WO2012104233A1 - Procédé de préparation de 2,3-butanediol par fermentation - Google Patents

Procédé de préparation de 2,3-butanediol par fermentation Download PDF

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WO2012104233A1
WO2012104233A1 PCT/EP2012/051406 EP2012051406W WO2012104233A1 WO 2012104233 A1 WO2012104233 A1 WO 2012104233A1 EP 2012051406 W EP2012051406 W EP 2012051406W WO 2012104233 A1 WO2012104233 A1 WO 2012104233A1
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production
strain
butanediol
klebsiella
gdh
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PCT/EP2012/051406
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German (de)
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Rupert Pfaller
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Wacker Chemie Ag
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    • 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
    • C12N9/0004—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
    • 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)
    • C12Y101/01047—Glucose 1-dehydrogenase (1.1.1.47)

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 which has the enzyraactivity of a glucose dehydrogenase (GDH) or an increased enzyme activity of a glucose dehydrogenase compared to the non-improved starting strain ,
  • GDH glucose dehydrogenase
  • Examples of basic chemical substances (so-called chemical synthesis building blocks) from renewable raw materials are ethanol (C2 building block), glycerol, 1,3-propanediol, 1,2-propanediol (C3 building blocks) or succinic acid, 1-butanol, 2-butanol 1 , 4-butanediol or 2, 3-butanediol ⁇ C4 building blocks ⁇ .
  • These chemical building blocks are the biogenic starting compounds from which further basic chemicals can be produced chemically. The prerequisite for this is the cost-effective fermentative production of the respective synthesis building blocks from renewable raw materials.
  • Decisive cost factors include the availability of suitable cheaper renewable raw materials as well as efficient microbial fermentation processes, which efficiently convert these raw materials into the desired chemical raw material.
  • microorganisms used produce the desired product in high concentration and with low by-product formation from the biogenic raw material.
  • the optimization of the productivity and cost-effectiveness of the microorganisms known as production strains is achieved, for example, through metabolic engineering.
  • a C4 building block accessible by fermentation is 2,3-butanediol.
  • the state of the art for the fermentative 2,3- Butanediol production is composed in Celinska and
  • 2, 3-butanediol is a possible starting material for petrochemical products with four O atoms (C4 building blocks) such as acetoin, diacetyl, 1,3-butadiene, 2-butanone (methyl ethyl ketone, MEK).
  • O atoms C4 building blocks
  • products with two carbon atoms C2 units
  • acetic acid DE 102010001399
  • acetaldehyde acetaldehyde
  • ethanol also ethylene
  • 3-butanediol The trodden by various microorganisms biosyntheti ⁇ cal way to 2, 3-butanediol is known (see review by Celins ⁇ ka and Grajek, Biotechnol Advances (2009). 27: 715-725) and leads from the central metabolite pyruvate over the three following enzymatic Steps to 2, 3-butanediol:
  • the object of the invention was to provide production of production of 2, 3-butanediol, which allow significantly higher 2,3-Butandiolausbeuten than the réellesstarnm.
  • the object has been achieved by a production strain which can be produced from a parent strain, characterized in that the production strain has a glucose dehydrogenase enzyme activity under conditions of 2,3-butanediol production which does not differ in the starting strain or only under physiological conditions different from the conditions of the 2, 3-butanediol production is present.
  • the glucose dehydrogenase enzyme activity is preferably the enzyme activity of an NAD- or NADP-dependent glucose dehydrogenase (GDH) from the enzyme class EC 1.1.1.47.
  • the parent strain may be a wild type strain that has not been further optimized but is capable of 2,3-butanediol production or a wild-type strain that has been further optimized. However, in an already optimized wild-type strain (e.g., by genetic engineering), the glucose dehydrogenase activity is not affected by the optimization.
  • a production strain is to be understood as meaning an initial strain which has been optimized with respect to the production of 2,3-butanediol and which has an enzyme activity of an NAD- or NADP-dependent glucose dehydrogenase (GDH), preferably from the enzyme class EC 1.1.1.47 either non-existent in the parent strain or only under specific physiological conditions other than the conditions of 2,3-butanediol production.
  • GDH NAD- or NADP-dependent glucose dehydrogenase
  • One such condition is, for example, sporulation.
