EP2670837A1 - Procédé de préparation de 2,3-butanediol par fermentation - Google Patents

Procédé de préparation de 2,3-butanediol par fermentation

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Publication number
EP2670837A1
EP2670837A1 EP12701152.6A EP12701152A EP2670837A1 EP 2670837 A1 EP2670837 A1 EP 2670837A1 EP 12701152 A EP12701152 A EP 12701152A EP 2670837 A1 EP2670837 A1 EP 2670837A1
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EP
European Patent Office
Prior art keywords
strain
production
butanediol
acetoin reductase
klebsiella
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EP12701152.6A
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German (de)
English (en)
Inventor
Rupert Pfaller
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Wacker Chemie AG
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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)
    • C12Y101/01004—R,R-butanediol dehydrogenase (1.1.1.4)

Definitions

  • the invention relates to a process for the fermentative preparation of 2,3-butanediol (2,3-BDL) by means of an improved microorganism strain which has a 3.6 to 34.8 fold increased acetoin reductase (equivalent to 2, 3-butanediol dehydrogenase) activity relative to the non-improved parent strain.
  • acetoin reductase equivalent to 2, 3-butanediol 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 (C 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. It is crucial that the microorganisms used produce the desired product in high concentration and with low by-product formation from the biogenic raw material. Optimization of the productivity and economic viability of the microorganisms known as production strains is achieved, for example, by 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 summarized in Celinska and
  • 3-Butanediol is a possible starting material for petrochemical products with four carbon atoms (C blocks) such as acetoin, diacetyl, 1,3-butadiene, 2-butanone (methyl ethyl ketone, EK).
  • C blocks carbon atoms
  • products with two carbon atoms (C2 units) such as acetic acid (DE 102010001399) and derived therefrom, acetaldehyde, ethanol and also ethylene are accessible.
  • C2 units such as acetic acid (DE 102010001399) and derived therefrom, acetaldehyde, ethanol and also ethylene are accessible.
  • C8 compounds are also conceivable, the use of e.g. as a fuel in the aviation sector.
  • acetoin reductase (2, 3-butanediol dehydrogenase): NADH-dependent reduction of acetoin to 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. But also yeasts are known as producers ⁇ eg bakers yeast ⁇ .
  • Production strains from the biological safety level S1 are strains of the species Klebsiella terrigena, Klebsiella planticola, strains of the genus Bacillus (or Paenibacillus) such as Bacillus polymyxa or Bacillus licheniformis.
  • Bacillus or Paenibacillus
  • the species Klebsiella terrigena and Klebsiella planticola synonymously as Raoultella terrigena and Raoultella are also called planticola due to a taxonomic designation , These are strains of the same species.
  • WO 2009/151342 discloses the production of 2,3-BDL by anaerobic fermentation with the bacterium Clostridium autoethanogenum and ohlenmonoxide as C source. Maximum achieved 2,3-BDL yields were 9.27 g / l after a fermentation period of 14 days. In the genetically unmodified strains, the level of expression of JolS was 2.3 réelle
  • Butanediol dehydrogenase in 2,3-BDL-producing batches (caused by excess carbon monoxide fumigation) compared to an acetate-producing batch (limited carbon monoxide fumigation) and an induction of the RNA of the wild-type gene of 2,3-butanediol dehydrogenase by a factor of 7.64 ( corresponding to a 2,3-BDL production of 8.67 g / l after 13 days).
  • the induction of gene expression was achieved by increased fumigation with carbon monoxide. It was not disclosed whether gene expression also involved an increase in acetoin reductase enzyme activity and how high the supposed increase in enzyme activity was compared to baseline.
  • autoethanogenum does not provide general guidance for production under aerobic or microaerobic conditions to increase 2,3-butanediol production due to the combination of fumigation with Carbon monoxide and an increased 2, 3-butanediol dehydrogenase activity derived.
  • 2,3-BDL yields of 8.67 g / l in 13 days are far from adequate for economical production, especially considering an increased engineering effort of large scale anaerobic fermentation.
  • WO 99/54453 discloses lactic acid bacteria with 10-fold increased activity of either diacetyl reductase, acetoin reductase, or 2,3-butanediol dehydrogenase, respectively. Surprisingly, only an influence on the diacetyl content of the lactic acid bacteria used as starter cultures in the food industry was observed. However, it was not disclosed whether an increased activity of acetoin reductase, or 2,3-butanediol dehydrogenase on the 2,3-BDL content of the lactic acid bacterial cultures had an effect.
