XP055010835, ISSN: 1096-7176, DOI: 10.1016/j.ymben.2011.03.004 *
JIAN WANG ET AL: "Succinate production from sucrose by metabolic engineered escherichia coli strains under aerobic conditions", BIOTECHNOLOGY PROGRESS, vol. 27, no. 5, 1 September 2011 (2011-09-01), pages 1242 - 1247, XP055010836, ISSN: 8756-7938, DOI: 10.1002/btpr.661 *
LABARRE ET AL., JOURNAL OF BACTERIOLOGY, vol. 175, 1993, pages 1001 - 1007
LOHAUS ET AL., BIOSPEKTRUM, vol. 5, 1998, pages 32 - 39
LOHAUS, MEYER, BIOSPEKTRUM, vol. 5, 1989, pages 32 - 39
LOTTSPEICH, ANGEWANDTE CHEMIE, vol. 111, 1999, pages 2630 - 2647
MALUMBRES ET AL., GENE, vol. 134, 1993, pages 15 - 24
MARTIN ET AL., BIO/TECHNOLOGY, vol. 5, 1987, pages 137 - 146
MENKEL ET AL., APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 64, 1989, pages 549 - 554
OHNISHI J ET AL.: "A novel methodology employing Corynebacterium glutamicum genome information to generate a new L-lysine- producing mutant.", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 58, no. 2, 2002, pages 217 - 223, XP002289984, DOI: doi:10.1007/s00253-001-0883-6
RAY ET AL., JOURNAL OF BACTERIOLOGY, vol. 182, no. 8, 2000, pages 2277 - 2284
REINSCHEID ET AL., APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 60, 1994, pages 126 - 132
SAMBROOK ET AL.: "Molecular Cloning: a Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SAMBROOK, J., FRITSCH, E. F., MANIATIS, T.: "Molecular cloning: a laboratory manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SCHÄFER ET AL., APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 60, 1994, pages 756 - 759
SCHÄFER ET AL., GENE, vol. 145, 1994, pages 69 - 73
SCHMID K ET AL: "PLASMID-MEDIATED SUCROSE METABOLISM IN ESCHERICHIA COLI K12: MAPPING OF THE SCR GENES OF PUR400", MOLECULAR MICROBIOLOGY, WILEY-BLACKWELL PUBLISHING LTD, GB, vol. 2, no. 1, 1 January 1988 (1988-01-01), pages 1 - 08, XP001024290, ISSN: 0950-382X, DOI: 10.1111/J.1365-2958.1988.TB00001.X *
SCHRUMPF ET AL., JOURNAL OF BACTERIOLOGY, vol. 173, pages 4510 - 4516
SCHWARZER, PUHLER, BIO/TECHNOLOGY, vol. 9, 1991, pages 84 - 87
SERWOLD-DAVIS ET AL., FEMS MICROBIOLOGY LETTERS, vol. 66, 1990, pages 119 - 124
SHUMAN, JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 269, 1994, pages 32678 - 84
SIMON ET AL., BIO/TECHNOLOGY, vol. 1, 1983, pages 784 - 791
SONNEN ET AL., GENE, vol. 107, 1991, pages 69 - 74
SPRATT ET AL., GENE, vol. 41, 1986, pages 337 - 342
STORHAS: "Bioreaktoren und periphere Einrichtungen", 1994, VIEWEG VERLAG
TAUCH ET AL., FEMS MICROBIOLOGY LETTERS, vol. 123, 1994, pages 343 - 347
THIERBACH ET AL., APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 29, 1988, pages 356 - 362
TSUCHIYA, MORINAGA, BIO/TECHNOLOGY, vol. 6, 1988, pages 428 - 430
WILSON ET AL., JOURNAL OF BACTERIOLOGY, vol. 183, 2001, pages 2151 - 2155
X. ZHANG ET AL: "Metabolic evolution of energy-conserving pathways for succinate production in Escherichia coli", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 106, no. 48, 1 December 2009 (2009-12-01), pages 20180 - 20185, XP055010824, ISSN: 0027-8424, DOI: 10.1073/pnas.0905396106 *

