EP1090126A1 - Microbial preparation of substances from aromatic metabolism/iii - Google Patents
Microbial preparation of substances from aromatic metabolism/iiiInfo
- Publication number
- EP1090126A1 EP1090126A1 EP99916080A EP99916080A EP1090126A1 EP 1090126 A1 EP1090126 A1 EP 1090126A1 EP 99916080 A EP99916080 A EP 99916080A EP 99916080 A EP99916080 A EP 99916080A EP 1090126 A1 EP1090126 A1 EP 1090126A1
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- EP
- European Patent Office
- Prior art keywords
- gene
- activity
- glucose
- process according
- increased
- Prior art date
- Legal status (The legal status 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 status listed.)
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- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
-
- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
- C12P13/222—Phenylalanine
Definitions
- the invention relates to a process for the microbial preparation of substances from aromatic metabolism according to Claims 1 to 19 and 29, to gene structures according to Claims 20 to 22, and to transformed cells according to Claims 23 to 28.
- Microbially prepared substances from aromatic metabolism, in particular aromatic amino acids, are of great economic interest, with the requirement for amino acids, for example, continuing to increase .
- L-phenylalanine for example, is used for preparing medicaments and, in particular, also in the preparation of the sweetener aspartame ( ⁇ -L- aspartyl-L-phenylalanine methyl ester) .
- L-Tryptophan is required as a medicament and as an additive for feedstuffs; there is likewise a need for L-tyrosine as a medicament and as a raw material in the pharmaceutical industry.
- biotechnological preparation is a very important method for obtaining amino acids, in the desired optically active form, under economically justifiable conditions. The biotechnological preparation is effected either enzymically or using microorganisms.
- amino acid analogues for example, have been employed for switching off the regulation of biosynthesis.
- mutants of Escherichia coli were obtained which made it possible to achieve an increased production of L-phenylalanine (GB-2 , 053 , 906) .
- a similar strategy also led to overproducing strains of Corynebacterium (JP-19037/1976 and JP-39517/1978) and Bacillus (EP-0 , 138 , 526) .
- EP-0,077,196 describes a process for producing aromatic amino acids in which a 3-deoxy- D-arabinoheptulosonate- 7 -phosphate synthase (DAHP synthase) which is no longer feedback- inhibited is overexpressed in E. coli .
- DAHP synthase 3-deoxy- D-arabinoheptulosonate- 7 -phosphate synthase
- E. coli strain in which, for producing L-phenylalanine, chorismate mutase/prephenate dehydratase is additionally overexpressed.
- PEP is an activated precursor of the glycolysis product pyruvate (pyruvic acid) ;
- Ery4P is an intermediate of the pentose phosphate pathway. - 3 -
- transketolase gene tktA resulted in an increased formation of the intermediate DAHP being observed (Flores et al . , nature Biotechnology 14 (1996) 620- 623) .
- German patent applications which have the reference numbers DE 196 44 566.3 and DE 196 44 567.1 and which have not yet been published, the applicants demonstrated that it was possible to produce phenylalanine, for example, in increased quantity by increasing the enzyme activities of transaldolase or transaldolase and transketolase in Escherichia coli or by increasing the activity of a glucokinase in Escherichia coli or of a glucokinase and a PEP- independent transport system for sugars in Escherichia coli or by combining the cited enzymes and the - 4 -
- the object of the invention is to make available a further process by which an improved microbial synthesis of substances from aromatic metabolism is achieved.
- a microorganism in which the formation of substances from aromatic metabolism is increased is also to be constructed.
- the object is achieved by glucose or glucose-containing substrates being transformed, in a microorganism producing substances from aromatic metabolism, by way of increasing the activity of a glucose-oxidizing enzyme.
- This pathway comprises oxidation of the free glucose to gluconolactone/gluconate, and the phosphorylation of the gluconate to form 6-phosphogluconate .
- substances from aromatic metabolism are understood as being all compounds whose biochemical synthesis is favoured by the increased provision of Ery4P or Ery4P and PEP.
- aromatic amino acids indigo, indoleacetic acid, adipic acid, melanin, shikimic acid, chorismic acid, quinone and benzoic acid, and also their potential derivatives and secondary products.
- Microorganisms producing substances from aromatic metabolism can metabolize glucose or glucose- containing substrates, i.e. glucose-containing disaccharides or oligosaccharides , in a variety of ways: thus, it is known that glucose is phosphorylated by ATP-dependent kinases (hexokinase and glucokinases) and thereby funnelled into glycolysis. In addition, many bacteria have available a PEP-dependent system for taking up glucose and phosphorylating it.