  • Butanediol included. It is therefore not obvious to the person skilled in the art that an increase in acetoin reductase activity necessarily results in an increased 2,3-BDL yield.
  • the object of the invention was to provide production strains for the production of 2, 3-butanedoxol, which allow significantly higher 2,3-Butandiolausbeuten than the parent strain.
  • the problem was solved by a production strain which can be produced from a parent strain, characterized in that the parent strain is selected from the genus Klebsiella, Raoultella, Paenibacillus and Bacillus and the production strain at least 3.6 to 34.8 times above the parent strain has underlying acetoin reductase activity.
  • 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.
  • an already further optimized wild-type strain ⁇ e.g. by genetic engineering
  • the acetoin reductase 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 increased activity of the enzyme acetoin reductase in comparison to the starting strain
  • the production strain is made from the parent strain. If an already optimized starting strain is to be further improved by an increase in the acetoin reductase activity, it is of course also possible first to increase the acetoin reductase activity in an unimpaired strain and then to introduce further improvements.
  • the increase in the acetoin reductase activity in the production strain can be caused by any mutation in the genome of the parent strain (eg a promoter activity-increasing mutation), an enzyme activity-increasing mutation in the acetoin reductase gene or by overexpression of a mologen or else heterologous Acetoinreduktasegens in the parent strain.
  • the acetoin reductase activity is preferably increased by at least the factor 3.6 to 20 and more preferably by the factor 3.6 to 10.
  • This increased acetoin reductase activity is particularly preferably achieved by an increased expression of a homologous or heterologous gene coding for an acetoin reductase enzyme compared with the starting strain.
  • the parent strain may be any 2,3-butanediol-producing strain of security level S1.
  • It is preferably 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 being even more preferred
  • it is a parent strain and a production strain classified in the security level Sl and of these, more preferably, strains of the species Klebsiella (Raoultella) terrigena or Klebsiella (Raoultella) planticola.
  • the yield of 2,3-butanediol can be significantly increased.
  • the increased acetoin reductase activity is achieved by recombinant overexpression of an acetoin reductase in a production strain.
  • an over-expression of the acetoin reductase by a factor of 3.6 up to a factor of 34.8 is suitable for the 2,3-butanediol yield (determined as volume yield 2,3 Butanediol in g / l) in the fermentation by more than 20%, preferably more than 30%, more preferably more than 40% and especially preferably more than 100%.
  • the recombinant overexpression of acetoin reductase also allows an increase of the 2, 3 -Butandiolausbeute in shake flasks by 20 - 29%.
  • the acetoin reductase (AR) may be any gene-encoded enzyme that, according to formula (III), causes the synthesis of 2,3-butanediol from acetoin by oxidation of the cofactor NADH (or NADPH) to NAD (or NADP) ,
  • the gene of the acetoin reductase is derived from a bacterium of the genus Klebsiella (Raoultella) or Bacillus (Paenibacillus).
  • the gene of the acetoin reductase is derived from a strain of the species Klebsiella terrigena, Klebsiella planticola, Bacillus polymyxa or Bacillus
  • strains including the strain Klebsiella terrigena DSM 2687 used in the present invention, 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 2,3-butanediol.
  • the invention thus, not only the production of 2,3-butanediol but also the production of other metabolites, which can be derived from the 2,3-butanediol.
  • These metabolites include diacetyl, acetoin, ethanol and acetic acid.
  • 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 an acetoin reductase in recombinant form with the result that its 2,3-BDL production (volume production expressed in g / 1 2,3-BDL) is increased by at least 20%, preferably 30%, more preferably 40% and especially preferably by 100% compared to the non-genetically optimized starting strain, wherein the 2,3-butanediol yield of the starting strain is 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 acetoin reductase structural gene operatively linked to a promoter upstream.
  • the gene construct may also comprise a terminator, which is connected downstream of the aceto-inuctuctase structural gene.
  • Preferred is a strong promoter, which leads to a strong transcription.
  • the strong promoters preferred is the so-called.
  • the expert from the molecular biology of E. coli 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, depending on their genetic
  • the gene construct can also be integrated in the genome of the production strain, with each gene location along the genome being suitable as integration site.
  • the gene construct either in plasmid form or with the aim of genomic integration, is introduced into the production strain in a manner known per se by genetic transformation.
