EP2670838A1

EP2670838A1 - Method for producing 2,3-butanediol by fermentation

Info

Publication number: EP2670838A1
Application number: EP12701748.1A
Authority: EP
Inventors: Rupert Pfaller
Original assignee: Wacker Chemie AG
Current assignee: Wacker Chemie AG
Priority date: 2011-01-31
Filing date: 2012-01-30
Publication date: 2013-12-11
Also published as: WO2012104234A1; DE102011003383A1; BR112013019525A2; US20130316419A1

Abstract

The invention relates to a microorganism strain which has an acetolactate decarboxylase activity 2-12.8 times higher than the non-improved original strain. The invention also relates to a method for producing 2,3-butanediol (2,3-BDL) by fermentation by means of said strain.

Description

Process for the fermentati en production of 2, 3-butanediol

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.

Due to rising crude oil prices, the manufacturing costs of the petrochemical raw materials for the chemical industry are also rising. This results in a growing interest in alternative production processes for chemical raw materials, especially based on renewable raw materials.

15 examples of chemical raw materials (so-called chemical synthesis building blocks) from renewable raw materials are ethanol (C2 block), glycerol, 1, 3-propanediol, 1, 2-propanediol (C3 ~

Building blocks) or succinic acid, 1-butanol, 1, 4-butanediol or 2, 3-butanediol (C blocks). These chemical building blocks

20 are the biogenic starting compounds, from which then further basic chemicals can be produced by chemical means. The prerequisite for this is the cost-effective fermentative production of the respective synthesis building blocks from renewable raw materials. Decisive cost factors are on the one hand the

On the other hand, efficient microbial fermentation processes, which efficiently convert these raw materials into the desired basic chemicals, are made available. It is crucial that the microorganisms used in the desired product in high Kon-

30 and produce 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.

35

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). In addition, products with two C atoms (C2 units) such as acetic acid (DE 102010001399) and derived therefrom, acetaldehyde, ethanol and also ethylene are available. By dimerization of 2, 3-butanediol and C8 compounds are conceivable, the use, for example, as a fuel in the aviation sector.

The biosynthetic pathway to 2,3-butanediol by various microorganisms is known (see review by Celinska and Grajek, Biotechnol. Advances (2009) 27: 715-725) and leads from the central metabolite pyruvate via the following three enzymatic steps to the 2, 3-butanediol:

Reaction of acetolactate synthase: Formation of acetolactate from two molecules of pyruvate with elimination of C0 ₂ .

Reaction of Acetolactate Decarboxylase: Decarboxylation of Acetolactate to Acetoin.

Reaction of acetoin reductase (2, 3-butanediol dehydrogenase): NADH-dependent reduction of acetoin υυ. 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) .Most bacterial producers are microorganisms of biological safety. Thus, 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) For a cost-efficient large-scale 2,3-BDL production, preference is given to production strains of the biological safety level S. For these, however, no sufficiently high 2,3-BDL yields have been described , 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. In the technical literature (Drancourt et al., Int J Syst. Evol Microbiol. (2001), 51: 925-932) 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. A common definition of the term "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),

Publisher: U.S. Dept. of Health and Human Services; 5, 5th edition, Revised December 2009 edition (March 15, 2010); ISBN 10: 0160850428.

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. 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. In

Blomqvist et al. , Appl. Environ. Microbiol. (1991) 57: 2796-2803 has extensively studied ALD activity and the product range of ALD overexpressing strains, including various esters, alcohols and diketones. While acetolactate was efficiently decarboxylated to acetoin in recombinant ALD strains, there is no suggestion that enhanced ALD expression would have been conducive to the production of 2,3-butanediol.

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.

In the present invention, a distinction is made between a parent strain and a production 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.

For the purposes of the present invention, 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. Preferably, 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.

Preferably, the overexpression of a homologous or heterologous acetolactate decarboxylase gene in the parent strain.

Preferably, 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.