- ATP-dependent kinases hexokinase and glucokinases
- Glucose can also be oxidized by various soluble or membrane-bound enzymes (to gluconic acid by way of gluconolactone) . These enzymes include glucose oxidases or glucose dehydrogenases . Glucose oxidases oxidize glucose to gluconolactone with the reduction of molecular oxygen. While glucose dehydrogenases also oxidize glucose to gluconolactone, they use other electron acceptors such as pyrroloquinoline quinone (PQQ) or other cofactors such as nicotine adenine dinucleotide (NAD) or NADP . It is known that membrane- bound glucose dehydrogenases can oxidize glucose using the cofactor pyrroloquinolone quinone (PQQ) , with the reaction taking place on the outer side of the membrane. In order to take up the product
- Glucose dehydrogenases using the cofactor NAD or NADP are soluble enzymes which are found within the cell.
- Known producers include Bacillus strains, some of which possess several isoenzymes of glucose dehydrogenase (e.g. glucose dehydrogenases I to IV in the case of Bacillus megaterium; Mitamura et al . , 1990, Journal of Fermentation and Bioengineering 70, 363- 369) .
- glucose dehydrogenases are strictly regulated and is known to occur, for example, only in prespore stages during the formation of endospores by Bacillus species; the physiological role of glucose dehydrogenase in association with growth on glucose is unclear, as has been reported by Lampel et al . , Journal of Bacteriology 166 (1986) 238-243, and recently by Steinmetz or Fortnagel ⁇ "Bacillus subtilis and other Gram-positive Bacteria" (Sonenshein, Hoch and Losick, eds . ) , M. Steinmetz pp. 157-170 and P. Fortnagel pp. 171-180; ISBN 1-55581-053-5; ASM Press, Washington, D.C., 1993).
- NAD (P) -dependent glucose dehydrogenases have so far been described in Escherichia coli , Corynebacteria or Brevibacteria, inter alia.
- Bacillus strain-derived genes for glucose dehydrogenase (s) have been cloned into Escherichia coli and expressed in this bacterium (Hilt et al . , Biochimica et Biophysica Acta 1076 (1991) 298-304) .
- the aim was, in particular, to obtain recombinant glucose dehydrogenase, which was employed as a detection system for glucose or for cofactor regeneration (Hilt et al .
- the glucose dehydrogenase derives from Bacillus megaterium, in particular the Bacillus znecjateriux ⁇ glucose dehydrogenase IV, as described by Mitamura et al . , Journal of Fermentation and Bioengineering 70 (1990) 363-369, and Nagao et al . , Journal of Bacteriology 15 (1992) 5013-5020.
- Gluconic acid relies on a gluconic acid- phosphorylating enzyme for its activation. It is known that such enzymes can, for example, be phosphoenolpyruvate-dependent enzymes II which have a specificity for gluconic acid, or ATP-dependent kinases having a specificity for gluconic acid.
- the Escherichia coli ATP-dependent gluconate kinase GntK as described by Izu et al., FEBS Letters 394 (1996) 14-16; Izu et al . Journal of Molecular Biology 267 (1997) 778-793, and Tong et al . , Journal of Bacteriology 178 (1996) 3260- 3269 is known, for example.
- the product, 6- phosphogluconate is an intermediate of both the oxidative branch of the pentose phosphate pathway and of the Entner-Douderoff pathway, as described by Fraenkel , pp. 189-198 in "Escherichia coli and Salmonella” , 2nd Edition (Neidhardt et al . , Eds.), ASM
- the production of substances can be improved by additionally increasing the activity of a gluconic acid (gluconate) -phosphorylating enzyme.
- gluconic acid-phosphorylating enzymes for example, from a variety of microorganisms are suitable, provided that they can be expressed functionally in the microorganisms producing substances from aromatic metabolism.
- an ATP-dependent gluconate kinase preferably an Escherichia coli gluconate kinase, in particular the gluconate kinase (GntK) from Escherichia coli K-12, is particularly advisable.
- genes for gluconic acid-phosphorylating enzymes whose gene products phosphorylate gluconic acid, are just as suitable for the process according to the invention.
- Genes from other enterobacteria, Zymomonas mobilis , Bacillus subtilis and Corynebacterium glutamicum may be mentioned by way of example.
- the effect of gluconate kinase is restricted to activating gluconic acid/gluconate, which is not, however, metabolized by Escherichia coli or other bacteria in the presence of glucose, for example.
- the gluconate kinase GntK is only of importance when the organism is growing on gluconic acid and does not contribute to the metabolism of glucose; indeed its formation is actually repressed in the presence of glucose, as described by Izu et al . , Journal of Molecular Biology 267 (1997) 778-793, and Tong et al . , Journal of Bacteriology 178 (1996) 3260-3269, and does not take place in the presence of glucose.