  • Various methods of genetic transformation are known to those skilled in the art (Aune and Aachmann, Ap, Microbiol, Biotechol., (2010) 85: 1301-1313), including, for example, US Pat
  • 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 complementary selection markers.
  • antibiotic resistance markers more preferably those which confer resistance to antibiotics selected from ampicillin, tetracycline, kanamycin, chloramphenicol or zeocin.
  • the gene construct integrated either in plasmid or in the genome and produces a Aceto "inreduktase enzyme in recombinant form.
  • the recombinant acetone reductase enzyme is able to produce 2,3-butanediol from acetoin under oxidation of NADH (or alternatively also NADPH) to NAD (or alternatively to NADP) .It has now surprisingly been found that by suitable recombinant overexpression of the Acetoxnreduktase enzyme in the production strain, the 2,3-butanediol yield compared to the parent strain can be significantly increased.
  • the parent strain may be a non-optimized wild type strain.
  • the parent strain may already be have been previously optimized, or further optimized as erfindungsgetnässer ace- toinreductase producing production strain.
  • the optimization of an inventive production of acetoin reductase by means of 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 and acetolactate decarboxylase.
  • genes can be expressed in a manner known per se as separate gene constructs in each case or also combined 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 2,3-butanediol production.
  • genes whose gene products are responsible for by-product formation include, for example, lactate dehydrogenase (formation of lactic acid), acetaldehyde dehydrogenase (formation of ethanol) or else phosphotrane acetylase, or 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 process is characterized in that cells of a production strain according to the invention, which produce acetoin reductase, are cultivated in a growth medium.
  • biomass of the production strain and on the other hand the Product 2,3-BDL 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).
  • 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 can be used by the production strain for 2,3-BDL product formation. These include all forms of monosaccharides, including CS sugars such as. Glucose, matmose or fructose, as well as 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 according to the invention furthermore also comprises all C sources in the form of higher saccharides, glycosides or carbohydrates with more than two sugar units, such as, for example, As maltodextrin, starch, cellulose, hemicellulose, pectin or by hydrolysis ⁇ enzymatically or chemically) released monomers or oligomers.
  • 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.
  • Other useful 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, to increase the economic efficiency, not further purified mixtures of the individual C sources, such as can be obtained as hydrolyzates by chemical or enzymatic digestion of the vegetable raw materials.
  • These include, B.
  • 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 formation. 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. B. KN0 3 , NaN0 3 , ammonium nitrate, Ca (N0 3 ) 2, Mg (N0 3 ) 2 and other N sources such as eg urea.
  • the N sources also include complex amino acid mixtures such as yeast extract, proteose peptone, malt extract, soy 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.
  • complex amino acid mixtures such as yeast extract, proteose peptone, malt extract, soy 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 growth 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 Feeäkomponenten can together as a mixture or separately in individual
  • 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.
  • 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.
  • Preferred N sources in the feed are ammonia, gaseous or in aqueous solution as NH 4 OH and its salts ammonium sulfate, ammonium phospha, ammonium acetate and ammonium chloride, furthermore urea, NO 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 Base.
  • 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 product ion 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, the oxygen supply being ensured by introduction of compressed air or pure oxygen. 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 directly further without further work-up or else be isolated from the cultivation mixture.
  • 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.
  • the figures show the plasmids mentioned in the examples. 1 shows the 3.65 kb acetoin reductase expression vector pBudCkt prepared in Example 1.
  • FIG. 2 shows the 3.67 kb acetoin reductase expression vector pARbl prepared in Example 1.
  • Fig. 3 shows the plasmid pACYC184 used in Example 1.
  • FIG. 4 shows the 5.1 kb expression vector pBudCkt-tet prepared in Example 1.
  • Fig. 5 shows the prepared in Example 1 5, 1 kb large exposition vector pARBL-tet.
  • the acetoin reductase genes from K. terrigena and B.licheniformis were used.
  • the DNA sequence of the acetoin reductase gene from K. terrigena is disclosed in the "GenBank” gene database under accession number L04507, bp 2671-3440. It was isolated 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 BUD7f and BUD9r as a DNA fragment of 0.78 kb size isolated. The DNA sequence of the B.
  • licheniformis acetoin reductase gene is disclosed in the "GenBank” gene database under accession number NC_006322.1, under which the entire genome sequence of B. licheniformis is disclosed
  • the acetoin reductase gene is there in a complementary form from bp 2007068 - It 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 BLar-lf and BLar-2r as DNA Fragment of 0.78 kb size isolated.