Particularly preferred is 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. Particularly preferred 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.

According to the Blomqvist et al. , Ap l. Environ. Microbiol. (1991) 57: 2796-2803 known acetolactate decarboxylase overexpressing brewery strains no 2,3-butanediol could be detected, it was understood by those skilled in the art that overexpression of the acetolactate decarboxylase can not effectuate 2, 3-butanediol production in a production strain. In the context of experiments leading to the present invention, it was surprisingly found that the yield of 2,3-butanediol can be significantly increased by a limited increase in ALD activity (2 to 12.8 times) in a production strain. Preferably, the increased ALD activity is achieved by recombinant overexpression of an acetolactate decarboxylase (EC 4.1.1.5) in a production strain. As shown in the examples of the present application, overexpression of the acetolactate decarboxylase by a factor of 2 to 12.8 (see Example 3) 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. It may be any gene-encoded enzyme which, according to formula (II), effects the synthesis of acetoin from acetolactate by cleavage of CO 2. In a preferred embodiment, the gene of the acetolactate decarboxylase originates from a bacterium of the genus Klebsiella (Raultella) or Bacillus.

In a particularly preferred embodiment, 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. Thus, 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. with the result that its 2,3-BDL production (volume production expressed in g / 1 2,3-BDL) compared to the non-genetically optimized starting strain by at least 20, preferably 30%, more preferably 40% and most preferably by 100% is increased, wherein the 2, 3-butanediol yield of the parent strain is at least 80 g / 1.

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. Optionally, 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.

However, 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, 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 the person skilled in the art (Aune and Aachmann, Appl. Microbiol. nol. (2010) 85: 1301-1313), including, for example, the

Electroporation.

For the selection of transformed production strains, the gene construct contains, likewise in a known manner, a so-called selection marker for the selection of transformants with the desired gene construct. Known selection markers are selected from so-called antibiotic resistance markers or from the auxotrophy complementing selection markers.

Preference is given to antibiotic resistance markers, more preferably those which confer resistance to antibiotics selected from ampicillin, tetracycline, kanamycin, chloramphenicol or zeocin.

Thus, in one preferred embodiment, 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. Now, it has surprisingly been found that by suitable recombinant overexpression of the Acetolactatdecarboxylase 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. However, 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. However, 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. These 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).

Furthermore, the production strain can be optimized by inactivating one or more genes whose gene products adversely affect the 2,3-butanediol production. Examples include genes whose gene products are responsible for by-product formation. These include z. B. the lactate dehydrogenase (lactic acid formation), the acetaldehyde dehydrogenase (ethanol formation) or else the phosphotransacetylase, or the acetate kinase (acetate formation).

Furthermore, 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. On the one hand 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).

Preference is given to a process on a production scale by fermentation, the production scale being a fermentation volume greater than 10 1 is particularly preferred and a fermentation volume greater than 300 1 is particularly preferred.

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. However, 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. 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). However, 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. purified mixtures of the individual C sources, as they can be obtained as hydrolysates by chemical or enzymatic digestion of vegetable raw materials. These include z, B.

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 , Galactose from hemicellulose and lignin not belonging to the carbohydrates.) 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. In addition, 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.

Preferred 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) .Aferobic cultivation with oxygen is preferred, 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. For the isolation of the 2,3-BDL product 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.

Various analytical methods for the identification, quantification and determination of the purity of the 2,3-BDL product are available, including NMR, gas chromatography, HPLC, mass spectrometry or a combination of these analytical methods. The figures show the plasmids mentioned in the examples. Figure 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 invention is further elucidated by the following examples: 1. Example:

Preparation of acetolactate decarboxylase expression vectors

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.

The DNA sequence of the acetolactate decarboxylase gene from B.

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).