- the additional, increased activity of a gluconic acid- phosphorylating enzyme in particular a gluconate kinase, best of all an Escherichia coli gluconate kinase, and, in particular, the gluconate kinase GntK from Escherichia coli K-12, provides an enzyme which enables a gluconic acid-phosphorylating enzyme to be made available in microorganisms in the absence of 9 -
- Gluconolactone is a reaction product of the glucose-oxidizing enzyme. While gluconolactone can convert spontaneously into gluconic acid, enzymes have also been described which catalytically accelerate this conversion ⁇ Zymomonas mobilis gluconolactonase, for example, as described by Kanagasundaram and Scopes,
- the gene for a gluconolactonase (e.g. from Zymomonas mobilis) is expressed, in addition to the glucose-oxidizing enzyme or in addition to the glucose-oxidizing enzyme and the gluconic acid-phosphorylating enzyme, in order to accelerate the conversion of gluconolactone to gluconic acid (or to 6-P-gluconate, respectively) .
- genes for glucose dehydrogenase and gluconate kinase can occur naturally in some Bacillus species; however, the genes for the enzymes are arranged in different operons and are evidently not used jointly for metabolizing glucose, as described by Steinmetz or Fortnagel ⁇ "Bacillus subtilis and other Gram-positive bacteria" (Sonenshein, Hoch and Losick, Eds.), Steinmetz pp. 157-170 and Fortnagel pp. 171-180; ISBN 1-55581-053-5; ASM Press, Washington, D.C., 1993).
- Steinmetz or Fortnagel ⁇ “Bacillus subtilis and other Gram-positive bacteria” (Sonenshein, Hoch and Losick, Eds.), Steinmetz pp. 157-170 and Fortnagel pp. 171-180; ISBN 1-55581-053-5; ASM Press, Washington, D.C., 1993).
- the activity of a transport protein for the PEP-independent uptake of a sugar is increased in addition to increasing the glucose-oxidizing enzyme or the glucose-oxidizing enzyme and the gluconic acid- phosphorylating enzyme.
- This embodiment also includes increasing the activity of a transport protein for the PEP- independent uptake of glucose or of glucose-containing substrates in a microorganism producing substances from aromatic metabolism which is able to take up the sugar by means of a PEP-dependent transport system.
- a PEP-independent transport system makes it possible to increase the provision of the sugar in the microorganism producing said substances.
- this sugar can be converted by an intracellular glucose-oxidizing enzyme into gluconolactone and subsequently gluconic acid. Gluconic acid is then the substrate for a gluconic acid-phosphorylating enzyme.
- PEP is not required as an energy donor for these reactions and is therefore available, on the basis of a constant flow of material in glycolysis and the pentose phosphate pathway, in greater quantity for the condensation with Ery4P to form the primary metabolite of the common - 11 -
- DAHP 3-deoxy-D-arabino-heptulosonate-7-phosphate
- activity is understood as being the protein-mediated uptake rate.
- transport proteins in particular a facilitator, that is a transport protein which acts in accordance with the principle of protein-mediated facilitated diffusion.
- the use of the glucose facilitator protein (Glf) from Zymomonas mobilis is particularly suitable.
- the gene, i.e. glf, encoding the protein is derived, for example, from Z . mobilis, in particular the facilitator gene glf isolated from Z. mobilis ATCC 31821, as described by Parker et al . , 1995, Molecular Microbiology 15 (1995) 795-802, and Weisser et al .
- genes for sugar transport systems such as HXT1 to HXT7 , which are derived from eukaryotic microorganisms such as Saccharomyces cerevisiae, Pichia stipi tis or Kluyveromyces lactis , or, quite generally, of sugar transport genes derived from other organisms provided that they can be expressed functionally in the microorganisms and that, at the same time, the gene products can operate without PEP for phosphorylating and/or transporting the - 12 -
- sugar transport genes can be expressed in amino acid producers .
- measures for increasing activity are to be understood as being all measures which are suitable for increasing the activity of a glucose-oxidizing enzyme or the activity of a glucose-oxidizing enzyme and, in addition, at least one activity from gluconic acid- phosphorylating enzyme, gluconolactonase and transport protein for the PEP-independent uptake of sugar.
- the following are particularly suitable for this purpose: introducing genes, for example using vectors or temperate phages; - increasing the gene copy number, for example using plasmids, with the aim of introducing the genes according to the invention into the microorganism in increased copy number, from slightly (e.g. 2 to 5 times) to greatly increased copy number (e.g.
- increasing gene expression for example by increasing the transcription rate, for example by using promoter elements such as Ptac, Ptet or other regulatory nucleotide sequences and/or by increasing the translation rate, for example by using a consensus ribosome binding site; increasing the endogenous activity of existing enzymes, for example by mutations which are generated in an undirected manner in accordance with classical methods, for example by UV irradiation or mutation-producing chemicals, or by mutations which are generated specifically by means of recombinant DNA methods such as deletion(s), insertion(s) and/or nucleotide exchange ( s ) ; - 13 -
- the endogenous activity can be increased, for example, by cloning the gene using abovementioned methods, for example, or by way of selecting mutants which exhibit an increased transport of substrates.