  • the production strain is preferably produced from the parent strain. If an already optimized starting strain is to be further improved by increasing the glucose dehydrogenase activity, then it is of course also possible first to increase the GDH activity in an unmodified strain and then to introduce further improvements.
  • the increase in glucose dehydrogenase activity in the production strain may be due to any mutation in the genome of the parent strain, e.g. a mutation that the
  • An increased activity of the GDH gene is preferably to be understood as meaning a GDH activity increased by a factor of 1000 to 10, more preferably by a factor of 100 to 10 and particularly preferably by a factor of 10 compared with the starting strain.
  • the parent strain can be any 2,3-butanediol producing strain. It is preferably a strain of the genus Klebsiella, Raoultella, 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.
  • GDHs glucose dehydrogenases
  • EC 1.1.99.17 contain pyrroloquinoline as enzyme-bound cofactor (Oubrie et al., EMBO J. 18 (1999): 5187-5194) for the enzymatic oxidation of glucose.
  • GDHs of the enzyme class EC 1.1.99.17 are partially membrane proteins and transfer the electrons obtained from the glucose oxidation to ubiquinone.
  • GDHs which are classified under the enzyme classification EC 1.1.1.47, oxidize glucose to ⁇ -gluconolactone and reduce NAD or NADP to NADH or NADPH. They have been described, for example, for strains of the genus Bacillus, where they are expressed in spores or sporulating cells (Fujita et al., J. Bacteriol 132 (1977): 282-293).
  • the enzyme stability of isolated GDB enzymes has been described as very low and mutants with increased stability have been isolated.
  • An example of a B. subtilis GDH with increased chemical stability is disclosed in DE102004059376.
  • GDH is suitable for increasing the 2,3-butanediol yield (determined as volume yield 2, 3-butanediol in g / l) in shake flasks by more than 25%, preferably more than 50% and in particular by more than 100% (see Example 4) and in the fermentation by more than 35%, preferably more than 45% and particularly preferably by more than 60% increase.
  • GDH is preferably an enzyme of En ⁇ zymstall EC 1.1.1.47. It may be any gene-encoded enzyme that oxidizes glucose, thereby reducing NAD or NADP to NADH or NADPH.
  • the gene of GDH is derived from a bacterium of the genus Bacillus. Particularly preferably, the gene of the GDH is derived from a bacterium of the species Bacillus subtilis.
  • the strain according to the invention contains the enzyme activity of a GDH, preferably a GDH obtained by recombinant expression of an NAD- or NADP-dependent GDH (EC 1.1.1.47), which makes it possible in an unexpected manner to significantly increase the yield of 2,3-butanediol increase.
  • GDH oxidizes glucose to gluconic acid and reduces NAD or NADP to NADH or NADPH.
  • the GDH enzyme Therefore, it can in principle change the energy budget of the cell by contributing to an increased regeneration of N ⁇ DH and NADPH cofactors. However, the extent of this response is dependent on the intracellular availability of free Glu TM cose.
  • the strain according to the invention also makes it possible to increase the yield of other products produced by fermentation, including acetolactate, acetoin, diacetyl, ethanol, acetic acid, but also pyruvate, lactate (lactic acid), 1,2-propanediol, 1, 3 ⁇ propanediol and 1,4-butanediol, 1-butanol and 2-butanol.
  • a production strain according to the invention is also characterized in a preferred embodiment by being prepared from a parent strain as defined in the application and producing a GDH in recombinant form with the result that its 2,3-BDL production (volume production expressed in g / 1 2,3-BDL) with respect to the non-genetically optimized from ⁇ gear stem by at least 30%, preferably 45%, particularly before ⁇ Trains t 60% and is particularly preferably increased by 100%, wherein the 2,3-BDL yield the parent strain at least 80 g / 1.
  • the production strain of the invention is preferably prepared by introducing a gene construct into one of said parent strains.
  • the gene construct in its simplest form is defined as consisting of the GDH structural gene which, operatively linked, is preceded by a promoter. If necessary, the gene structur also includes a terminator downstream of the GDH structural gene.
  • the function of the promoter is defined as a genetic element that effects the transcription of the GDH gene in the host organism.
  • the function of the terminator is defined as a genetic element that stops the transcription of the GDH gene in the host organism.
  • Preferred is a strong promoter, which leads to a strong transcription. Among the strong promoters preferably of the skilled man ⁇ known. Familiar Tac promoter.
