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
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
• • 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.)

Definitions

• the invention relates to a process for the fermentative production of 2,3-butanediol (2,3-BDL) by means of an improved microorganism strain having a 2 to 12.8 times increased acetolactate decarboxylase activity compared to the non-improved starting strain.
• a C4 building block accessible by fermentation is 2,3-butanediol.
• the state of the art for fermentative 2,3-butanediol production is summarized in Celinska and Grajek (Biotechnol. Advances (2009) 27: 715-725).
• 2, 3 Butanediol is a possible starting material for petrochemical products with four carbon atoms (C4 building blocks) such as acetoin, diacetyl, 1,3-butadiene, 2-butanone (methyl ethyl ketone, MEK).
• products with two C atoms (C2 units) such as acetic acid (DE 102010001399) and derived therefrom, acetaldehyde, ethanol and also ethylene are available.
• dimerization of 2, 3-butanediol and C8 compounds are conceivable, the use, for example, as a fuel in the aviation sector.
• acetoin reductase (2, 3-butanediol dehydrogenase): NADH-dependent reduction of acetoin ⁇ . 2, 3-butanediol.
• 2,3-butanediol Various natural producers of 2,3-butanediol are known, e.g. B. from the genera Klebsiella, Raoultella, Enterobacter, Aerobacter, Aeromonas, Serratia, Bacillus, Paenibacillus, Lactobacillus, Lactococcus etc.
• yeasts are known as producers (eg bakers yeast) .Most bacterial producers are microorganisms of biological safety.
• stage S2 can not be used on a large scale without elaborate and expensive technical safety measures (for this and for other microbial 2, 3-butanediol producers, see the review by Celinska and Grajek, Biotechnol Advances (2009) 27: 715-725)
• 3-BDL production strains from the biological safety level Sl are strains of the species Klebsiella terrigena, Klebsiella planticola, strains of the genus Bacillus (resp.
• Paenibacillus such as Bacillus polymyxa or Bacillus licheniformis.
• the species Klebsiella terrigena and Klebsiella planticola are also synonymously referred to as Raoultella terrigena and Raoultella planticola as a result of a taxonomic renaming , These are strains of the same species.
• 2,3-butanediol yields of not more than 57 g / l (637 mM, production time 60 h) have so far been reported for these strains classified in safety level Sl (Nakashimada et al., J. Bioscience and Bioengineering (2000) 90: 661- These yields are far too low for economic production, and the minimum fermentation yield for economical production is> 80 g / l 2, 3-butanediol (fermentation time maximum 72 h), preferably> 100 g / l 2, 3-butanediol viewed.
• biosafety level and the division into different security levels can be found for example in "Biosafety in Microbiological and Bio- medical Laboratorie n, p 9ff. (Table 1), Centers for Disease Control and Prevention (US) ( Editor), Public Health Service (US) (Editor), National Institutes of Health (Editor),
• acetolactate decarboxylase ALD
• Klebsiella terrigena and Enterobacter aerogenes in brewing yeast The heterologous expression of acetolactate decarboxylase (ALD) genes from Klebsiella terrigena and Enterobacter aerogenes in brewing yeast is described in Blomqvist et al. , Ap l. Environ. Microbiol. (1991) 57: 2796-2803. The aim of these investigations, however, was not the production of 2, 3-butanediol, but the reduction of occurring during brewing as tasting by-product diacetyl.
• ALD acetolactate decarboxylase
• Diacetyl by-produced in brewing yeast from acetolactate, is degraded during beer maturation, and increased ALD expression is expected to reduce intracellular levels of acetolactate and prevent the formation of diacetyl by directing acetolactate directly to aceto-2,3 ' Butanediol precursor is decarboxylated.
• the object of the invention was to provide production strains for the production of 2,3-butanediol, which enable significantly higher 2,3-butanediol yields than the parent strain.
• the problem was solved by a production strain which can be produced from an initial strain, characterized in that the production strain has a minimum of 2 to 12.8-fold over acetolactate decarboxylase activity exhibited by the parent strain.
