DE102013205986A1 - Microorganism strain and process for fermentative production of C4 compounds from C5 sugars - Google Patents

Abstract

The invention relates to a production strain for the fermentative production of C4 products which, as a C source during fermentation, is able to utilize biomass containing lignocellulose and the sugar obtained from it, hemicellulose, which can be produced from a starting strain, characterized in that the starting strain is a wild type strain which is naturally capable of producing 2,3-butanediol and the production strain at the same time has an increased expression of a gene coding for a transketolase (EC 2.2.1.1) and a gene coding for a transaldolase (EC 2.2.1.2) compared to the starting strain.

Description

The invention relates to a microorganism strain and a process for the fermentative production of C4 compounds from C5 sugars (pentoses).

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

Examples of chemical raw materials (so-called chemical synthesis building blocks or platform chemicals called) from renewable resources are ethanol (C2-block), glycerol, 1,3-propanediol, 1,2-propanediol, lactic acid, 3-hydroxypropionic acid (C3 building blocks) or Succinic acid, 1-butanol, 2-butanol, 1,4-butanediol, acetoin, diacetyl 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. It is crucial for the economy of these fermentative processes that the microorganisms used can efficiently convert the cheapest available raw materials into the desired product.

A C4 building block accessible by fermentation (C4 platform chemical) 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 and in Ji et al., Biotechnol. Adv. (2011) 29: 351-364) ,

From the microbial 2,3-butanediol biosynthetic pathway (see equations (I) to (III)), in addition to 2,3-butanediol, further compounds having four carbon atoms are fermentatively available, namely acetoin and, derived therefrom by oxidation, also diacetyl. 2,3-butanediol, acetoin and diacetyl are therefore collectively also referred to as C4 products.

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 carbon atoms (C2 building blocks) such as acetic acid ( US 2012288908 AA ) and derived therefrom, acetaldehyde, ethanol and also ethylene accessible. By dimerization of 2,3-butanediol and C8 compounds are conceivable, the use z. B. as a fuel in the aviation sector.

Reaktion der Acetolactatdecarboxylase: Decarboxylierung von Acetolactat zu Acetoin.

Reaktion der Acetoinreduktase (2,3-Butandioldehydrogenase): NADH-abhängige Reduktion von Acetoin zu 2,3-Butandiol.

The biosynthetic pathway to 2,3-butanediol followed by various microorganisms is known (see Reviews by Celinska and Grajek, Biotechnol. Advances (2009) 27: 715-725 and Ji et al., Biotechnol. Adv. (2011) 29: 351-364 ) and leads from the central metabolite pyruvate via the following three enzymatic steps to 2,3-butanediol: Reaction of Acetolactatsynthase: Formation of acetolactate from two molecules of pyruvate with elimination of CO ₂ .

Reaction of Acetolactate Decarboxylase: Decarboxylation of Acetolactate to Acetoin.

Acetoin reductase reaction (2,3-butanediol dehydrogenase): NADH-dependent reduction of acetoin to 2,3-butanediol.

The prior art discloses processes for the preparation of 2,3-butanediol based mainly on the use of glucose as a fermentation raw material (C source) (see e.g. WO 12104234 A1 ). In order to produce 2,3-butanediol and also other C4 and C2 platform chemicals such as acetoin, ethanol and acetic acid economically, ie competitive for production from petrochemical raw materials, glucose is not the preferred fermentation raw material. Glucose is one of the basic products in the food industry (mainly made of starch, sugar cane or sugar beet) and is now increasingly used for bioethanol production. The resulting increase in demand is expected to lead to a steady increase in the price of glucose. As the fermentation raw material is one of the cost-determining factors in the production of biogenic platform chemicals, more and more alternatives are being sought. Since large-scale fermentation processes based on glucose as a fermentation raw material represent an unwanted competition to its use as a foodstuff, there are also important socio-politically motivated reasons for the search for alternative fermentation raw materials.

Among the alternative fermentation raw materials lignocellulosic biomass is of central importance. In agriculture or forestry, it occurs as straw, bagasse or as a waste of wood processing in large quantities without corresponding exploitation possibilities. Accordingly, there is a great effort to find value-increasing uses for components of lignocellulose.

Lignocellulosic biomass consists of the three main components cellulose, hemicellulose and lignin. The focus in the utilization of lignocellulose is the recovery of glucose from cellulose, v. a. for the fermentative production of bioethanol. The recovery of glucose from cellulose is a relatively expensive and expensive process that currently can not compete with conventional production from food plants.

Hemicellulose is, after cellulose, the most abundant naturally occurring polysaccharide ( Saha, J. Ind. Microbiol. Biotechnol. (2003) 30: 279-291 ) and therefore as a source for obtaining sugar for the fermentation of great interest.

The sugars contained in hemicellulose are comparatively easy to access, especially the most abundant xylose and also arabinose. Various methods of extracting sugars from hemicellulose are known in the art, including those based on hydrolysis of the hemicellulose hydrothermally, thermally at acidic pH or enzymatically with xylanases ( Saha, J. Ind. Microbiol. Biotechnol. (2003) 30: 279-291 ).

Xylose (mainly D-xylose, CAS number 58-86-6), the major constituent of hemicellulose, is one of the most abundant naturally occurring carbohydrates. Xylose is in itself a waste which finds only limited use in the food industry (eg Preparation of the sweetener Xylitol, Saha, J. Ind. Microbiol. Biotecanol. (2003) 30: 279-291 ) and because of lack of metabolism of xylose by the industrially used fermentation yeasts not suitable for the production of bioethanol on a large scale.

These factors, cheap and abundant raw material, comparatively simple isolation and low demand for alternative utilization make xylose an attractive fermentation raw material in the production of biogenic platform chemicals, especially in the fermentative production of the C4 products 2,3-butanediol or acetoin and here in particular when using the fermentatively produced 2,3-butanediol or acetoin to obtain acetic acid by gas phase oxidation.

Various natural producers of 2,3-butanediol are known, for. 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 level S2, so can not be used on a large scale without expensive and costly technical safety measures (see also and to other microbial 2,3-butanediol producers see Review by Celinska and Grajek, Biotechnol. Advances (2009) 27: 715-725 ). This would also apply to hitherto undescribed genetically optimized production strains based on these strains.