  • 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, which can replicate autonomously depending on their genetic structure in a given production strain.
  • 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. Biotechol. (2010) 85: 1301-1313), including, for example, the
  • Gene construct also in a known manner, a so-called selection markers 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.
  • an antibiotic resistance marker particularly preferably those which are resistant to antibiotics selected from ampicillin lin, 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 a GDH enzyme, preferably a GDH from the enzyme class EC 1.1.1.47, in recombinant form.
  • the recombinant GDH enzyme is able to oxidize glucose and NAD, or NADP to NADH, respectively
  • the parent strain may be a non-optimized wild type strain.
  • the parent strain may have been previously optimized or further optimized as the GDH TM producing production strain of the present invention.
  • the optimization of the production strain encompassed by the invention can on the one hand be carried out 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 biosynthesis genes acetolactate synthase, acetolactate decarboxylase and acetoin reductase.
  • genes can be expressed in a manner known per se as separate gene constructs or else combinedly as an expression unit ⁇ 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, the genes of acetolactate synthase and acetolactate decarboxylase are organized in an operon (Renna et al., J. Bacteriol (1993) 175: 3863-3875). Furthermore, the production strain can be optimized such that one or more genes are inactivated whose Genproduk ⁇ te negatively on the 2, 3 TM butanediol production effect.
  • 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 r 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 GDH-producing production strain according to the invention are cultivated in a growth medium.
  • the biomass of the producing strain and on the other hand, the product 2,3-BDL is one hand ge ⁇ forms.
  • the formation of biomass and 2,3-BDL can be correlated in time or decoupled in time.
  • 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). Preference is given to a production-scale process by fermentation, with a fermentation volume greater than 10 l being particularly preferred as the production scale and a fermentation volume greater than 300 l being particularly preferred.
  • Cultivation 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, which optimize cell growth and 2,3-BDL product formation.
  • C sources are those that originate from the production base
  • 2,3-BDL product formation can be availed.
  • C6 sugars such as.
  • cose f mannose or fructose and C5 sugars such as xylose, arabinose, ribose or galactose.
  • the production process according to the invention also encompasses 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.
  • C sources other than sugars or carbohydrates are acetic acid (or derived acetate salts), ethanol, glycerol, citric acid (and its salts), lactic acid (and its salts), or pyruvate (and its salts)
  • the C sources which are affected by the production process according to the invention comprise both the isolated pure substances and, for reasons of greater economic efficiency, not further purified mixtures of the individual C sources, such as hydrolyzates can be obtained by chemical or enzymatic digestion of the vegetable raw materials.
  • Hydrolysates of starch (monosaccharide glucose), sugar beet (monosaccharides glucose, fructose and arabinose), sugar cane (disaccharide sucrose), pectin (monosaccharide galacturonic acid) or lignocellulose (monosaccharide glucose from cellulose, monosaccharides xylose, arabinose , Mannose, hemicellulose galactose, and non-carbohydrate lignin).
  • C sources can also be used as fall products from the digestion of vegetable raw materials are used, such.
  • molasses sucgar beet
  • bagasse bagasse
  • 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 OH or else its salts such. For example, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium acetate or ammonium nitrate. Furthermore, suitable N-source are the known nitrate salts such. As KNO 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 be carried out in the so-called batch mode, wherein the An ⁇ breeding medium is inoculated with a starter culture of the production strain and then the cell growth takes place without further feeding of nutrient sources. The growth can also be done in the so-called.
  • Fed-batch mode and after an initial period of growth in batch mode to ⁇ additionally nutrient sources may be fed (feed) 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.
  • others can Media components and specifically the 2,3-BDL production-enhancing additives to 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 NH 4 OH and its salts ammonium sulfate, ammonium phosphate, ammonium acetate and ammonium chloride, furthermore urea, KNO 3 , NaNO 3 and ammonium nitrate, yeast extract, proteose peptone, malt extract, soya peptone, casamino acids Corn steep liquor (Corn Steep Liquor) as well as NZ-Amine and Yeast Nitrogen B ⁇ 3.