• the parent strain may be a wild-type strain that is not further optimized but capable of generating 2,3-butanediol or an already optimized wild-type strain. However, in an already further optimized wild type strain (e.g., by genetic engineering), the acetolactate decarboxylase activity is not affected by the optimization.
• a production strain is to be understood as meaning a starting strain which has been optimized with respect to the production of 2,3-butanediol and which is characterized by an increased activity of the enzyme acetolactate decarboxylase (ALD) in comparison to the starting strain.
• ALD acetolactate decarboxylase
• the production strain is made from the parent strain. If an already optimized starting stamina is to be improved even further by an increase in the acetolactate decarboxylase activity, then it is of course also possible first to increase the acetolactate decarboxylase activity in an unimpaired strain and then to introduce further improvements.
• the increase in acetolactate decarboxylase activity in the production strain can be caused by any mutation in the genome of the parent strain (eg a promoter activity-enhancing mutation), an enzyme activity-enhancing mutation in the acetolactate decarboxylase gene or by expression of a homologous or heterologous acetolactate decarboxylase gene in the starting strain.
• the overexpression of a homologous or heterologous acetolactate decarboxylase gene in the parent strain is preferably, the overexpression of a homologous or heterologous acetolactate decarboxylase gene in the parent strain.
• the acetolactate decarboxylase activity is increased by a factor of 2 to 10 and more preferably by a factor of 2 to 5.
• This increased acetolactate decarboxylase activity is particularly preferably achieved by increased expression of a homologous or heterologous gene coding for an acetolactate-decarboxylase enzyme compared with the starting strain.
• the parent strain may be any 2,3-butanediol-producing strain. It is preferably a strain of the genus Klebsiella (Raoultella) or Bacillus (Paenibacillus) or Lactobacillus.
• a strain of the species Klebsiella (Raoultella) terrigena, Klebsiella (Raoultella) planticola, Bacillus (Paenibacillus) polymyxa or Bacillus licheniformis with a strain of the species Klebsiella (Raoultella) terrigena or Klebsiella (Raoultella) planticola is again preferred.
• the increased ALD activity is achieved by recombinant overexpression of an acetolactate decarboxylase (EC 4.1.1.5) in a production strain.
• an acetolactate decarboxylase EC 4.1.1.5
• overexpression of the acetolactate decarboxylase by a factor of 2 to 12.8 is suitable for the 2,3-butanediol yield (determined as the volume yield of 2,3-butanediol in g / l ) in the fermentation by more than 20%, preferably more than 30% and most preferably by more than 40% increase.
• Acetolactate decarboxylase (ALD) is an enzyme from the enzyme class EC 4.1.1.5.
• the gene of the acetolactate decarboxylase originates from a bacterium of the genus Klebsiella (Raultella) or Bacillus.
• the gene of Acetolactatdecarboxylase derived from a strain of the species Klebsiella terrigena, Klebsiella planticola, Bacillus polymyxa or Bacillus licheniformis and in particular from a strain of the species Klebsiella terrigena, Klebsiella planticola or Bacillus licheniformis.
• These strains are all commercially available, e.g. at the DSMZ German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig).
• the strain according to the invention thus makes it possible to increase the fermentative production of acetoin.
• the invention not only enables the production of 2,3-butanediol but also the production of other metabolites which can be derived from acetoin, such as 2,3-butanediol. These metabolic products include diacetyl, ethanol and acetic acid.
• a production strain according to the invention in a preferred embodiment is also characterized in that it was prepared from a starting strain as defined in the application and comprises an acetolactate decarboxylase in recombinant form.
• the production strain according to the invention is preferably prepared by introducing a gene construct into one of the said starting strains.
• the gene construct in its simplest form is defined as consisting of the acetolactate decarboxylase structural gene operatively linked to a promoter upstream.
• the gene construct may also comprise a terminator downstream of the acetolactate decarboxylase structural gene.