A common definition for the term "biological security level" and the division into different security levels can be found z. In "Biosafety in Microbiological and Biomedical Laboratories", p. 9 et seq. (Table 1), Centers for Disease Control and Prevention (US) (Editor), Public Health Service (US) (Editor), National Institutes of Health (Editor), Publisher: US Dept. of Health and Human Services; 5, 5th edition, Revised December 2009 edition (March 15, 2010); ISBN 10: 0160850428 ,

For cost-effective large-scale production of C4 products, in particular 2,3-butanediol, production strains of biological safety level S1 are preferred. For these, however, no sufficiently high product yields have been described so far. Known 2,3-butanediol production strains from the biological safety level S1 are strains of the species Raoultella terrigena or strains of the genus Bacillus (or Paenibacillus) such as Paenibacillus polymyxa or Bacillus licheniformis. In the literature ( Drancourt et al., Int. J. Syst. Evol. Microbiol. (2001), 51: 925-932 ) various bacterial species originally belonging to the genus Klebsiella were genetically analyzed. Due to the heterogeneity found (see in Drancourt et al., Int. J. Syst. Evol. Microbiol. (2001), 51: 925-932 ) tribes of the originally Klebsiella terrigena named species were taxonomically rearranged and renamed within the newly created genus Raoultella in the species Raoultella terrigena. Bacterial strains of the species Klebsiella terrigena and Raoultella terrigena thus designate strains of the same species, but are different from other strains of the genus Klebsiella.

Only a few of the known 2,3-butanediol-producing strains a fermentative production of xylose is described. For Klebsiella pneumoniae, a microorganism of biological safety level S2, the fermentative production of the C4 products 2,3-butanediol and acetoin from the C sources xylose and glucose was compared ( Yu and Saddler, Appl. Environ. Microbiol. (1983) 46: 630-635 ). The C4 yields obtained were 88 g / l for xylose as the C source and 112 g / l for glucose as the C source. The fermentation time to obtain the maximum C4 yield was 6-8 days unusually long, corresponding to a particularly for xylose comparatively low space-time yield of C4 products 2,3-butanediol and acetoin of about 0.6 gl ^-1 h ^-1 (see 3 and 4 in Yu and Saddler, Appl. Environ. Microbiol. (1983) 46: 630-635 ). Thus, on the one hand, significantly lower 2,3-butanediol yields were achieved with xylose than with glucose as the fermentation raw material, and, moreover, the space-time yield, which is important for the economy of the process, was low.

Due to the stoichiometry of the biochemical pathways, glycolysis in the case of glucose, pentose phosphate pathway in the case of xylose, the maximum achievable yield in each case 0.5 g of 2,3-butanediol per g of glucose, or xylose ( Jansen et al., Biotechnol. Bioeng. (1984) 26: 362-369 ). Obviously, the metabolism of xylose via the pentose phosphate pathway does not lead as efficiently to the C4 products acetoin and 2,3-butanediol as the metabolism of glucose via glycolysis.

US 7998722 discloses recombinant strains of the genera Zymomonas and Zymobacter with improved xylose utilization. The improved xylose utilization is achieved primarily by expression of a gene encoding a xylose isomerase. By additional expression of the genes of a xylulokinase, as well as the genes for a transketolase and a transaldolase, the xylose utilization is further improved. The thus optimized strains can be used for the production of the C2 compound ethanol from xylose. Gene constructs are disclosed which, above all, improve the expression of xylose isomerase and thereby lead to improved xylose utilization. US 7998722 discloses that in strains of the genera Zymomomonas and Zymobacter, at least four genes, including the transketolase and transaldolase genes, must be overexpressed to enhance xylose utilization and use such strains for ethanol production, the production of C4 products is not addressed ,

The object of the present invention is to provide a production strain for the fermentative production of C4 products, which is able to utilize as C source in the fermentation of lignocellulose-containing biomass and the sugar which can be isolated from hemicellulose.

The problem has been solved by a production strain which can be produced from an initial strain, characterized in that the parent strain naturally results in 2,3-butanediol production is capable of wild-type strain and the production strain at the same time an increased compared to the parent strain expression of a gene encoding a transketolase (EC 2.2.1.1) and a gene encoding a transaldolase (EC 2.2.1.2).

In the present invention, a distinction is made between a parent strain and a production strain. The parent strain is a wild-type strain naturally capable of 2,3-butanediol production. Such wild-type strains are z. B. disclosed in Celinska and Grajek, Biotechnol. Advances (2009) 27: 715-725 (see Chapter 4, "Microorganisms") ,

For the purposes of the present invention, a production strain is thus to be understood as meaning an initial strain which is optimized with regard to 2,3-butanediol production and which is characterized by an expression of the genes coding for the enzymes transketolase and transaldolase in comparison to the starting strain. The production strain is preferably produced from the parent strain.

Preferred starting strains are microorganism strains selected from the genera Raoultella, Klebsiella, Bacillus, Paenibacillus, Serratia or Lactobacillus.

Particularly preferred starting strains are microorganism strains selected from the genus Raoultella or Klebsiella, particularly preferably Raoultella terrigena. These strains are commercially available for. B. in the DSMZ German Collection of Microorganisms and Cell Cultures GbmH (38124 Braunschweig, Inhoffenstraße 7B). This also applies to the strain Raoultella terrigena DSM 2687 used in the examples, which is a particularly preferred starting strain.

The C4 products are preferably 2,3-butanediol, acetoin or diacetyl, preferably 2,3-butanediol or acetoin and more preferably 2,3-butanediol.

The sugars obtained from lignocellulose-containing biomass and the hemicellulose isolable therefrom are preferably pentoses (C5-sugars) and particularly preferably xylose.

Transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2) are both enzymes from the non-oxidative part of the pentose phosphate pathway. Among other things, they are involved in the metabolism of D-xylose. D-xylose is first converted to D-xylulose-5-phosphate by the enzymes xylose isomerase and xylulokinase. Starting from D-xylulose-5-phosphate then takes place the further metabolism in addition to the enzymes ribulose-5-phosphate epimerase and ribose-5-phosphate isomerase and transketolase and transaldolase are involved. The following reactions described in equations (IV) to (VIII) proceed.