Primers BUD3f (SEQ ID NO: 1) and BUD4r (SEQ ID NO: 2) had the following DNA sequences: SEQ ID NO: 1:

5'-GAA TTC ATG AAT CAT ACT CCT GAA TGC ACC-3 ^Y

SEQ ID NO: 2:

5'-AAG CTT AGT TTT CTA CCG CAC GAA TAG C-3 ⁴

Primers BLALD-lf (SEQ ID NO: 3) and BLALD-2r {SEQ ID NO: 4) had the following DNA sequences:

SEQ ID NO: 3:

5'-GAA TTC ATG AAA AGT GCA AGC AAA CAA AAA AT-3 * SEQ ID NO: 4:

5'-AAG CTT TTA CTC GGG ATT GCC TTC GGC CG-3 The PCR products were post-digested with Eco RI (contained in primer BUD3f and BLALD-lf) and Hind III (contained in primer B0D4r and BLALD2r) and in the expression vector p Kj cloned. This resulted in the respective 4.9 kb acetolactate decarboxylase expression vectors pBudAkt (FIG. 1) and pALDbl (FIG. 2).

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).

1947 of the DNA sequences disclosed in M77749.1), resulting in the 2.9 kb expression vector pKKj. The expression vectors pBudAkt and pALDbl were modified by incorporation of an expression cassette for the tetracycline resistance gene. For this purpose, 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.) By PCR (Taq DNA polymerase, Qiagen) with the primers tetlf and tet2r and subsequent digestion with 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- As shown in Fig. 4 and Fig. 5, 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:

SEQ ID NO: 5:

5'-TCA TGA GAT CTC AGT GCA ATT TAT CTC TTC-3 SEQ ID NO: 6:

5 '~ TCA TGA GAT CTG CCA AGG GTT GGT TTG CGC ATT C-3' 2nd Example:

Expression analysis in 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. As control served Ξ. coli JM105 transformed with the vector pACYC184. 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). 2 ml 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-β-thiogalactoside, 0.4 mM final concentration) was used. added and shaken overnight at 30 ° C and 180 rpm.

For analysis of 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.

"French ^® Press" high-pressure homogenizer (SLM-AMINCO) disrupted and a cell extract by centrifugation (15 min 15,000 rpm, Sorvall SS34 rotor) was isolated. Aliquots of the cell extracts were used for the spectrophotometric determination of the acetolactate decarboxylase activity. While in the control strain, no activity could be measured, was the specific Aceto ^¬ lactatdecarboxylase activity in cell extracts, depending on the respective Kon trukt, between 5 and 20 U / mg protein,

Spectrophotometric determination of acetolactate decarboxylase activity:

The determination of the Acetolactatdecarboxylase activity was carried out in a conventional manner (US 4617273). Here, 1 U acetolactate decarboxylase activity is defined as the amount of enzyme which produces 1 μτηοΐ acetoin / min under test conditions.

An appropriate amount of enzyme extract, together with assay buffer (30 mM NaCitrate, pH 6.0), was adjusted to a volume of 10 ml, mixed with 0.4 ml of acetolactate substrate and incubated at 25 ° C. The chemically unstable acetolactate substrate had previously been freshly prepared from the starting compound 2-acetoxy-2-methyl-acetoacetic acid ethyl ester ("acetolactate diester", Sigma Aldrich) by alkaline saponification by adding 30 μΐ

Acetolactate diester mixed with 240 μΐ H20 and 330 μΐ 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.

At 0 ', 10', 30 'and 60' incubation time, 1 ml each of the batch was pipetted into 0.2 ml of 1 M NaOH and kept on ice to stop the reaction. Subsequently, the samples (1.2 ml volume) were mixed with 0.2 ml of 0.5% creatine and 0.2 ml of 5% alpha-naphthol in 2.5 M NaOH and incubated for 60 min at room temperature 524 nm, whereby the zero adjustment of the photometer was carried out by a test batch only with buffer, without enzyme.

The amount of acetoin formed was determined from a standard curve previously prepared with acetoin.

To determine the specific activity, the protein concentration of the cell extracts was determined in a manner known per se using the so-called "BioRad protein assay" from BioRad.