• Preferred is a strong promoter, which leads to a strong transcription.
• Preferred among the strong promoters is the so-called "Tac promoter" which is familiar to the person skilled in the art from the molecular biology of E. coli.
• the gene construct can be present in a manner known per se in the form of an autonomously replicating plasmid, it being possible for the copy number of the plasmid to vary.
• a variety of plasmids are known to those skilled in the art, depending on their genetic
• Structure in a given production strain can autonomously replicate.
• the gene construct can also be integrated in the genome of the production strain, with each gene locus along the genome being suitable as an integration site.
• the gene construct is introduced into the production strain in a manner known per se by genetic transformation.
• Various methods of genetic transformation are known to the person skilled in the art (Aune and Aachmann, Appl. Microbiol. nol. (2010) 85: 1301-1313), including, for example, the
• the gene construct contains, likewise in a known manner, a so-called selection marker for the selection of transformants with the desired gene construct.
• selection markers are selected from so-called antibiotic resistance markers or from the auxotrophy complementing selection markers.
• antibiotic resistance markers Preference is given to antibiotic resistance markers, more preferably those which confer resistance to antibiotics selected from ampicillin, tetracycline, kanamycin, chloramphenicol or zeocin.
• a production strain according to the invention contains the gene construct, either in plasmid form or integrated into the genome, and produces an acetolactate decarboxylase enzyme in recombinant form.
• the recombinant acetolactate decarboxylase enzyme is capable of producing acetoin with elimination of C0 2 from acetolactate.
• the parent strain may be a non-optimized wild type strain.
• the starting strain may have already been optimized beforehand, or it may be further optimized as a production strain producing acetolactate decarboxylase according to the invention.
• the optimization of a production strain according to the invention which comprises acetolactate decarboxylase according to the invention can on the one hand be achieved by mutagenesis and selection of mutants with improved production properties.
• the optimization can also be carried out by genetic engineering by additional expression of one or more genes which are suitable for improving the production properties. Examples of such genes are the already mentioned 2, 3-butanediol Biosynthetic genes acetolactate synthase and acetoin reductase.
• genes can be expressed in a manner known per se as separate gene constructs or in combination as an expression unit (as a so-called operon) in the production strain. It is known, for example, that in Klebsiella terrigena all three biosynthesis genes of 2,3-butanediol (so-called BUD-Operon, Blomqvist et al., J. Bacteriol. ⁇ 1993 ⁇ 175: 1392-1404), or in strains of the genus Bacillus genes of acetolactate synthase and acetolactate decarboxylase are organized in an operon (Renna et al., J. Bacteriol (1993) 175: 3863-3875).
• the production strain can be optimized by inactivating one or more genes whose gene products adversely affect the 2,3-butanediol production.
• genes whose gene products are responsible for by-product formation include z. B. the lactate dehydrogenase (lactic acid formation), the acetaldehyde dehydrogenase (ethanol formation) or else the phosphotransacetylase, or the acetate kinase (acetate formation).
• the invention comprises a process for the production of 2,3-butanediol with the aid of a production strain according to the invention.
• the method is characterized in that cells of a production strain according to the invention which produces acetolactate decarboxylase are cultured in a growth medium.
• biomass of the production strain and on the other hand the product 2,3-BDL are formed.
• the formation of biomass and 2,3-BDL can be time correlated or temporally decoupled from each other.
• the cultivation takes place in a manner familiar to the person skilled in the art. This can be done in shake flasks (laboratory scale) or by fermentation (production scale).
• Seed media are familiar to those skilled in the practice of microbial cultivation. They typically consist of a carbon source (C source), a nitrogen source (N source), and additives such as vitamins, salts, and trace elements that optimize cell growth and 2,3-BDL product formation.
• C sources are those that can be used by the production strain for 2,3-BDL product formation. These include all forms of monosaccharides, including C6 ⁇ sugars such as. Glucoses, mannose or fructose, and C5 sugars, e.g. Xylose, arabinose, ribose or galactose.