- the increase in activity is effected by the gene or the genes being integrated into a gene structure or into several gene structures, with the gene or the genes being introduced into the gene structure as single copies or in increased copy number.
- gene structure is to be understood as being a gene or any nucleotide sequence which carries the genes according to the invention.
- Appropriate nucleotide sequences can, for example, be plasmids, vectors, chromosomes, phages or other nucleotide sequences which are not closed in a circular manner.
- PEP for producing the first intermediate of aromatic amino acid metabolism
- PPS sugar phosphotransferase system
- the activity of the PTS can also be influenced by adding inducers or inhibitors of the relevant promoter during culture.
- microorganisms in which one or more enzymes, which are additionally involved in the synthesis of these substances, are deregulated and/or have had their activity increased.
- enzymes are, in particular, the enzymes of aromatic amino acid metabolism and especially DAHP synthase, shikimate kinase and chorismate mutase/prephenate dehydratase, and also all other enzymes, in particular transaldolase, transketolase and glucokinase as well, which are involved in the synthesis of substances from aromatic metabolism.
- DAHP synthase which is of importance for preparing substances such as adipic acid, bile acid - 15 -
- shikimate kinase should be deregulated and have its activity increased in order to achieve excess synthesis of, for example, L-tryptophan, L-tyrosine, indigo, and derivatives of hydroxy- and aminobenzoic acid and naphtho- and anthraquinones , and their secondary products.
- a deregulated and overexpressed chorismate mutase/prephenate dehydratase is additionally of particular importance for efficient production of phenylalanine and phenylpyruvic acid and their derivatives. However, this is also intended to encompass all other enzymes whose activities contribute to the biochemical synthesis of substances whose production is promoted by the provision of Ery4P or Ery4P and PEP.
- the process according to the invention is suitable for preparing aromatic amino acids, in particular L-phenylalanine.
- L-phenylalanine preference is given to increasing, at the same time, the gene expression and/or enzyme activity of a deregulated DAHP synthase (e.g. in E. coli AroF or AroH) and/or a likewise deregulated chorismate mutase/prephenate dehydratase (PheA) .
- a deregulated DAHP synthase e.g. in E. coli AroF or AroH
- PheA deregulated chorismate mutase/prephenate dehydratase
- Suitable production organisms are Escherichia species and also microorganisms of the genera Serratia, Bacillus, Corynebacterium or Brevibacterium, and other strains known from classical amino acid methods. Likewise bacteria from the families 16
- Nocardiaceae and Actinomycetales Nocardiaceae and Actinomycetales .
- Escherichia coli is particularly suitable.
- the invention also relates to the provision of suitable gene structures and transformed cells which carry these gene structures and which make it possible to implement the process particularly successfully.
- novel gene structures are now made available, which gene structures, in recombinant form, either contain a gene which encodes a glucose-oxidizing enzyme a) together with a gene which encodes a gluconic acid- phosphorylating enzyme or b) together with a gene which encodes a transport protein for the PEP-independent uptake of a sugar or c) together with at least two of the three following genes encoding a gluconic acid- phosphorylating enzyme, a gluconolactonase or a transport protein for the PEP-independent uptake of a sugar.
- the gene for the glucose- oxidizing enzyme encodes a glucose dehydrogenase and the gene for the gluconic acid-phosphorylating enzyme encodes a gluconate kinase.
- the gene for the glucose dehydrogenase is preferably derived from Bacillus megaterium, while the gene for the gluconate kinase is preferably derived from Escherichia coli and the gene for gluconolactonase and the transport protein are preferably derived from Zymomonas mobilis.
- the transformation of the cells are effected in accordance with current methods:
- the polymerase chain reaction (PCR) method for specifically amplifying the gene is, for example, suitable using chromosomal DNA from Escherichia coli K-12 ⁇ gntK) , Bacillus megaterium ⁇ gdhlV) and the Zymomonas mobilis strains ATCC 29191 or ATCC 31821 ⁇ gnl , glf) , respectively.
- the host cell After amplifying the DNA and recombining it in vitro with known vectors (pGEM7, pUCBM20, pUC19 or others) , the host cell is transformed by means of chemical methods, electroporation, transduction or conjugation.
- known vectors pGEM7, pUCBM20, pUC19 or others
- the complete nucleotide sequences of the gntK, gdhlV, gnl and glf genes from the 3 donor organisms are known and obtainable from generally accessible sources, for example deposited in databases such as in the EMBL/HUSAR in Heidelberg under access numbers D 84362 ⁇ gntK) , D 10626 ⁇ gdhlV) , X 67189 ⁇ gnl) and M 60615 ⁇ glf) .