Ribulose-5-phosphate epimerase:

• D-xylulose-5-phosphate → D-ribulose-5-phosphate (IV)

Ribose 5-phosphate isomerase:

• D-ribulose-5-phosphate → D-ribose-5-phosphate (V)

transketolase:

• D-xylulose-5-phosphate + D-ribose-5-phosphate → D-sedudeptulose-7-phosphate + glyceraldehyde-3-phosphate (VI)

transaldolase:

• D-Sedudeptulose-7-phosphate + glyceraldehyde-3-phosphate → fructose-6-phosphate + erythrose-4-phosphate (VII)

transketolase:

• Erythrose-4-phosphate + D-xylulose-5-phosphate → fructose-6-phosphate + glyceraldehyde-3-phosphate (VIII)

In sum, in the transketolase and transaldolase-containing reaction sequence, three molecules of D-xylose (C5 sugar) are formed into two molecules of fructose-6-phosphate (C6 sugar) and one molecule of glyceraldehyde-3-phosphate (C3 compound), both intermediates Glycolysis and thus the central metabolism available.

The simultaneously overexpressed genes of transketolase (Tkt gene) and transaldolase (Tal gene) are genes which code for enzymes of the enzyme classes EC 2.2.1.1 (transketolase) and EC 2.2.1.2 (transaldolase) and according to the equations (VI ), (VII) and (VIII) are involved in the metabolism of D-xylose to fructose-6-phosphate and glyceraldehyde-3-phosphate. The overexpressed genes of transketolase and transaldolase may be either homologous or heterologous genes. Preferably, it is homologous genes.

As disclosed in Examples 4 and 6, the sole expression of either a transketolase or a transaldolase is not sufficient to increase the yield of C4 products. An improvement in the yield of C4 products is only achieved by simultaneous overexpression of a transketolase and transaldolase.

In a preferred embodiment, the Tkt gene (transketolase) and the Tal gene (transaldolase) are derived from a bacterium of the genus Raoultella.

In a particularly preferred embodiment, the Tkt gene (transketolase) and the Tal gene (transaldolase) are derived from a strain of the species Raoultella terrigena.

Especially preferred are the Tkt gene (transketolase) and the Tal gene (transaldolase) from the strain Raoultella terrigena DSM 2687. The Tkt gene has the SEQ ID NO: 1 and encodes a transketolase protein having the amino acid sequence SEQ ID NO: 2.

The gene has SEQ ID NO: 3 and encodes a transaldolase protein having the amino acid sequence SEQ ID NO: 4.

The simultaneous overexpression of the transketolase and transaldolase genes leads to production strains with increased enzyme activities of transketolase and transaldolase, as described in Example 3. Strains are preferred in which the simultaneous overexpression of the transketolase and transaldolase genes, the enzyme activity of transketolase and transaldolase each by at least a factor of 2, preferably by at least the factor 5, more preferably by at least a factor of 10 and more preferably by at least the factor 20 increased compared to the enzyme activity in the parent strain.

As shown in the examples of the present application (5th and 7th examples), the simultaneous overexpression of the genes of a transketolase and a transaldolase in the production strain according to the invention is suitable when using xylose as C source of fermentation or culture in the shake flask the yield of C4 products 2,3-butanediol and acetoin, and preferably 2,3-butanediol, to increase more than 20%, preferably 40% and most preferably more than 60% compared to the starting strain. At the same time, the proportion of by-product acetate in the fermentation decreases by at least 30%, preferably by at least 60%, and more preferably by 80%, compared to the starting strain.

The increase in the expression of the transketolase and transaldolase genes in the production strain can be caused by any mutation in the genome of the parent strain (eg the promoter activity-enhancing mutations) or by simultaneous overexpression of homologous or even heterologous transketolase and transaldolase genes in the parent strain.

Preferably, the simultaneous overexpression of homologous or heterologous transketolase and transaldolase genes in the parent strain.

Particularly preferred is the simultaneous overexpression of homologous transketolase and transaldolase genes in the parent strain.

The parent strain is a wild type strain naturally capable of 2,3-butanediol production (such as summarized in US Pat Celinska and Grajek, Biotechnol. Advances (2009) 27: 715-725, Chapter 4, "Microorganisms" ), which in addition already elsewhere with respect to the 2,3-butanediol Production may have been optimized. In an already z. B. optimized by a genetic engineering wild-type strain, however, the expression of transketolase and transaldolase is not affected by the optimization.

The increase in the expression of the transketolase and transaldolase genes in the production strain can be caused by any mutation in the genome of the parent strain (eg the promoter activity-enhancing mutations) or by simultaneous overexpression of homologous or even heterologous transketolase and transaldolase genes in the parent strain.

The production strain of the invention is preferably prepared by introducing a gene construct into one of said parent strains.

In its simplest form, the gene construct consists of the structural genes coding for a transketolase and a transaldolase, which are operatively linked and preceded by a promoter. Optionally, the gene construct may also comprise a terminator, which is connected downstream of the transketolase or transaldolase structural gene. Preferred is a strong promoter, which leads to a strong transcription. Preferred among the strong promoters is the "Tac promoter" familiar to the person skilled in the art from the molecular biology of E. coli.

The structural genes of transketolase and transaldolase can also be arranged together in a gene construct in a so-called operon form (construct pTkt-Tal, see 6 ).

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.

However, 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 is introduced into the production strain in a manner known per se by genetic transformation. Various methods of genetic transformation are known in the art ( Aune and Aachmann, Appl. Microbiol. Biotechnol. (2010) 85: 1301-1313 ), including, for example, electroporation.

For the selection of transformed production strains, the gene construct contains, likewise in a known manner, a selection marker for the selection of transformants with the desired gene construct. Known selection markers are selected from 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, neomycin or zeocin.

Particularly preferred are antibiotic resistance markers conferring resistance to the antibiotics ampicillin and tetracycline.