• the production process according to the invention also comprises all C sources in the form of disaccharides, in particular sucrose, lactose, maltose or cellobiose.
• the production process of the invention further comprises all C sources in the form of higher saccharides, glycosides or carbohydrates with more than two sugar units such.
• C sources in the form of higher saccharides, glycosides or carbohydrates with more than two sugar units such.
• the hydrolysis of the higher C sources may precede the production process according to the invention or take place in situ during the production process according to the invention.
• C sources other than sugars or carbohydrates are acetic acid (or acetate salts derived therefrom), ethanol, glycerol, citric acid (and its salts), lactic acid (and salts thereof) or pyruvate (and its salts).
• gaseous carbon sources such as carbon dioxide or carbon monoxide are also conceivable.
• the C sources which are affected by the production process according to the invention comprise both the isolated pure substances or else, for the sake of economic efficiency, not further specified.
• C sources can also be waste products from the digestion of vegetable raw materials, such as molasses (sugar beet) or bagasse (sugar cane).
• Preferred C sources for the production of the production strains are glucose, fructose, sucrose, mannose, xylose, arabinose and vegetable hydrolysates, which can be obtained from starch, lignocellulose, sugar cane or sugar beet.
• a particularly preferred C source is glucose, either in isolated form or as part of a vegetable hydrolyzate.
• N sources are those that can be used by the production strain for biomass production. These include ammonia, gaseous or in aqueous solution as NH 4 0H or else its salts such. As ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium acetate or ammonium nitrate. Furthermore, suitable N-source are the known nitrate salts such. B. KN0 3 , NaN0 3 , ammonium nitrate, Ca ⁇ N0 3 ) 2 , Mg (N0 3 ) 2 and other N sources such as urea.
• the N sources also include complex amino acid mixtures such as yeast extract, proteose peptone, malt extract, soya peptone, casamino acids, corn steep liquor (corn steep liquor, liquid or dried as so-called CSD) as well as NZ amines and Yeast Nitrogen Base.
• the cultivation can take place in the so-called batch mode, wherein the culture medium is inoculated with a starter culture of the production strain and then the cell growth takes place without further feeding of nutrient sources. Cultivation can also take place in the so-called fed-batch mode, with additional nutrient sources being fed in after an initial phase of growth in batch mode (feed) in order to compensate for their consumption.
• the feed can consist of the C source, the N source, one or more important for the production of vitamins, or trace elements or a combination of the aforementioned.
• the feed components can be used together as a mixture or else separately
• Feed lines are added.
• other media components as well as specific 2,3-BDL production enhancing additives may be added to the feed.
• the feed can be fed continuously or in portions (batchwise) or else in a combination of continuous and discontinuous feed. Preference is given to the cultivation according to the fed-batch mode.
• Preferred C sources in the feed are glucose, sucrose, molasses, or vegetable hydrolysates, which can be obtained from starch, lignocellulose, sugar cane or sugar beet.
• N sources in the feed are ammonia, gaseous or in aqueous solution as NHOH and its salts aramonium sulfate, ammonium phosphate, ammonium acetate and ammonium chloride, furthermore urea, KN0 3 , NaN0 3 and ammonium nitrate, yeast extract, proteose peptone, malt extract, soya peptone, casamino acids, corn steep liquor (Com Steep Liquor) as well as NZ-Amine and Yeast Nitrogen Base.
• Particularly preferred N sources in the feed are ammonia, or ammonium salts, urea, yeast extract, soya peptone, malt extract or corn steep liquor (in liquid or in dried form).
• the cultivation takes place under pH and temperature conditions which favor the growth and the 2,3-BDL production of the production strain.
• the useful pH range is from pH 5 to pH 8.
• Preferred is a pH range of pH 5.5 to pH 7.5.
• Particularly preferred is a pH range of pH 6.0 to pH 7.
• the preferred temperature range for the growth of the production strain is 20 ° C to 40 ° C.