- the isolated glucose dehydrogenase IV gene can be integrated, together with one or more of the genes described within the context of the invention, in any combination, into a gene structure or into several gene structures. Without considering the precise allocation to gene structures, this leads to combinations such as gdhlV + gntK, gdhlV + glf, gdhlV + gntK + glf; gdhlV + gntK + gnl ; gdhlV + gnl + glf; gdhlV + gntK + gnl + glf .
- any gene structure which additionally includes one or more of the genes encoding for transketolase, transaldolase, glucokinase, DAHP- synthase, chorismate mutase / prephenate dehydratase, chorismate mutase / prephenate dehydrogenase, or for other enzymes positively influencing the synthesis of substances from aromatic metabolism.
- Gene structures which contain at least one regulatory gene sequence, which is assigned to one of the genes, are advantageous.
- regulatory elements can preferably be reinforced at the transcriptional level by, in particular, reinforcing the transcription signals. This can be effected, for example, by increasing the activity of the promoter or the promoters by altering the promoter sequences which are located upstream of the structure genes, or by completely replacing the promoters with more active promoters. Transcription can also be reinforced by appropriately influencing a regulatory gene which is assigned to the genes; in addition to that, however, it is also possible to reinforce the translation by, for example, improving the stability of - 19
- mRNA messenger RNA
- transformed cells which harbour a gene structure according to the invention in replicatable form are also made available.
- a transformed cell is to be understood as being any microorganism which carries a gene structure according to the invention which brings about the increased formation in the cell of substances from aromatic metabolism.
- the host cells can be transformed by chemical methods (Hanahan J. Mol . Biol. 166 (1983) 557-580) , and also by electroporation, conjugation or transduction.
- a microorganism strain in particular Escherichia coli , which according to the invention produces an aromatic amino acid or another substance from aromatic metabolism is transformed with the gene structure which contains the relevant genes.
- transformed cells are made available which are able to produce an aromatic amino acid, with the aromatic amino acid preferably being
- a process for microbially preparing substances from aromatic metabolism is consequently made available, which process employs transformed cells, as described above, which harbour gene - 20 -
- transformed cells which, apart from Ery4P, also contain other metabolites of central metabolism in increased availability.
- these metabolites are ⁇ -oxoglutarate or oxaloacetate , which result from intracellular synthetic processes, or else are made available to the growing cells by feeding in the corresponding compounds, or their precursors, such as fumarate or malate, as metabolites of the citric acid cycle .
- the strain Escherichia coli AT247l/pGEM7gntKgdhIV was deposited in the DSMZ (German Collection of Microorganisms and Cell Cultures) on
- the host organism employed i.e. AT2471, was deposited by Taylor and Trotter (Bacteriol. Rev. 13 (1967) 332-53) in the CGSC under number 4510 and can be obtained without payment .
- E. coli strains were, unless otherwise mentioned, cultured on LB medium consisting of Difco Bacto tryptone (10 g-1 "1 ), Difco yeast extract (5 g-1 "1 ) and NaCl (10 g-1 "1 ) .
- ampicillin 100 mg-1 "1
- chloramphenicol 17.34 mg-1 "1
- ampicillin was 21 -
- Plasmid DNA from E. coli was isolated by means of alkaline lysis using a commercially available system (Qiagen, Hilden) . Chromosomal DNA was isolated from E. coli and Bacillus megaterium DSM 319 using the method of Chen and Kuo (Nucl . Acid Res. 21 (1993)
- the cells were centrifuged down and taken up in a tenth of the volume of TSS (LB medium containing 10% (w/v) PEG 8000, 5% (v/v) DMSO and 50 mM MgCl 2 ) . After having been incubated for 30 minutes at 4°C with from 0.1 to 100 ng of DNA and subsequently incubated at 37°C for 1 h, the cells were plated out on LB medium containing the appropriate antibiotic.
- TSS LB medium containing 10% (w/v) PEG 8000, 5% (v/v) DMSO and 50 mM MgCl 2
- PCR polymerase chain reaction
- Primer 2 (Sad) consisted of 5' GCC AGA GCT CTT TTT TCC ACA TCG ATT AAA AAC TAT 3 ' , and was complementary to the 3 ' end of the gdhlV gene.
- the resulting DNA amplification product of approx. 800 base pairs, was restricted with BamHI plus Sad and then ligated into the vector pGEM7 , which had been treated in the same way (see Tab. 1) . Transformation was effected into the strain JM109DE3, with selection on LB agar plates containing X-Gal and ampicillin. Successful cloning was detected by determining the DNA sequence of the cloned gdhlV gene.
- This vector enabled glucose dehydrogenase IV activity to be expressed even in the absence of the T7 polymerase system (strain JM109DE3) (see Tab. 2) .