In the present invention, the production strain of the present invention simultaneously contains the gene constructs of a transketolase and a transaldolase, either as single constructs in a two-plasmid system or in a single-plasmid system. In a single-plasmid system, the transketolase and transaldolase gene constructs can be separated or in operon structure (see example 1 and 2) 6 , Plasmid pTkt valley). In a separate arrangement, the transcription of the two genes is carried out in each case by its own promoter, in an operon structure, both genes are transcribed together by a promoter.

It is well known to those skilled in the art that in a two-plasmid system the transketolase and transaldolase gene constructs must contain different selection markers, preferably different antibiotic resistance markers to select for strains which simultaneously contain the transketolase and transaldolase gene constructs.

Thus, in a preferred embodiment, a production strain according to the invention simultaneously contains the transketolase and transaldolase gene constructs, either as individual constructs or in Operonform. These constructs are either present in the production strain in plasmid form or integrated into the genome and simultaneously produce a transketolase enzyme and a transaldolase enzyme, each in recombinant form. The plasmid form is preferred.

The recombinant transketolase and transaldolase enzymes are able to convert C-5 sugars, preferably xylose, into C6 and C3 compounds via the pentose phosphate pathway and to infect the glycolysis pathway. It has now surprisingly been found that by simultaneous recombinant expression of the transketolase and transaldolase enzymes in the production strain, the yield of C4 products, in particular 2,3-butanediol, compared to the parent strain can be significantly increased.

The invention further relates to a fermentative process for the production of C4 products from C5 sugars.

The method is characterized in that a production strain according to the invention is cultured in a growth medium. On the one hand, biomass of the production strain and, on the other hand, the C4 products, including preferably 2,3-butanediol, are formed. The formation of biomass and the C4 products can be correlated in time or decoupled from each other in terms of 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 process on a production scale 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 C4 product formation, including preferably 2,3-butanediol.

Preferred C sources for growing the production strains are pentoses such. B. xylose, arabinose, or ribose and pentoses containing plant hydrolysates, which consist of lignocellulosic biomass such. B. sugar cane bagasse, cereal straw or waste of maize or wood processing, or the hemicellulose contained therein can be obtained. In this case, the hydrolysis of the lignocellulose-containing biomass can be connected upstream of the production process according to the invention or else take place in situ during the production process according to the invention.

Particularly preferred C source is xylose, either in isolated form or as part of a vegetable hydrolyzate.

C sources which are suitable for the production process according to the invention thus 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 thermal, chemical or enzymatic digestion of the vegetable raw materials ,

The use of the preferred and particularly preferred C sources thus does not exclude that the C sources according to the invention occur in admixture with other non-inventive C sources in the growth medium. These include all forms of monosaccharides, including C6 sugars such as. As glucose, mannose or fructose. However, the C sources also include disaccharides, in particular sucrose, lactose, maltose or cellobiose. Further affected are all C sources in the form of higher saccharides, glycosides or carbohydrates with more than two sugar units such. As maltodextrin, starch, cellulose, pectin or by hydrolysis (enzymatically or chemically) released monomers or oligomers.

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 , NaNO ₃ , ammonium nitrate, Ca (NO ₃ ) ₂ , Mg (NO ₃ ) ₂ and other N-sources such. B. 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.

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 feed components can be added together as a mixture or else separately in individual feed sections. In addition, other media components as well as specific C4 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 xylose or xylose-containing plant hydrolysates which can be obtained from lignocellulosic biomass waste.

Particularly preferred C source in the feed is xylose.

Preferred 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 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 suitable 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). Preferred is the aerobic cultivation with oxygen, wherein the oxygen supply is ensured by entry of compressed air or pure oxygen. Particularly preferred is the aerobic cultivation by entry of compressed air.

The cultivation time for C4 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 C4 product, including preferably 2,3-butanediol, preferably in the culture supernatant. The C4 product contained in the cultivation mixtures can either be used directly again without further work-up or else be isolated from the cultivation batch. For isolation of the C4 product, including preferably 2,3-butanediol, known process steps are available, including centrifugation, decantation, filtration, extraction (see, for example, US Pat. DE 10 2011 004 915 ), Distillation or crystallization, or precipitation. These process steps can be combined in any desired form in order to isolate the C4 product in the desired purity. The degree of purity to be achieved depends on the further use.

Various analytical methods for the identification, quantification and determination of the purity of the C4 products, including preferably 2,3-butanediol, are available, including NMR, gas chromatography, HPLC, mass spectroscopy or even a combination of these analysis methods.

The figures show the plasmids mentioned in the examples.

1 shows the 2.9 kb exposition vector pKKj used in Example 1.

2 shows the 5 kb transketolase expression vector pTkt prepared in Example 1.

3 shows the 3.8 kb transaldolase epitope pTal prepared in Example 1.

4 shows the 4.3 kb transaldolase epitope pTal-tet prepared in Example 1.

5 shows the 3.3 kb expression vector pKKj-tet used in Example 1.

6 shows the 6 kb vector pTkt-valley produced in Example 1 for the simultaneous expression of the transketolase and transaldolase gene.

The invention is further illustrated by the following examples:

Example 1: Production of transketolase and transaldolase expression vectors

Raoultella (Klebsiella) terrigena DSM 2687 (commercially available from the DSMZ German Collection of Microorganisms and Cell Cultures GmbH) was used as a starting strain for gene isolation and for strain development.

The transketolase and transaldolase genes from R. terrigena were used. The DNA sequences of the genes from R. terrigena are disclosed in SEQ ID NO: 1 (tansketolase), coding for a protein with the amino acid sequence SEQ ID NO: 2, and in SEQ ID NO: 3 (transaldolase), coding for a protein with the amino acid sequence SEQ ID NO: 4.

Genes of transketolase (Tkt gene) and transaldolase (Tal gene) were isolated in PCR reactions (Taq DNA polymerase, Qiagen) from genomic R. terrigena DNA (strain DSM 2687,).

The genomic DNA used for the PCR reactions was previously in a conventional manner with a DNA isolation kit (Qiagen) from cells of the R. terrigena DSM 2687 culture in LB medium (10 g / l tryptone, 5 g / l yeast extract , 5 g / l NaCl).