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10786064B2 (en) 2010-02-11 2020-09-29 Cj Cheiljedang Corporation Process for producing a monomer component from a genetically modified polyhydroxyalkanoate biomass
US10006058B2 (en) 2010-06-18 2018-06-26 Butamax Advanced Biofuels Llc Recombinant host cells comprising phosphoketalase
US8871488B2 (en) 2010-06-18 2014-10-28 Butamax Advanced Biofuels Llc Recombinant host cells comprising phosphoketolases
US9914941B2 (en) 2011-11-09 2018-03-13 Amyris, Inc. Production of acetyl-coenzyme a derived isoprenoids
DE102012201360A1 (en) 2012-01-31 2013-08-01 Evonik Industries Ag New cell capable of forming rhamnolipid compound, which is useful for the production of cosmetic, dermatological or pharmaceutical formulations, of plant protection formulations and of care and cleaning agents and surfactant concentrates
US9765366B2 (en) 2012-02-22 2017-09-19 Evonik Degussa Gmbh Biotechnological method for producing butanol and butyric acid
WO2013134167A1 (en) * 2012-03-05 2013-09-12 E. I. Du Pont De Nemours And Company Variant sucrose transporter polypeptides
CN104245723A (en) * 2012-03-05 2014-12-24 纳幕尔杜邦公司 Variant sucrose transporter polypeptides
WO2013142033A1 (en) * 2012-03-20 2013-09-26 Metabolix, Inc. Genetically engineered microorganisms for the production of poly-4-hydroxybutyrate
US9765370B2 (en) 2012-04-02 2017-09-19 Evonik Degussa Gmbh Method for aerobically producing alanine or a compound produced using alanine
US9850192B2 (en) 2012-06-08 2017-12-26 Cj Cheiljedang Corporation Renewable acrylic acid production and products made therefrom
CN102876760B (en) * 2012-10-22 2014-07-30 南京工业大学 Application of Bacillus subtilis in high-yield adenosine and acetoin co-production
CN102876760A (en) * 2012-10-22 2013-01-16 南京工业大学 Application of Bacillus subtilis in high-yield adenosine and acetoin co-production
US9725746B2 (en) 2012-12-21 2017-08-08 Evonik Degussa Gmbh Producing amines and diamines from a carboxylic acid or dicarboxylic acid or a monoester thereof
FR3002774A1 (en) * 2013-03-04 2014-09-05 Agronomique Inst Nat Rech MUTANT YEAS HAVING INCREASED PRODUCTION OF LIPIDS AND CITRIC ACID
WO2014136028A1 (en) * 2013-03-04 2014-09-12 Institut National De La Recherche Agronomique Mutant yeasts having an increased production of lipids and of citric acid
WO2015158716A1 (en) * 2014-04-16 2015-10-22 Novamont S.P.A. Process for the production of 1,4-butanediol
US10227616B2 (en) 2014-04-16 2019-03-12 Novamont S.P.A. Process for the production of 1,4-butanediol
US10174353B2 (en) 2014-05-26 2019-01-08 Evonik Degussa Gmbh Methods of producing rhamnolipids
WO2018091525A1 (en) 2016-11-15 2018-05-24 Danmarks Tekniske Universitet Bacterial cells with improved tolerance to diacids
CN110651037A (en) * 2017-05-19 2020-01-03 巴斯夫欧洲公司 Method for producing organic compounds
CN111139193A (en) * 2019-12-05 2020-05-12 天津科技大学 Grape juice yeast strain with low yield of higher alcohol and strong degradation malic acid and application thereof

Also Published As

Publication number Publication date
DE102010029973A1 (en) 2011-12-15

Similar Documents

Publication Publication Date Title
WO2011154503A1 (en) Microbiological production of c4 bodies from saccharose and carbon dioxide
EP2175024B1 (en) Microbiological production of 3-hydroxypropionic acid
EP2152659B1 (en) A process for preparing methacrylic acid or methacrylic esters
US10100342B2 (en) Method for producing 3-hydroxypropionic acid and other products
EP2291530B1 (en) Recombinant cell producing 2-hydroxyisobutyric acid
Wang et al. Anaerobic fermentation for production of carboxylic acids as bulk chemicals from renewable biomass
Joo et al. Production of 5-aminovaleric acid in recombinant Corynebacterium glutamicum strains from a Miscanthus hydrolysate solution prepared by a newly developed Miscanthus hydrolysis process
DE102007015583A1 (en) An enzyme for the production of methylmalonyl-coenzyme A or ethylmalonyl-coenzyme A and its use
WO2007141208A2 (en) Microbiological production of 3-hydroxyisobutyric acid
EP2354235B1 (en) Method for the fermentative production of L-amino acids involving the use of coryneform bacteria
US8435768B2 (en) Method for producing succinic acid using a yeast belonging to the genus Yarrowia
WO2008148640A1 (en) Microbiological production of aldehydes, especially of 3-hydroxypropionaldehyde
DE102012201360A1 (en) New cell capable of forming rhamnolipid compound, which is useful for the production of cosmetic, dermatological or pharmaceutical formulations, of plant protection formulations and of care and cleaning agents and surfactant concentrates
KR102430878B1 (en) Method for fermentative production of a molecule of interest by a microorganism comprising a gene encoding a sugar phosphotransferase system (PTS)
CN112204146A (en) Acid-tolerant yeast having inhibited ethanol production pathway and method for producing lactic acid using the same
WO2020016287A1 (en) Biotechnological production of cannabinoids
US20160326554A1 (en) Recombinant microorganisms for producing organic acids
US20160348082A1 (en) Microorganisms for Succinic Acid Production
DE102007059248A1 (en) Recombinant cell capable of converting bicarbonate to organic compounds utilizes a 16-step metabolic pathway starting with acetyl coenzyme A
JP5651989B2 (en) Method for producing succinic acid
WO2009053489A1 (en) Fermentative production of alpha-ketoglutaric acid
Peters-Wendisch et al. Use of Corynebacterium glutamicum for the Production of High-Value Chemicals from New Carbon Sources

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11724632

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11724632

Country of ref document: EP

Kind code of ref document: A1