Expression of acetolactate decarboxylase in Klebsiella terrigena

Starting strain was Klebsiella terrigena DSM 2687 (commercially available from DSMZ GmbH). The transformation with the plasmids pBudAct-tet and pALDbl-tet was carried out in a manner known per se

Analogous to the methods known to those skilled in the transformation of E. coli. 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 (determined as described in Example 2) is shown in Table 1.

Table 1: Comparison of acetolactate decarboxylase activity in recombinant K. terrigena strains

4th example:

2, 3 -BDL production by shake flask culture of recombinant K. terrigena strains The cultivation of the K. terrigena strains transformed with the plasmids pBudAkt-tet and pALDbl-tet was carried out as described in example 3. However, the cultivation time was 96 hours. The glucose concentration was determined at intervals of 24 h (glucose analyzer 7100MBS from YSI) and glucose was re-fed from a 40% (w / v) stock solution as needed. In from- At 48 h, samples were analyzed for their 2,3-BDL content. The result of the 2,3-BDL production is shown in Table 2, Table 2: BDL production in Klebsiella terrigena transformants

As shown in Table 2, overexpression of the acetolactate decarboxylase genes from K. terrigena and B. licheniformis, respectively, in Klebsiella terrigena resulted in a 12-32% increase in 2,3-BDL production in shake flask culture. The increase in 2,3-butanediol production is apparently caused by overexpression of the acetolactate decarboxylase.

The determination of the 2,3-butanediol content in culture supernatants was carried out in a manner known per se by ¹ H-NMR. For this purpose, 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) ropionsäure-2, 3, 3 -d ₄ sodium salt) standard solution defined content (internal standard, typically 5 g / 1) were mixed in D ₂ 0. ¹ H-NMR analysis incl. peak integration was carried out according to the prior art with an Avance 500 NMR instrument from. Bruker. For quantitative analysis were the NMR signals of the analytes are integrated in the following ranges:

TSP: 0.140 - - 0.145 ppm (9H)

2, 3-butanediol: 1.155 - - 1.1110 ppm (6H)

Ethanol: 1.205 - 1.157 ppm (3H)

Acetic acid: 2,000 - - 1,965 PP (3H)

Acetoin: 2, 38 - 2,200 PP ™ (3H) 5th example:

2,3-BDL production with acetolactate decarboxylase overexpressing K. terrigena strains by fed-batch fermentation "Labfors II" fermenters from Infors were used, the working volume was 1.5 l (3 l fermenter volume) The state of the art was equipped with electrodes for measuring the pQ ₂ and the pH, and with a foam probe which regulated the addition of an antifoam solution, if necessary.The values of the measuring probes were recorded by a computer program and represented graphically. 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. To control foam formation, 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).

1.35 L of 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 permenter was sampled periodically to analyze the following parameters:

The cell density OD600 as a measure of the biomass formed was determined photometrically at 600 nm (Bio ad photometer SmartSpec ™ 3000).

To determine the dry biomass, 1 ml of fermentation mixture was centrifuged per measurement point in a tripple determination, the cell pellet was washed with water and dried at 80 ° C. to constant weight.

The glucose content was determined as described in Example 4.

The 2, 3-BDL content was determined by NMR as described in Example 4.

After the glucose present in the batch medium was consumed, a 74% (w / v) glucose solution was fed in via a pump (Watson Marlow peristaltic pump 101 U / R). The feeding rate was determined by the current glucose consumption rate. Table 3 shows the time-dependent 2, 3 -BDL formation in the K. terri ™ gena control strain and in the acetolactate decarboxylase overproducing recombinant Klebsiella terrigena strains.

As already observed in the shake flask experiments (4th example), the overexpression of acetolactate decarboxylase with the expression plasmids pBudAkt-tet and pALDbl-tet leads to a significant increase of 2, 3-butanediol production by 20% (heterologous ALD gene) - 30% ( homologous ALD gene), based on the maximum yield at a fermentation time of 72 hours The production curves are shown in Table 3.