The following primers were used for the PCR reactions:
Transketolase (Tkt gene): primer Tkt-1f (SEQ ID NO: 5) and Tkt-2r (SEQ ID NO: 6)

Transaldolase (Tal-Gen): Primer Tal-1f (SEQ ID NO: 7) und Tal-2r (SEQ ID NO: 8)The primers had the following DNA sequences:

Transaldolase (Tal gene): Primer Tal-1f (SEQ ID NO: 7) and Tal-2r (SEQ ID NO: 8)

The primers had the following DNA sequences:

The tansketolase gene was isolated in the PCR reaction as a 2100 bp DNA fragment. For subsequent cloning into the expression vector pKKj cut with Eco RI and Hind III, the Tkt PCR product was post-digested with Mfe I (contained in primer Tkt-1f, compatible with Eco RI) and Hind III (contained in primer Tkt-2r). Expression vector pKKj is disclosed in DE 10 2011 003 394 , pKKj is a derivative of the expression vector pKK223-3. The DNA sequence of pKK223-3 is disclosed in the "GenBank" gene database under the access number M77749.1. Approximately 1.7 kb were removed from the 4.6 kb plasmid (bp 262-1947 of the DNA sequence disclosed in M77749.1), whereby the 2.9 kb expression vector pKKj (FIG. 1 ) originated.

When cloning the transketolase gene into the pKKj vector, the 5 kb vector pTkt ( 2 ), in which the transketolase gene is functionally linked to the tac promoter and which is suitable for expression of the transketolase gene in R. terrigena.

The Tanzaldolase gene was isolated in the PCR reaction as a 950 bp DNA fragment. For subsequent cloning into the pKKj expression vector cut with Eco RI and Pst I, the valley PCR product was digested with Eco RI (contained in primer Tal-1f) and Pst I (contained in primer Tal-2r). The result was the 3.8 kb vector pTal ( 3 ) in which the transaldolase gene is functionally linked to the tac promoter and which is suitable for expression of the transaldolase gene in R. terrigena.

Expression vector pTal-tet:

The post-digested with Eco RI and Pst I 950 bp PCR product of the transaldolase gene was additionally cloned into the previously cut with Eco RI and Pst I expression vector pKKj-tet. This resulted in the 4.3 kb expression vector pTal-tet ( 4 ).

pKKj-tet is revealed in DE 10 2011 003 386 , To produce it, the ampicillin resistance gene was first removed from pKKj as a 1 kb fragment by digestion with Ex HI. In the remaining 1.9 kb vector fragment, the tetracycline resistance gene was cloned as a 1.45 kb fragment. The tetracycline resistance gene had previously been isolated from the plasmid pACYC184 by PCR (Taq DNA polymerase, Qiagen). The DNA sequence of pACYC184 is available in the "Genbank" gene database under accession number X06403.1. The result was the 3.3 kb expression vector pKKj-tet ( 5 ).

Expression vector pTkt-Tal:

The expression vector pTal was digested with Eco RI and the 3.8 kb linearized vector fragment isolated. The Tkt gene fragment was, as described in the expression vector pTkt, in a PCR reaction from genomic R. terrigena DNA with the primers Tkt-1f (SEQ ID NO: 5) and Tkt-3r (SEQ ID NO: 9) as 2.1 kb fragment isolated. Primer Tkt-3r, in addition to an Mfe I site (compatible with Eco RI for subsequent cloning into the vector pTal) and the 3 'end of the Tkt gene, also contained a ribosome binding site (RBS, see 6 ) for the production of the subsequent in the vector pTkt valley Tal gene (so-called artificial operon).

Primer Tkt-3r (SEQ ID NO: 9) had the following DNA sequence:

The 2.1 kb Tkt gene fragment was digested with Mfe and cloned into the Eco RI cut vector pTal. The result was the 6 kb expression vector pTkt-Tal ( 6 ). Of the clones obtained, those were selected in which the transketolase gene was functionally linked to the tac promoter, ie the transketolase gene was incorporated in the same orientation as the transaldolase gene (see 6 ).

In pTkt-Tal, the transketolase and the transaldolase gene are arranged as an artificial operon. Their expression is under the control of the tac promoter.

Example 2: Comparative fermentation of the initial strain Raoultella (Klebsiella) terrigena DSM 2687 with glucose and xylose as C source

"Labfors II" fermenters from Infors were used. The working volume was 1.5 l (3 l fermentor volume). The fermenters were equipped according to the prior art with electrodes for measuring the PO ₂ and the pH and with a foam probe, which regulated, if necessary, the addition of an antifoam solution. The values of the probes were recorded by a computer program and graphically displayed. The fermentation parameters stirrer speed (rpm), aeration (supply with compressed air in vvm, volume of compressed air per volume of fermentation medium per minute), pO ₂ (oxygen partial pressure, on 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. Feed medium (73% glucose, w / v or 75% xylose, w / v) was metered in according to the consumption of glucose or xylose 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.

The strain used in the fermentation was Raoultella (Klebsiella) terrigena DSM 2687 (control strain R.t.-WT). Batch fermentation media were FM2-G (C source glucose) and FM2-X (C source xylose).

FM2-G medium contained glucose 40 g / L; CSD (Corn Steep Liquor dried) 10 g / l; Ammonium sulfate 5 g / l; NaCl 0.5 g / l; FeSO ₄ × 7H ₂ O 75 mg / l; Na ₃ citrate × 2H ₂ O 1 g / l; CaCl ₂ × 2H ₂ O 14.7 mg / L; MgSO ₄ .7H ₂ O 0.3 g / l; KH ₂ PO ₄ 1.5 g / l; Trace element mix 10 ml / l. The pH of the FM2-G medium was adjusted to 6.0 before starting the culture.

FM2-X medium contained the same composition as FM2-G medium. However, glucose was replaced by xylose (40 g / L).

The trace element mix had the composition H ₃ BO ₃ 2.5 g / l; CoCl ₂ × 6H ₂ O 0.7 g / l; CuSO ₄ × 5H ₂ O 0.25 g / l; MnCl ₂ × 4H ₂ O 1.6 g / l; ZnSO ₄ .7H ₂ O 0.3 g / l and Na ₂ MoO ₄ .2H ₂ O 0.15 g / l.