• Particularly preferred is the temperature range of 25 ° C to 35 ° C.
• the growth of the production strain can optionally take place without oxygen supply (anaerobic cultivation) or else with oxygen supply (aerobic cultivation) .
• oxygen supply being ensured by introduction of compressed air or pure oxygen the aerobic cultivation by entry of compressed air.
• the cultivation time for 2,3-BDL production is between 10 h and 200 h. Preferred is a cultivation period of 20 h to 120 h. Particularly preferred is a cultivation time of 30 h to 100 h.
• Cultivation batches obtained by the method described above contain the 2,3-BDL product, preferably in the culture supernatant.
• the 2,3-BDL product contained in the cultivation mixtures can either be used further directly without further work-up or else be isolated from the cultivation batch.
• known process steps are available, including centrifugation, decantation, filtration, extraction, distillation or crystallization, or precipitation. These process steps can be combined in any desired form in order to isolate the 2,3-BDL product in the desired purity. The degree of purity to be achieved depends on the further use of the 2,3-BDL product.
• FIG. 1 shows the 3.65 kb acetolactate decarboxylase epitope pBudAkt prepared in Example 1.
• Figure 2 shows the 3.64 kb acetolactate decarboxylase epitope pALDbl prepared in Example 1.
• Fig. 3 shows the plasmid pACYC184 used in Example 1.
• FIG. 4 shows the 5.1 kb acetolactate decarboxylase expression vector pBudAkt-tet prepared in Example 1.
• FIG. 5 shows the 5.1 kb acetolactate decarboxylase expansion vector pALDbl-tet prepared in Example 1.
• the acetolactate decarboxylase genes were used. terrigena and B. licheniformis.
• the DNA sequence of the acetolactate decarboxylase gene from K. terrigena is disclosed in the "GenBank” gene database under accession number L04507, bp 179-958. It was obtained in a PCR reaction (Taq DNA polymerase, Qiagen) from genomic K. terrigena DNA (strain DSM 2687, commercially available from the DSMZ German Collection of Microorganisms and Cell Cultures GmbH) with the primers BUD3 f and BUD4r as a DNA fragment of 0.78 kb size isolated.
• licheniformis is disclosed in the "GenBank” gene database under accession number NC_006270, under which the entire genome sequence of B. licheniformis is disclosed
• the acetolactate decarboxylase gene is found there in a complementary form from bp 3674476-3675237 and was isolated in a PCR reaction ( Taq DNA polymerase, Qiagen) from genomic B. licheniformis DNA (strain DSM 13, commercially available from DSMZ GmbH) with the primers BLALD-lf and BLALD-2r as a DNA fragment of 0.76 kb size isolated.
• the genomic DNA used for the PCR reactions was previously isolated in a manner known per se with a DNA isolation kit (Qi agen) from cells of the culture of K. terrigena DSM 2687 and B. licheniformis DSM 13 in LB medium (10 g / l). 1 tryptone, 5 g / 1 yeast extract, 5 g / 1 NaCl).
• the expression vector pKKj is a derivative of the expression vector pKK223-3.
• the DNA sequence of pKK223-3 is disclosed in the "GenBank” gene database under accession number M77749.1, and approximately 1.7 kb were removed from the 4.6 kb plasmid (bp 262).
• the expression vectors pBudAkt and pALDbl were modified by incorporation of an expression cassette for the tetracycline resistance gene.
• the tetracycline resistance gene was first isolated from the plasmid pACYC184 (FIG. 3).
• the DNA sequence of pACYC184 is available in the "Genbank” gene database under accession number X06403.1.)
• PCR Transcription DNA polymerase, Qiagen
• Bgl II cleavage contained in the primers tetlf and tet2r
• the tetracycline expression cassette was isolated as a 1.45 kb fragment from pACYC184 and then cloned into the pBudAkt and pALDbl vectors respectively cut with Bam HI, resulting in the respectively 6 kb expression vectors pBudAct- tet ( Figure 4) and pALDbl-
• a clone was selected in each case, in which the Te tracyclin- and the Acetolactatdecarboxylase expression cassettes were oriented in each case in the same direction.