  • the genomic DNA used for the PCR reactions was previously in a conventional manner with a DNA isolation kit (Qiagen) from cells of the culture of. terrigena DSM 2687 and B. licheniformis DSM 13 in LB medium (10 g / l tryptone, 5 g / l yeast extract, 5 g / l NaCl).
  • the PCR products were post-digested with Eco RI (contained in primer BUD7f and BLar-lf) and Hind III (contained in primer BUD9r) or Pst I (contained in primer BLar-2r) and expressed in the expression cloned onsvektor pKKj.
  • Eco RI obtained in primer BUD7f and BLar-lf
  • Hind III obtained in primer BUD9r
  • Pst I obtained in primer BLar-2r
  • the expression vector pKKj is a derivative of the expression vector pKK223 ⁇ 3.
  • the expression vectors pBudCkt and pARbl 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 sites 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 pBudCkt and pARbl vectors respectively cut with Bam HI, resulting in the expression vectors pBudCkt-tet (FIG.
  • a clone was selected in each of which the tetracycline and the acetoin reductase expression cassettes were oriented in the same direction, respectively.
  • Primer tetlf (SEQ ID NO: 5) and tet2r (SEQ ID NO: 6) had the following DNA sequence:
  • Plasmid DNA of the expression vectors pBudCkt-tet and pARbl-tet was transformed according to methods known per se into E. coli strain JM105.
  • the control was E. coli JM105 transformed with the vector pACYClS4.
  • One clone each was selected and cultured in a ScblinlkolJoenanzucht.
  • a preculture was prepared in LB tet- edium (10 g / l tryptone, 5 g / l yeast extract, 5 g / l NaCl, 15 pg / ml tetracycline) (cultivation at 37 ° C. and 120 rpm overnight).
  • acetoin reductase expression 50 ml of the E. coli cells were centrifuged (10 min 15000 rpm, Sorvall RC5C centrifuge equipped with a SS34 rotor), the cell pellet in 2 ml KPi buffer (0.1 M potassium phosphate, 0.1 M NaCl, pH 7.0), in a manner known per se with a so-called.
  • KPi buffer 0.1 M potassium phosphate, 0.1 M NaCl, pH 7.0
  • acetoin reductase activity The determination of the acetoin reductase activity was carried out in a manner known per se. The substrate acetoine is reduced by AR in an NADH-dependent reaction. It produces 2, 3-butanediol and NADH is consumed in stoichiometric amounts. The consumption of NADH is determined photometrically at 340 nm. 1 U acetoin reductase activity is defined as the amount of enzyme that reduces 1 pmol acetoin / min under test conditions.
  • acetoin reductase in Klebsiella terrigena starting strain was Klebsiella terrigena DSM 2687.
  • the transformation with the plasmids pBudC, pBudCkt-tet, pARbl and pARbl TM tet was carried out in a manner known per se analogously to the methods for the transformation of E. coli known to the person skilled in the art.
  • the control strain used was the non-transformed strain strain Klebsiella terrigena DSM 2687.
  • Tran formants were isolated and tested for acetoin reductase activity by shake flask culture.
  • 50 ml of FM2amp medium (plasmids pBudCkt and pARbl), or FM2tet medium (plasmids pBudCkt-tet and pARbl-tet), or FM2 medium without antibiotics (K. terrigena wild-type control strain) were inoculated with a transformant and Incubated for 24 h at 30 ° C and 140 rpm (Infors shaker).
  • FM2 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, im Case of FM2amp, ampicillin 100 mg / 1, or in the case of
  • FM2tet, tetracycline 15 mg / l 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 X 5 H 2 O 0.25 g / 1; MnCl 2 ⁇ 4 H 2 O 1.6 g / 1; ZnS0 4 ⁇ 7 H 2 0 0.3 g / 1 and Na 2 Mo0 4 ⁇ 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 the "French Press ®” and examines the cell extracts on Acetoinreduktase activity. The specific Acetoinreduktase activity in crude extracts of different strains (determined as described in Example 2) is shown in Table 1.
  • Table 2 BDL production in the shake flask of acetoin reductase over-expressing Klebsiella terrigena transformants
  • the determination of the 2,3-butanediol content in culture supernatants was carried out in a manner known per se by .sup.1 H-NMR.