  • Particularly preferred N sources in the feed are ammonia, or ammonium salts, urea, yeast extract, soya peptone, malt extract or corn steep liquor (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). Preference is given to aerobic cultivation with oxygen, oxygen supply being ensured by introduction of compressed air or pure oxygen. tet is. Particularly preferred is 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 4.1 kb GDH expression vector pGDBS-E96Atet (+) prepared in Example 1.
  • FIG. 2 shows the 2.9 kb expansion vector pKKj used in Example 1.
  • FIG. 3 shows the 3.3 kb expression vector pKKj-tet prepared in Example 1.
  • GDBS-E96Atet The mutant GDB gene from B. subtilis named GDBS-E96A was used as disclosed in DE102004059376.
  • GDBS-E96A was isolated in a PCR reaction (Taq DNA polymerase, Qiagen) from the plasmid pGDBS-E96Ayexl disclosed in DE102004059376 with the primers gdbslf and gdbs2r as a DNA fragment of 0.8 kb size.
  • the PCR product was digested with Eco RI (contained in primer gdbsl) and Pst I (contained in primer gdbs2r) and cloned into the expression vector pKKj-tet. This resulted in the 4.1 kb GDH expression vector pGDBS -E96Atet (+) ( Figure 1).
  • pKKj-tet is a derivative of the expression vector pK j (FIG. 2).
  • the expression vector pKKj is a derivative of the expression vector pKK223 ⁇ 3.
  • the DNA sequence of p K223-3 is disclosed in the "GenBank" gene database under accession number M77749.1 From the 4.6 kb plasmid approximately 1.7 kb were removed (bp 262-1947 of those disclosed in M77749.1 From the pKKj, the ampicillin resistance gene was removed as a 1 kb fragment by digestion with Ex HI and the tetracycline resistance gene was expressed as 1.45 kb in the remaining 1.9 kb vector agent Fragment cloned. The tetracycline resistance gene was previously removed from the plasmid
  • P ⁇ CYC184 was isolated by PCR (Taq DNA polymerase, Qiagen) with the primers tet3f and tet4r and subsequent digestion with Nco I (cleavage sites contained in the primers tet3f and tet4r).
  • the DNA sequence of pACYC184 is accessible in the "Genbank” gene database under the accession number X06403.1, resulting in the 3.3 kb expression vector pKKj-tet (FIG.
  • Plasmid DNA of the expression vector pGDBS-E96Atet (+) was transformed according to methods known per se into E. coli strain JM105.
  • the control was E. coli JM105, with the pKKj-tet
  • the measurement batch of 1 ml volume for the photometric determination of GDH activity was composed of measurement buffer (0.1 M potassium phosphate, pH 7.0, 0.1 M NaCl), 10 mg / ml glucose, 2 mM NAD and GDH-containing cell extract , Measurement temperature was 25 ° C.
  • One unit of GDH activity is defined as the formation of 1 pmol of NADH / min under test conditions. in order to determine the specific activity of the protein concentration of the cell extracts was in so-called per se known manner to the.”
  • BioRad protein assay the company BioRad determined.
  • the strain Klebsiella (Raoultella) terrigena DSM 2687 is commercially available from the DSMZ German Collection of Microorganisms and Cell Cultures GmbH.
  • the transformation with the plasmid pGDBS-E96Atet ⁇ +) was carried out in a manner known per se analogously to the methods familiar to the skilled worker for the transformation of E. coli.
  • Klebsiella terrigena, with which pKKj-tet vector was transformed was used as a control strain.
  • Transmonauts were isolated and tested for GDH activity by shake flask culture. For this purpose, in each case 50 ml FM2tet medium 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; 10g / 1; 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; KH 2 P0 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 BO 3 2.5 g / l; C0Cl 2 x 6 H 2 0 0.7 g / 1; CuSO 4 ⁇ 5 H 2 O 0.25 g / 1; MnCl 2 ⁇ 4 H 2 O 1.6 g / 1; nS0 x 7 H 2 0 0, 3 g / 1 and Na 2 o0 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 disrupted with French®Press and the cell extracts were analyzed for GDH activity The specific GDH activity in crude extracts (determined as described in Example 2) was between 0.9 and 1.6
  • Example 1 The cultivation time was 96 hours.
  • the glucose concentration was determined at intervals of 24 h (glucose analyzer 7100MBS from YSI) and glucose if necessary from a 40% (w / v) stock solution re-fed. At intervals of 48 h, samples were tested for their 2,3-BDL content. The result of 2,3-BDL production is shown in Table 1.