- the gntK gene for the Escherichia coli K-12 gluconate kinase was cloned by specific DNA amplification using the strain E. coli K-12 W3110 as the chromosomal template. The sequence of the gntK gene has been described by Tong et al . Journal of Bacteriology 178 (1966) 3260-3269.
- oligonucleotide primers were selected which were additionally provided with restriction cleavage sites for EcoRI (5') and BamHI (3') .
- Primer 1 consisted of 5 ' CCG AAT TCT TGT ATT GTG GGG GCA C 3 ' and binds 5 ' upstream of the gntK gene;
- primer 2 consisted of 5' CCG GAT CCG TTA ATG TAG TCA CTA CTT A 3' and is complementary to the 3' end of the gntK gene.
- amplification product of approx. 600 base pairs was purified, restricted with EcoRI plus BamHI and ligated into vector pGEM7 , which had been opened as well. Transformation was effected into the strain JM109DE3, with selection on LB agar plates containing X-Gal and ampicillin. Successful cloning was detected by determining the DNA sequence of the cloned gntK gene.
- This vector (pGEM7gntK) enabled the gluconate kinase activity to be expressed even in the absence of the T7 polymerase system (strain JM109DE3) (see Tab. 2) .
- the gntK and gdhlV genes were combined by opening vector pGEM7gntK by subjecting it to double restriction with BamHI plus Sad .
- the new gene structure pGEM7gntKgdhIV in accordance with the invention, mediates T7 polymerase- independent expression of the enzyme activities for glucose dehydrogenase IV and for gluconate kinase GntK (see Tab. 2) .
- the transformants which had been obtained were stored on LB medium in the form of glycerol cultures (30%) at -80°C. When required, the glycerol cultures were thawed directly before use.
- the E . coli cells, and the cells of plasmid-harbouring mutants were cultured in mineral medium.
- This medium consisted of sodium citrate-3H 2 0 (1.0 g-1 "1 ), MgS0 4 • 7H 2 0 (0.3 g-1 "1 ), KH 2 P0 4 - 24
- Vitamin Bl (5.0 mg-1 "1 ) was dissolved in water and was added, after having been sterilized by filtration, to the medium after the latter had been autoclaved, just as were ampicillin and/or ampicillin and chloramphenicol when required.
- Glucose (30 g-1 "1 ) was autoclaved separately and likewise added to the medium after the latter had been autoclaved.
- the harvested cells were washed in 100 mM tris/HCl buffer (pH 8.0).
- the cells of the sediment were disrupted by ultrasound (Branson sonifier 250 fitted with a microtip) , in a sonication cycle of 25% and with an intensity of 40 watts, for 4 min per ml of cell suspension. After centrifuging for 30 min at 18,000 g and 4°C, the supernatant (crude extract) was used for measuring the activity of the glucose dehydrogenase and/or gluconate kinase.
- the activity of the glucose dehydrogenase was determined in accordance with Harwood & Cutting, Molecular Biological Methods for Bacillus, John Wiley & sons. Glucose dehydrogenase catalyzes the oxidation of glucose to gluconolactone. The activity of the enzyme was determined photometrically at a wavelength of 340 nm by means of the increase in the concentration of the reduced cofactor NADH + H + . The determination was performed in quartz cuvettes having a total volume of 1 ml . The reaction mixture consisted of Tris HC1 (final - 25
- the gluconate kinase was determined in the crude extract as described by Izu et al . , FEBS Letters 394 (1996) 14-16. Gluconate kinase catalyzes the ATP- dependent phosphorylation of gluconate to 6- phosphogluconate .
- the 6- phosphogluconate formed is determined photometrically at a wavelength of 340 nm by means of the increase in NADPH concentration when using the NADP-dependent auxiliary enzyme 6-phosphogluconate dehydrogenase (Boehringer Mannheim, No. 108 405) .
- the formation of 1 ⁇ mol of NADPH corresponds to the phosphorylation of 1 ⁇ mol of gluconate.
- the enzymic detection was carried out at 25 °C in quartz cuvettes having a total volume of 1 ml.
- the reaction mixture contained 50 mM Tris HC1, pH 8.0, 100 mM ATP, 0.25 mM NADP, 1.2 units of the auxiliary enzyme 6-phosphogluconate dehydrogenase and variable quantities of crude extract.
- the mixtures were preincubated at 25 °C for 5 minutes, and the reaction was started by adding gluconic acid (pH 6.8; final concentration in the mixture, 10 mM) . Mixtures to which gluconic acid was not added served as the controls.
- the protein concentration in the crude 26 The reaction mixture contained 50 mM Tris HC1, pH 8.0, 100 mM ATP, 0.25 mM NADP, 1.2 units of the auxiliary enzyme 6-phosphogluconate dehydrogenase and variable quantities of crude extract.