• Primer tetlf (SEQ ID NO: 5) and tet2r (SEQ ID NO: 6) had the following DNA sequences:
• E. coli plasmid DNA of the expression vectors pBudAkt-tet and pALDbl-tet was transformed into E. coli strain JM105 by methods known per se.
• One clone each was selected and cultured in a shake flask culture. From the E. coli strains a preculture was prepared in LBtet medium (10 g / 1 tryptone, 5 g / 1 yeast extract, 5 g / 1 NaCl, 15 pg / ml tetracycline) (cultivation at 37 ° C and 120 rpm Night).
• each preculture was inoculum of a main culture of 100 ml LBtet medium (300 ml Erlenmeyer flask) was used and the main cultures were shaken at 30 ° C. and 180 rpm until a cell density OD 600 of 2.0 was reached, then the inducer IPTG (isopropyl- ⁇ -thiogalactoside, 0.4 mM final concentration) was used. added and shaken overnight at 30 ° C and 180 rpm.
• inducer IPTG isopropyl- ⁇ -thiogalactoside, 0.4 mM final concentration
• acetolactate decarboxylase expression 50 ml of the E. coli cells were centrifuged ⁇ 10 min 15000 rpm, Sorvall centrifuge RC5C equipped with a SS34 rotor), the cell pellet in 2 ml of Pi buffer (0.1 M potassium phosphate, 0 , 1 M NaCl, pH 7.0), in a conventional manner with a so-called.
• Pi buffer 0.1 M potassium phosphate, 0 , 1 M NaCl, pH 7.0
• Acetolactatdecarboxylase activity was carried out in a conventional manner (US 4617273).
• 1 U acetolactate decarboxylase activity is defined as the amount of enzyme which produces 1 ⁇ acetoin / min under test conditions.
• Acetolactate diester mixed with 240 ⁇ H20 and 330 ⁇ IM NaOH and incubated on ice for 15 min. Subsequently, 5.4 ml H 2 O was added and the acetolactate substrate kept on ice for testing.
• the amount of acetoin formed was determined from a standard curve previously prepared with acetoin.
• the protein concentration of the cell extracts was determined in a manner known per se using the so-called “BioRad protein assay” from BioRad.
• the control strain used was the untransformed wild-type strain Klebsiella terrigena DSM 2687. Transformants were isolated and tested for acetolactate decarboxylase activity by shake flask culture. For this purpose, in each case 50 ml of FM2tet medium (without tetracycline in the K. terrigena wild-type control strain) were inoculated with a transformant and incubated for 24 h at 30 ° C. and 140 rpm (Infors shaker).
• FM2tet medium contained glucose 60 g / l; 10 g / l yeast extract (Oxoid) 2.5 g / 1; Ammonium sulfate 5 g / 1; NaCl 0.5 g / 1; FeS0 4 x 7 H 2 O 75 mg / l; Na 3 citrate x 2 H 2 0 1 g / 1; CaCl 2 ⁇ 2 H 2 O 14.7 mg / 1; MgS0 4 x 7 H 2 0 0.3 g / 1; H 2 PO 4 1.5 g / 1; Trace element mix 10 ml / 1 and tetracycline 15 mg / 1.
• the pH of the FM2tet medium was adjusted to 6.0 before starting the culture.
• the trace element mix had the composition H 3 B0 3 2.5 g / l; CoCl 2 x 6 H 2 O 0.7 g / 1; CuSO 4 ⁇ 5 H 2 O 0.25 g / 1; MnCl 2 ⁇ 4 H 2 O 1.6 g / 1; ZnS0 4 x. 7 H 2 0 0.3 g / 1 and Na 2 Mo0 4 x 2 H 2 0 0.15 g / 1.