Table 3 Production of 2,3-BDL in K. terrigena strains by fed-batch fermentation

6th example:

2, 3 -BDL fermentation in 330 l scale The strain Klebsiella terrigena pBudAkt- tet was fermented (see 4th and 5th examples).

Preparation of an inoculum for the prefermenter: 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 ^¬ weils 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). To control foaming, Struktol J673 (20-25% v / v in water) was used. After fermentation for 18 h (cell density OD ₆₀ 0 / l 30 to 40 l), 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). To control foaming, Struktol J673 (20-25% v / v in water) was used. In the course of fermentation, glucose consumption was reduced by off-line glucose

Measurement with a glucose analyzer from the company YSI determined (see 4th example). Once the glucose concentration of the fermentation mixtures was about 20 g / l {8-10 h after inoculation), the metered addition of a 60% w / w glucose feed solution was started. The flow rate of the feed was chosen so that during the production phase a glucose concentration of 10 - 20 g / 1 could be maintained. After completion of the fermentation, the volume in the fermenter was 330 1. The analysis of the fermentation parameters was carried out as described in the 5th example. The production process is shown in Table 4. The yield of 126 g / 1 2, 3-butanediol achieved with the strain Klebsiella terrigena pBudAkt-tet according to the invention was unexpectedly high and was about 40% higher than the yield of about 90 g usually achieved with a Klebsiella terrigena wild-type strain / 1 (see 5th example). Table 4

Production of 2,3-BDL by fed-batch fermentation in the 330 1 scale

Claims

Claims:

1. production strain for the production of 2,3-butanediol preparable from a parent strain, characterized in that the production strain has a 2 to 12.8 times over the parent strain acetolactate decarboxylase activity.

2. Production strain according to claim 1, characterized in that it has a by a factor of 2 to 10, more preferably by a factor of 2 to 5 increased Acetolactatdecarboxylase activity.

3. production strain according to claim 1 or 2, characterized in that the Acetolactatdecarboxylase activity is achieved by a relation to the parent strain increased expression of a coding for the Acetolactatdecarboxylaseenzens gene.

4. production strain according to one of claims 1 to 3,

characterized in that it is a strain of the genus Klebsiella (Raoultella), Bacillus (Paenibacillus) or Lactobacillus, with a strain of the kind Klebsiella (Raoultella) terrigena, Klebsiella (Raoultella) planticola, Bacillus (Paenibacillus) polymyxa or Bacillus licheniformis preferred and a strain of the species Klebsiella (Raoultella) terrigena or Klebsiella (Raoultella) planticola is particularly preferred, and more preferably is a strain of safety level Sl.

5. production strain according to one of claims 1 to 4,

characterized in that the gene of the acetolactate decarboxylase is derived from a bacterium of the genus Klebsiella (Raoultella) or Bacillus.

6. production strain according to one of claims 1 to 5,

characterized in that it consists of a non-GM nisch optimized starting strain was produced and a Acetolactatdecarboxylase produced in recombinant form with the result that its 2,3-BDL production (volume production, expressed in g / 1 2,3-BDL) over the non-genetically optimized starting strain by at least 20%, preferred 30%, more preferably 40% and particularly preferably increased by 100%, wherein the 2,3-butanediol yield of the parent strain is at least 80 g / 1.

7. A process for the preparation of 2, 3-butanediol, characterized in that a production strain according to claim 1 to 6 is cultured in a seed medium.

8. The method according to claim 7, characterized in that a cultivation in a pH range of pH 5 to pH 8 and a temperature range of 20 ° C to 40 ° C and anaerobic or aerobic with an oxygen supply by entry of compressed air or pure oxygen and a cultivating time for 2,3-BDL production between 10 h and

200 h is present.

9. The method according to claim 7, or 8, characterized in that a cultivation in a fermentation volume greater than 300 1 takes place.