  • Strains used in the fermentation were the Klebsiella wild type strain transformed with the vector pKKj-tet (control strain from the 3rd example) and the GDH-producing strain K.t. GDBS-E96A # 3 (see 4th example).
  • Fermentation medium was FM2tet medium (see 3rd example).
  • the medium was inoculated with 150 ml of preculture.
  • the preculture of the strains to be fermented was prepared by 24 h shake flask growing in batch fermentation medium.
  • the fermentation conditions were: temperature 30 ° C, stirrer speed 1000 rpm, aeration with 1 vvm, pH 6.0.
  • the fermenter was sampled periodically to analyze the following parameters:
  • the cell density OD 60 o as a measure of the biomass formed was determined photometrically at 600 nm (BioRad photometer SmartSpec TM 3000).
  • the glucose level was determined as beschrie ⁇ ben in the fourth example. was determined by NMR as described in Example 4.
  • the product was pumped by means of a pump (peristaltic pump 101 U / R of the Fa.
  • the strain Klebsiella terrigena pGDBS-E96A # 3 was fermented (see 4th and 5th examples).
  • An inoculum of Klebsiella terrigena pGDBS-E96A # 3 in LBtet medium was prepared by mixing 2 ⁇ 100 ml LBtet medium, in each case in a 1 liter Erlenmeyer flask, each with 0, 25 ml of a glycerol culture (overnight culture of the strain in LBtet medium, treated 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 6oo l 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.
  • Pre-fermenter The fermentation was carried out in two Biostat® C-DCU 3 fermenters from Sartorius BBI Systems GmbH. Fermentation medium was FM2tet (see 3rd example). The Fermen ⁇ tation took place in the so-called. Batch mode. 2 x 8 1 FM2tet 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% NH 4 OH, or 6 NH 3 PO 4 . Aeration was carried out with compressed air at a constant flow rate of 1 vvm. The oxygen partial pressure p02 was set at 50% saturation.
  • Fermentation medium was FM2tet (see 3rd example). The Fermen ⁇ tation took place in the so-called. Batch mode. 2 x 8 1 FM2tet 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
  • the regulation of the partial pressure of oxygen was effected by the stirring speed (stirrer speed 450-1000 rprn) .Structol.RTM. J673 (20-25% v / v in water) was used to control the foam formation After 18 h fermentation period (cell density OD 60 %) from 30-40) the two pre-fermenters were used as inoculum for the main fermenter.
  • Main fermenter The fermentation was carried out in a Biostat® D 500 fermenter (working volume 330 l, boiler volume 500 l) from Sartorius BBI Systems GmbH. Fermentation medium was FM2tet (see Example 3) The fermentation was carried out in the so-called fed-batch mode: 180 l of FM2tet were inoculated with 16 l of inoculum, fermentation temperature was 30 ° C. The pH of the fermentation was 6.0 and was corrected using the correction means 25 % NH 4 OH or 6 NH 3 PO 4. The aeration was carried out with compressed air at a constant flow rate of 1 vvm (see Example 4, based on the initial volume) The oxygen partial pressure pO 2 was set to 50% saturation .
  • the regulation of the oxygen partial pressure was made about the rate of stirring (stirrer speed 200-500 RPRN) to control foaming ® Struktol J673 was used (20-25% v / v in water) in the course of the fermenter tation of glucose consumption was.. off-line glucose

Abstract

L'invention concerne une souche de production pour préparer du 2,3-butanediol. Cette souche de production peut être obtenue à partir d'une souche de départ et possède dans des conditions de production de 2, 3-butanediol une activité enzymatique glucose déshydrogénase qui n'est pas présente dans la souche de départ, ou uniquement dans des conditions physiologiques différentes des conditions de production de 2, 3-butanediol. L'invention concerne en outre un procédé de préparation de 2,3-butanediol (2, 3-BDL) par fermentation au moyen d'une souche de production de ce type.
PCT/EP2012/051406 2011-01-31 2012-01-30 Procédé de préparation de 2,3-butanediol par fermentation WO2012104233A1 (fr)

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CN102936603A (zh) * 2012-10-31 2013-02-20 上海昊海生物科技股份有限公司 尿苷二磷酸-葡萄糖脱氢酶的表达及酶活测定

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