- the mixtures were preincubated at 25 °C for 5 minutes, and the reaction was started by adding gluconic
- the synthetic efficiency of Escherichia coli AT2471 and Escherichia coli AT247l/pGEM7ghdIV was determined in the mineral medium described in Example II. For this, shaking flasks (1000 ml containing 100 ml of medium) were inoculated with 2 ml of glycerol culture and incubated on an orbital shaker at 37 °C and 150 rpm for 72 h. The pH of the cultures was measured at intervals of approximately 12 h and, as required, restored to the starting value of 7.2 by adding KOH
- the concentration of phenylalanine was ascertained by means of high pressure liquid chromatography (HPLC, Hewlett Packard, Kunststoff, Germany) in combination with detection by fluorescence (extinction 335 nm, emission 570 nm) , A nucleosil-120-8 C18 column (250-4.6 mm) was used as the solid phase,- the elution was performed using a gradient (eluent A: 27
- the Zymomonas mobilis glf gene was amplified using the plasmid pZY600 (Weisser et al . , J. Bacteriol 177 (1995) 3351-3345) as the template. At the 28 -
- the choice of the primers resulted in a BamHI cleavage site and a Kpnl cleavage site being introduced.
- the gene was inserted into the vector pUCBM20 (Boehringer Mannheim) , which had likewise been opened with BamHI and Kpnl.
- a DNA fragment of 1.5 kb in size was isolated from this vector (pBM20glf) by restricting it with BamHI and Hindlll and ligated to the vector plasmid pZY507 (Weisser et al . , J. Bacteriol 177 (1995) 3351- 3345) , which had likewise been opened with the restriction enzymes BamHI and Hindlll.
- the recombinant plasmid pZY507glf was obtained after transforming E. coli and cloning the transformants .
- This vector confers resistance to chloramphenicol , contains the lacl q -tac promoter system and has a low copy number.
- Vector pZY507glf was transformed into the host strain AT2471 together with the gene structures of the invention obtained as described in Example I . Following the experimental conditions described for Example III, the mutants E. coli
- AT2471glf/pGEM7gntKgdhIV were in each case cultured in two parallel mixtures. After 48 h, the concentration of L-phenylalanine in the medium was determined.
- glucose dehydrogenase and gluconate kinase in E. coli AT2471glf/pGEMgntKgdhlV, made it possible to achieve a further increase, to a phenylalanine concentration-representing index value of 195.
- the plasmid pPTSl was digested with Bgll l and treated with Klenow fragment .
- the unique cleavage site lies in the ptsl gene.
- the glf gene was isolated as a BamHI /Kpnl fragment from the plasmid pBM20glfglk and likewise treated with Klenow fragment. Clones which carry the glf gene in the same orientation as the ptsHI genes were obtained by blunt end ligation.
- the vector moiety can be recombined out in a second homologous crossover, leading to loss of the resistance to carbenicillin. Since the pts genes are interrupted by the insertion of the glf gene in this case, the PTS is not functionally expressed in these mutants.
- the desired PTS " mutants were selected as follows: After repeatedly subinoculating the still PTS + transformants onto LB medium without antibiotics, aliquots of the cell suspension were plated out on LB plates containing 100 ⁇ g-1 "1 phosphomycin. PTS " mutants are able to grow on these plates . Growing clones were streaked out on LB plates containing either phosphomycin or 20 ⁇ g-1 "1 carbenicillin.
- the mutant AT2471glfintPTS " /pGEM7gntKgdhIV achieved an integral biomass-specific productivity which had an index value of 133.