• the cells were analyzed as described in Example 2 for E. coli. Klebsiella cells were digested with the " French® Press" and the cell extracts were analyzed for acetolactate decarboxylase activity
• the specific acetolactate decarboxylase activity in crude extracts of the various strains is shown in Table 1.
• Aeration supply of compressed air in vvm, volume of compressed air per volume of fermentation medium per minute
• p0 2 oxygen partial pressure, to an initial value of 100% calibrated relative oxygen content
• pH and fermentation temperature were controlled and recorded via a computer program provided by the fermentor manufacturer Medium (74% glucose, w / v) was entspr the glucose consumption is metered in via a peristaltic pump.
• a vegetable based alkoxylated fatty acid ester commercially available under the name Struktol J673 from Schill & Seilacher (diluted 20-25% v / v in water) was used.
• Strains used in the fermentation were the Klebsiella terrigena wild type strain (control strain from Example 3) and the acetolactate decarboxylase overproducing strains Klebsiella terrigena pBudAkt-tet and Klebsiella terrigena-pALDbl-tet (see Example 4).
• Batch fermentation medium was FM2tet medium (see 3rd example, medium without tetracycline for the K. terrigena wild-type control strain).
• the cell density OD600 as a measure of the biomass formed was determined photometrically at 600 nm (Bio ad photometer SmartSpec TM 3000).
• the glucose content was determined as described in Example 4.
• the 2, 3-BDL content was determined by NMR as described in Example 4.
• An inoculum of Klebsiella terrigena-pBudAkt-tet in LBtet medium ⁇ see Example 2) was prepared by mixing 2 x 100 ml of LBtet medium, depending ⁇ wells in a 1 1 Erlenmeyer flasks, each with 0.25 ml of a glycerol culture (overnight culture of the strain in LBtet medium, mixed with glycerol in a final concentration of 20% v / v and stored at -20 ° C) were inoculated.
• the cultivation was carried out for 7 h at 30 ° C and 120 rpm on an infus orbital shaker (cell density OD SO o / ml of 0.5 - 2.5). 100 ml of the preculture were used to inoculate 8 liters of fermentation medium. Inoculated were two Vorfermenter with 8 1 fermenter medium. Prefermenters: The fermentation was performed three fermenters the company Sartorius BBI Systems GmbH in two Biostat ® C-DCU. Fermentation medium was FM2tet (see 3rd example). The fermentation took place in the so-called batch mode. 2 x 8 1 F 2tet were inoculated with 100 ml of inoculum. Fermentation temperature was 30 ° C.
• pH of the fermentation was 6.0 and was kept constant with the correction agents 25% NHOH, or 6 NH 3 P0 4 .
• Aeration was carried out with compressed air at a constant flow rate of 1 vvm.
• the oxygen partial pressure pO 2 was set at 50% saturation.
• the regulation of the oxygen partial pressure was carried out via the stirring speed (stirrer speed 450-1,000 rpm).
• Struktol J673 (20-25% v / v in water) was used.
• the two pre-fermenters were used as inoculum for the main fermenter.
• Main fermenter The fermentation was a Biostat ® in D 500 fermenter (working volume 330 1, boiler volume 500 1 ⁇ of the company Sartorius BBI Systems GmbH carried out the fermentation medium was FM2tet ⁇ see 3rd example.). The fermentation took place in the so-called fed-batch mode. 180 l of FM2tet were inoculated with 16 l of inoculum. Fermentation temperature was 30 ° C. pH of the fermentation was 6.0 and was kept constant with the correction agents 25% NH 4 OH, or 6 NH 3 P0 4 . The ventilation was carried out with compressed air at a constant flow rate of 1 vvm (see 4th example, based on the initial volume). The oxygen partial pressure pO 2 was set at 50% saturation.
• the regulation of the oxygen partial pressure was carried out via the stirring speed ⁇ stirrer speed 200-500 rpm).
• Struktol J673 (20-25% v / v in water) was used.
• glucose consumption was reduced by off-line glucose

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