1.35 l of FM2-G or FM2-X medium were inoculated with 150 ml of preculture. The preculture of the strains to be fermented was prepared by 24 h shake flask growing in glucose containing FM2-G medium. The fermentation conditions were: temperature 30 ° C, stirrer speed 1000 rpm, aeration with 1 vvm, pH 6.3.

The fermenter was sampled periodically to analyze the following parameters:
The cell density OD ₆₀₀ as a measure of the biomass formed was determined photometrically at 600 nm (BioRad photometer SmartSpec ^™ 3000).

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

The glucose or xylose content of fermenter samples was determined using the analyzer 7100MBS from YSI equipped with measuring stations for both glucose and xylose.

The content of C4 products (2,3-butanediol and acetoin) and of C2 products (acetate and ethanol) in culture supernatants was determined in a manner known per se by ¹ H-NMR (see, for example, US Pat. DE 10 2011 003 394 ). For this purpose, in each case an aliquot of the culture was centrifuged (10 min 5,000 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 / l) mixed in D ₂ O. The ¹ H-NMR analysis including peak integration was carried out according to the prior art with an Avance 500 NMR instrument from Bruker. For quantitative analysis, the NMR signals of the analytes were integrated in the following ranges: TSP: 0.140-0.145 ppm (9H) 2,3-butanediol: 1.155-1.110 ppm (6H) ethanol: 1.205-1.157 ppm (3H) Acetic acid: 2,000-1,965 ppm (3H) acetoin: 2,238-2,200 ppm (3H)

After the glucose or xylose present in the batch medium had been consumed, feed solutions of glucose (73% w / v) or xylose (75% w / v) were each pumped (Watson Marlow peristaltic pump 101 U / R). v) fed. The feeding rate was determined by the current glucose or xylose consumption rate.

Table 1 shows the time-dependent formation of C2 and C4 products in the R. terrigena control strain Rt-WT with glucose and xylose as the C source of the fermentation. Table 1: Production process in fed-batch fermentation with glucose and xylose as C source C source glucose C source xylose Time (h) product (G / l) (G / l) 24 2,3-BDL 72.5 44.6 acetoin 2.6 13.1 acetate 1.5 9.1 ethanol 6.3 2.2 48 2,3-BDL 109.7 61.8 acetoin 6.3 16.0 acetate 3.5 19.9 ethanol 5.3 1.7 72 2,3-BDL 117.1 62.1 acetoin 6.9 17.7 acetate 3.7 23.0 ethanol 5.4 1.6

When using xylose as the C source of the fermentation instead of glucose, a significant shift in the product spectrum was observed. With xylose as the C source, significantly less 2,3-butanediol was formed while the proportion of the second C4 product acetoin (the third possible C4 product diacetyl was undetectable) increased. However, with Xylose as the C source, the space-time yield of the C4 products 2,3-butanediol and acetoin was significantly higher after 72 h at 1.1 gl ^-1 h ^-1 than in the prior art Yu and Saddler, Appl. Environ. Microbiol. (1983) 46: 630-635 (about 0.6 gl ^-1 h ^-1 , as out there 3 and 4 removed).

In the C2 products, a large increase of acetate was observed and a decrease of ethanol. The metabolism of xylose via the pentose phosphate pathway obviously leads to a significantly different product spectrum than that of glucose via glycolysis.

3. Example: Expression of transketolase and transaldolase genes in Raoultella terrigena

Starting strain was Raoultella (Klebsiella) terrigena DSM 2687. The transformation with the plasmids pKKj (empty vector), pTkt, pTal, pTal-tet and pTkt-Tal was carried out in a manner known per se (see, for example, US Pat. DE 10 2011 003 394 ), analogous to the methods known to those skilled in the transformation of E. coli. The strains obtained were designated Rt-pKKj (control strain), Rt-pTkt (transketolase), Rt-pTal (transaldolase), Rt-pTal-tet (transaldolase), Rt-pTkt-Tal (transketolase-transaldolase operon construct) and Rt -pTkt-ptal. The strain Rt-pTkt-pTal was a two-plasmid system for the simultaneous expression of transketolase and transaldolase in a strain obtained by transformation of R. terrigena with the vectors pTkt (ampicillin selection) and pTal-tet (tetracycline selection).

Transformants were isolated and the recombinant expression of the Tkt and Tal genes by Schüttelkolbenanzucht examined by on the one hand the respective enzyme activity and on the other hand, the production of the C4 products 2,3-butanediol and acetoin and the C2 products acetate and ethanol was determined. In each case 50 ml of FM2-G or FM2-X medium were inoculated and incubated at 30 ° C and 140 rpm (Infors shaker). Depending on the strain, the media contained as selective antibiotics either ampicillin (FM2-G-amp, or FM2-X-amp) or tetracycline (FM2-G-tet, or FM2-X-tet), and ampicillin and tetracycline ( FM2-G-tet + amp or FM2-X-tet + amp). FM2-G medium and FM2-X medium are described in Example 2. Depending on the expression vector, the media contained ampicillin (200 mg / L, FM2-G and FM2-X-amp), tetracycline (15 mg / L, FM2-G-tet and FM2-X-tet), and both antibiotics together (FM2-G-tet + amp and FM2-X-tet + amp).

In a first experiment, the expression of the transketolase and transaldolase genes was exemplarily checked for R. terrigena DSM 2687, transformed either with pKKj (strain Rt-pKKj, control strain), pTkt (strain Rt-pTkt) or with pTal (strain Rt-pTal ). 50 ml FM2-G-amp were inoculated with the control strain Rt-pKKj (negative control) as well as with Rt-pTkt and Rt-pTal.

After 24 h incubation at 30 ° C and 140 rpm (Infors shaker) cell growth was determined photometrically at 600 nm wavelength (OD ₆₀₀ ) and in a conventional manner, the protein composition of the three strains determined by SDS-PAGE (SDS-PAGE: sodium Dodecyl sulfate polyacrylamide gel electrophoresis). The commercially available SDS-PAGE system Novex ^® NuPAGE 12% was used Bis-Tris gels Fa. Life Technologies.