- JM109DE3 A (pro- lac) /F'pro + Promega Co . lacZ ⁇ M15; carries gene for T7 RNA polymerase
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Application Number | Priority Date | Filing Date | Title |
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DE19818541A DE19818541C2 (en) | 1998-04-24 | 1998-04-24 | Microbial production of substances from the aromatic metabolism / III |
DE19818541 | 1998-04-24 | ||
PCT/NL1999/000232 WO1999055877A1 (en) | 1998-04-24 | 1999-04-22 | Microbial preparation of substances from aromatic metabolism/iii |
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EP1090126A1 true EP1090126A1 (en) | 2001-04-11 |
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EP99916080A Withdrawn EP1090126A1 (en) | 1998-04-24 | 1999-04-22 | Microbial preparation of substances from aromatic metabolism/iii |
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EP (1) | EP1090126A1 (en) |
JP (1) | JP2002512802A (en) |
KR (1) | KR100567120B1 (en) |
CN (1) | CN1289675C (en) |
AU (1) | AU3445699A (en) |
CA (1) | CA2328598A1 (en) |
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DE19958159A1 (en) | 1999-12-02 | 2001-06-07 | Degussa | New nucleotide sequences coding for the glk gene |
DE10047403A1 (en) * | 2000-09-26 | 2002-04-11 | Degussa | New nucleotide sequences coding for the ppgK gene |
AU2002314738A1 (en) | 2001-04-04 | 2002-10-21 | Genencor International, Inc. | Methods for the production of products in host cells |
EP2055773B1 (en) * | 2001-04-04 | 2011-06-15 | Danisco US Inc. | Uncoupled productive and catabolic host cell pathways |
KR100433134B1 (en) * | 2002-03-05 | 2004-05-27 | 김병기 | Novel thermophilic microorganism and methods for producing l-type aromatic amino acids by using the same |
US7572607B2 (en) | 2002-04-23 | 2009-08-11 | Cargill, Incorporated | Polypeptides and biosynthetic pathways for the production of monatin and its precursors |
US8372989B2 (en) | 2002-04-23 | 2013-02-12 | Cargill, Incorporated | Polypeptides and biosynthetic pathways for the production of monatin and its precursors |
CN1890377B (en) | 2003-10-21 | 2013-06-05 | 嘉吉有限公司 | Production of monatin and monatin precursors |
RU2004105179A (en) * | 2004-02-25 | 2005-08-10 | Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО АГРИ) (RU) | 6-PHOSPHOGLUCONOLACTONASE FROM ESCHERICHIA COLI, DNA FRAGMENT, BACTERIA BELONGING TO THE GENUS ESCHERICHIA, PRODUCER OF L-AMINO ACID, AND METHOD FOR PRODUCING L-AMINO ACID |
EP1718731B2 (en) * | 2004-02-25 | 2016-10-19 | Ajinomoto Co., Inc. | Microorganism expressing 6-phosphogluconolactonase and its use in the production of L-amino acids. |
US8158389B2 (en) | 2005-04-20 | 2012-04-17 | Cargill, Incorporated | Products and methods for in vivo secretion of monatin |
WO2006113897A2 (en) | 2005-04-20 | 2006-10-26 | Cargill, Incorporated | Products and methods for in vivo secretion of monatin |
US7582455B2 (en) | 2005-04-26 | 2009-09-01 | Cargill, Incorporated | Polypeptides and biosynthetic pathways for the production of stereoisomers of monatin and their precursors |
US8076108B2 (en) | 2005-04-26 | 2011-12-13 | Cargill, Incorporated | Polypeptides and biosynthetic pathways for the production of stereoisomers of monatin and their precursors |
CN102197135B (en) * | 2008-11-05 | 2014-08-13 | 三井化学株式会社 | Bacterium capable of producing 2-deoxy-scyllo-inosose (DOI), and process for producing 2-deoxy-scyllo-inosose (DOI) by using same |
KR102105532B1 (en) * | 2013-10-17 | 2020-04-29 | (주)아모레퍼시픽 | Method for derivation of inducible Pluripotent stem cells and inducible Pluripotent stem cells produced using the same |
KR102134418B1 (en) * | 2019-06-17 | 2020-07-16 | 씨제이제일제당 주식회사 | A microorganism producing L-tyrosine and a method for producing L-tyrosine using thereof |
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DE3711881A1 (en) * | 1987-04-08 | 1988-10-27 | Merck Patent Gmbh | METHOD FOR PRODUCING GLUCOSEDEHYDROGENASE FROM BACILLUS MEGATERIUM |
US5032514A (en) * | 1988-08-08 | 1991-07-16 | Genentech, Inc. | Metabolic pathway engineering to increase production of ascorbic acid intermediates |
JPH0286779A (en) * | 1988-09-22 | 1990-03-27 | Amano Pharmaceut Co Ltd | Improved type recombinant dna, transformant containing the same and production of heat-resistant glucose dehydrogenase therewith |
US5437083A (en) | 1993-05-24 | 1995-08-01 | Advanced Cardiovascular Systems, Inc. | Stent-loading mechanism |
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1998
- 1998-04-24 DE DE19818541A patent/DE19818541C2/en not_active Expired - Fee Related
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1999
- 1999-04-22 KR KR1020007011806A patent/KR100567120B1/en not_active IP Right Cessation
- 1999-04-22 EP EP99916080A patent/EP1090126A1/en not_active Withdrawn
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- 1999-04-22 AU AU34456/99A patent/AU3445699A/en not_active Abandoned
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AU3445699A (en) | 1999-11-16 |
CN1289675C (en) | 2006-12-13 |
KR20010042974A (en) | 2001-05-25 |
CN1298448A (en) | 2001-06-06 |
WO1999055877A1 (en) | 1999-11-04 |
CA2328598A1 (en) | 1999-11-04 |
JP2002512802A (en) | 2002-05-08 |
DE19818541A1 (en) | 1999-11-11 |
KR100567120B1 (en) | 2006-03-31 |
DE19818541C2 (en) | 2003-04-10 |
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