  • an aliquot of the culture was centrifuged (10 min 5000 rpm, Eppendorf Labofuge) and 0.1 ml of the culture supernatant with 0.6 ml of TSP (3- (trimethylsilyl) propionic acid 2,2,3,3-d 4 sodium salt) standard solution defined content (internal standard, typically 5 g / 1) in D 2 0 mixed.
  • TSP trimethylsilyl
  • Strains used in the fermentation were the Klebsiella terrigena wild type strain ⁇ control strain of the 3rd and 4th examples) and the acetoin reductase overproducing strains
  • Klebsiella terrigena pBudCkt-tet and Klebsiella terrigena pARbl-tet were prepared from pBudCkt-tet and Klebsiella terrigena pARbl-tet (see 3rd and 4th examples).
  • 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 (BioRad 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.
  • Table 3 shows the time-dependent 2, 3 -BDL formation in the K. terrigena control strain and in the acetoin reductase overproducing, recombinant Klebsiella terrigena strains.
  • the strain Klebsiella terrigena-pBudCkt-tet was fermented (see 4th and 5th examples).
  • Preparation of an inoculum for the pre-fermenter An inoculum of Klebsiella terrigena pBudCkt ⁇ tet in LBtet medium (see Example 2) was prepared by adding 2 ⁇ 100 ml LBtet medium, each in a 1 1 Erlenmeyer flask, each containing 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 S00 / 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.
  • 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
  • the analysis of the fermentation parameters was carried out as described in the 5th example.
  • the content of 2,3-butanediol and the by-products acetoin, ethanol and acetate was determined by NMR (see Example 4)
  • the lactate content was determined in a manner known per se (see, for example, US 2010/0112655) by HPLC.
  • the production process of the fermentation is shown in Table 4.
  • the yield of 128.8 g / l of 2,3-butanediol achieved with the strain Klebsiella terrigena pBudCkt ⁇ tet according to the invention was unexpectedly high and was more than 40% higher than the yield of about 10% customarily achieved with a Klebsiella terrigena wild-type strain. 90 g / 1 (see 5th example).

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WO2023094010A1 (fr) 2021-11-29 2023-06-01 Wacker Chemie Ag Procédé pour la production de taurine
WO2023232233A1 (fr) 2022-05-31 2023-12-07 Wacker Chemie Ag Procédé de production d'acide l-cystéique et son utilisation
WO2025087508A1 (fr) 2023-10-24 2025-05-01 Wacker Chemie Ag Production de biotine dans des micro-organismes exprimant des gènes pour des pantothénate kinases ayant une inhibition de produit réduite par la coenzyme a

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DE102013204728A1 (de) 2013-03-18 2014-09-18 Wacker Chemie Ag Mikroorganismenstamm und Verfahren zur fermentativen Herstellung von C4-Verbindungen aus C5-Zuckern
DE102013205986A1 (de) * 2013-04-04 2014-10-23 Wacker Chemie Ag Mikroorganismenstamm und Verfahren zur fermentativen Herstellung von C4-Verbindungen aus C5-Zuckern
DE102013216658A1 (de) 2013-08-22 2015-02-26 Wacker Chemie Ag Verfahren zur fermentativen Herstellung von C4-Produkten und dafür geeignete Mikroorganismenstämme
DE102013223176A1 (de) 2013-11-14 2015-05-21 Wacker Chemie Ag Verfahren zur fermentativen Herstellung von C4-Produkten und dafür geeignete Mikroorganismenstämme
DE102013224909A1 (de) 2013-12-04 2015-06-11 Wacker Chemie Ag Verfahren zur Isolierung von 2,3-Butandiol aus Fermentationsansätzen
JP6486020B2 (ja) * 2014-05-30 2019-03-20 三菱ケミカル株式会社 ２，３−ブタンジオールの製造方法
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Publication number Priority date Publication date Assignee Title
WO2022184255A1 (fr) 2021-03-03 2022-09-09 Wacker Chemie Ag Procédé de production d'un hydroxytyrosol
WO2023094010A1 (fr) 2021-11-29 2023-06-01 Wacker Chemie Ag Procédé pour la production de taurine
WO2023232233A1 (fr) 2022-05-31 2023-12-07 Wacker Chemie Ag Procédé de production d'acide l-cystéique et son utilisation
WO2025087508A1 (fr) 2023-10-24 2025-05-01 Wacker Chemie Ag Production de biotine dans des micro-organismes exprimant des gènes pour des pantothénate kinases ayant une inhibition de produit réduite par la coenzyme a

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