For sample preparation for SDS-PAGE, respectively, an aliquot corresponding to a cell density of OD was calculated from the shake flask cultures of 2 ₆₀₀ removed (approximately 0.2 ml) (in 0.4 ml of SDS and β-mercaptoethanol-containing sample buffer Novex ^® "Tris Glycine SDS Sample Buffer "from the Fa. Invitrogen), incubated for 3 min at 94 ° C and then applied 10 .mu.l of each sample on adjacent tracks of a polyacrylamide gel and separated in a known manner by electrophoresis. As size standard, a mix of proteins of known size was applied in another lane. After completion of the electrophoresis, the proteins separated in the gel according to their size were stained in a known manner by the Coomassie Brilliant Blue dye.

A comparison of the protein patterns revealed that over the control strain Rt-pKKj (negative control) in the strains Rt-pTkt, Rt-pTkt-Tal and Rt-pTkt-pTal overexpression of the transketolase gene to amplify a band at a molecular weight of about 70 kD (relative molecular weight of 70,000 daltons) and in the strains Rt-pTal-tet, Rt-pTkt-Tal and Rt-pTkt-pTal overexpression of the transaldolase gene to amplify a band at a molecular weight of about 35 kD (molecular weight of 35,000 daltons ).

The remaining cells of the shake flask cultures (approximately 50 ml volume) were used in a second experiment to determine the enzyme activities of the recombinantly expressed transketolase and transaldolase compared to the control strain. The determination of the enzyme activities of transketolase and transaldolase was carried out according to the prior art ( Josephson and Fraenkel, J. Bacteriol. (1969) 100: 1289-1295 ).

The transketolase enzyme assay is based on equation (VI), in which D-xylulose-5-phosphate and D-ribose-5-phosphate as one of the reaction products form glyceraldehyde-3-phosphate. Glyceraldehyde-3-phosphate is converted to dihydroxyacetone phosphate by the triosephosphate isomerase enzyme, which is finally reduced by the enzyme glycerol-3-phosphate dehydrogenase, also contained in the test, with stoichiometric consumption of NADH.

The transaldolase enzyme assay is based on the reverse reaction of equation (VII), in which glyceraldehyde-3-phosphate is formed from fructose-6-phosphate and erythrose-4-phosphate as one of the reaction products. Glyceraldehyde-3-phosphate is converted by the enzyme Triosephosphatisomerase contained in the test to dihydroxyacetone phosphate, which is finally reduced by the enzyme also contained in the test glycerol-3-phosphate dehydrogenase with stoichiometric consumption of NADH.

50 ml of the cells from the shake flask were centrifuged (10 min 15000 rpm, Sorvall SS34 rotor), the cell pellet in 2 ml Tris buffer (0.05 M Tris-HCl, pH 7.6, 0.01 M MgCl ₂ × 6H ₂ O) was added, so-called in a known manner with a. digested "French Press ^®" high-pressure homogenizer (SLM-AMINCO) 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 transketolase or transaldolase activity.

Spectrophotometric determination of transketolase activity:

The measurement batch of 1 ml volume for the photometric determination of transketolase activity (components of the enzyme assay all from Sigma-Aldrich) was composed of Tris buffer, 0.5 mM ribose-5-phosphate, 0.5 mM xylulose-5 Phosphate, 0.2 mM NADH and 10 μg of an enzyme mixture consisting of α-glycerol-3-phosphate dehydrogenase and triosephosphate isomerase (Sigma-Aldrich) and transketolase-containing cell extract. Measurement temperature was 25 ° C. The reaction was started by adding a mix of the substrates ribose-5-phosphate and xylulose-5-phosphate (mix each 20-fold concentrated) and the Extinction decrease due to the consumption of NADH measured at a wavelength of 340 nm (extinction coefficient of NADH: ε = 0.63 × 10 ⁴ l × mol ^-1 × cm ^-1 ). A unit transketolase activity is defined as the consumption of 1 μmol NADH / min under assay conditions.

Spectrophotometric determination of transaldolase activity:

The measurement batch of 1 ml volume for the photometric determination of transaldolase activity (components of the enzyme assay all from Sigma-Aldrich) was composed of Tris buffer, 0.3 mM erythrose-4-phosphate, 0.4 mM fructose-6 Phosphate, 0.2 mM NADH and 10 μg of an enzyme mixture consisting of α-glycerol-3-phosphate dehydrogenase and triosephosphate isomerase (Sigma-Aldrich) and transaldolase-containing cell extract. Measurement temperature was 25 ° C. The reaction was started by adding a mixture of the substrates erythrose-4-phosphate and fructose-6-phosphate (mix each 20-fold concentrated) and the absorbance decrease due to the consumption of NADH measured at a wavelength of 340 nm (extinction coefficient of NADH: ε = 0.63 x 10 ⁴ l x mol ^-1 x cm ^-1 ). A unit transaldolase activity is defined as the consumption of 1 μmol NADH / min under assay conditions.

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.

The enzyme activities determined under measurement conditions are listed in Tables 2 and 3. Table 2: Enzyme activity of recombinant, transketolase overexpressing R. terrigena strains tribe construct Transketolase (U / mg) relative activity Rt-pKKj pKKj, control 0.06 1 Rt-PTKT PTKT 1.63 27.2 Rt-PTKT Valley PTKT Valley 1.47 24.5 Rt-PTKT-ptal pTkt + pTal-tet 1.58 26.3 Table 3: Enzyme activity of recombinant, transaldolase overexpressing R. terrigena strains tribe construct Transaldolase (U / mg) relative activity Rt-pKKj pKKj, control 0.12 1 Rt-ptal-tet ptal-tet 2.94 24.5 Rt-PTKT Valley PTKT Valley 2.27 18.9 Rt-PTKT-ptal pTkt + pTal-tet 2.51 20.9

As shown in Tables 2 and 3, transketolase activity in recombinant R. terrigena strains is increased by a factor of 24.5-27.2 by overexpression, transaldolytic activity in recombinant R. terrigena strains is increased by a factor of 18.9-24.5 ,

Example 4: Comparative shake flask culture of recombinant R. terrigena strains with glucose and xylose as C source

The cultivation of the strains Rt-pKKj, Rt-pTkt and Rt-pTal was carried out as described in Example 3. Culture media (see Example 2) were each FM2-G-amp (glucose medium), or FM2-X-amp (xylose medium). The culture time was 96 h. The glucose and xylose concentrations were determined at intervals of 24 h (analyzer 7100MBS from YSI, equipped with measuring stations for both glucose and xylose) and glucose, or xylose, if necessary, from 40% (w / v) stock solutions. At intervals of 24 h samples were examined for their content of C4 and C2 products. The production curves for the C source glucose are shown in Table 4, for the C source xylose in Table 5. Table 4: Production process in recombinant Raoultella terrigena strains with glucose as C source Time (h) product Rt-pKKj (g / l) Rt-ptkt (g / l) Rt-pTal (g / l) 48 2,3-BDL 26.4 21.0 29.0 acetoin 6.8 7.5 5.2 acetate 2.0 1.8 1.7 ethanol 4.0 2.8 4.2 72 2,3-BDL 43.5 37.0 39.5 acetoin 8.1 7.5 6.9 acetate 2.4 2.0 2.1 ethanol 5.2 3.7 5.0 96 2,3-BDL 57.1 57.2 50.8 acetoin 7.6 6.4 6.9 acetate 2.3 2.0 2.1 ethanol 6.1 5.0 5.7 Table 5: Production process in recombinant Raoultella terrigena strains with xylose as C source Time (h) product Rt-pKKj (g / l) Rt-ptkt (g / l) Rt-pTal (g / l) 48 2,3-BDL 25.0 24.2 24.1 acetoin 3.3 4.5 3.1 acetate 3.5 3.8 2.9 ethanol 3.6 3.9 3.7 72 2,3-BDL 26.8 26.7 28.6 acetoin 3.9 5.1 4.2 acetate 5.0 5.1 4.8 ethanol 3.3 3.2 3.5 96 2,3-BDL 26.1 26.6 25.1 acetoin 4.5 5.7 6.4 acetate 5.3 5.9 5.4 ethanol 3.0 3.0 3.1

As can be seen from Tables 4 and 5, the sole overexpression of the transketolase gene as well as the transaldolase gene resulted in no significant change in the spectrum of C4 products (2,3-butanediol and acetoin) and C2 products (acetate and ethanol) , This was independent of which C source was used. However, xylose as a C source in shake flask cultivation resulted in significantly lower yields of C4 products, in particular of 2,3-butanediol, than glucose (see also Example 2).

5. Example: Shake flask culture of the recombinant R. terrigena strain R.t. pTkt valley with glucose and xylose as C source

The cultivation of the strains Rt-pKKj and Rt-pTkt-Tal (Operon construct, see Example 1) was carried out as described in Example 4. Culture media (Example 2) were FM2-G-amp (glucose medium) and FM2-X-amp (xylose medium), respectively. The culture time was 96 h. The glucose and xylose concentrations were determined at intervals of 24 h (analyzer 7100MBS from YSI, equipped with measuring stations for both glucose and xylose) and glucose, or xylose, if necessary, from 40% (w / v) stock solutions. At intervals of 24 h samples were examined for their content of C4 and C2 products. The production curves for both the C source glucose and xylose are shown in Table 6. Table 6: Production process of the recombinant Raoultella terrigena strains Rt-pKKj and Rt-pTkt-valley with glucose, or xylose as C source C source glucose xylose Time (h) product Rt-pKKj (g / l) Rt-ptkt valley (g / l) Rt-pKKj (g / l) Rt-ptkt valley (g / l) 48 2,3-BDL 26.6 25.9 20.9 18.5 acetoin 6.1 7.6 2.2 4.8 acetate 2.2 2.4 3.1 2.4 ethanol 3.3 3.2 3.7 3.4 72 2,3-BDL 38.5 38.9 25.2 29.2 acetoin 9.1 9.1 3.4 3.7 acetate 2.3 2.6 4.2 3.9 ethanol 4.0 4.0 4.0 3.3 96 2,3-BDL 50.8 49.1 28.2 37.7 acetoin 9.3 10.1 3.8 3.9 acetate 2.6 2.6 4.6 4.1 ethanol 4.7 4.8 3.6 3.3

As shown in Table 6, the yields of C4 and C2 products of the transketolase and transaldolase were simultaneously recombinantly producing strain R.t. pTkt valley comparable to the control strain R.t.-pKKj, if glucose was used as C source. On the other hand, when xylose was used as the C source, the strain Rt-pTkt-Tal, in which the transketolase and transaldolase genes were overexpressed simultaneously (operon construct), faced a significant increase in the 2,3-butanediol yield of 30 to 40% to observe the control strain Rt-pKKj. This unexpected increase was particularly pronounced for 2,3-butanediol production as the yields of the other products, acetoin, acetate and ethanol, were barely altered under these conditions.

6th example:

Production of C4 and C2 compounds with transketolase and transaldolase overexpressing R. terrigena strains by fed-batch fermentation with xylose as C source

The fermentation was carried out as described in Example 2. Strains used in the fermentation were the R.t. pKKj (control strain) described in Example 3, R.t. pTkt (transketola strain) and R.t. pTal (transaldolas strain). Batch fermentation medium was the FM2-X-amp medium described in Example 2.

1.35 l of FM2-X-amp 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 glucose-containing FM2-G-amp (see Example 2). The fermentation conditions were: temperature 30 ° C, stirrer speed 1000 rpm, aeration with 1 vvm, pH 6.3.

After the xylose present in the batch medium had been consumed, a 75% (w / v) xylose feed solution was fed in via a pump (Watson Marlow peristaltic pump 101 U / R). The feeding rate was determined by the current xylose consumption rate.

Table 7 shows the time-dependent formation of C2 and C4 products in the R. terrigena control strain Rt-pKKj and in the recombinant, transketolase or transaldolase overproducing Raoultella terrigena strains Rt-pTkt, or Rt-pTal. Table 7: Production process of recombinant R. terrigena strains in fed-batch fermentation with xylose as C source Time (h) product Rt-pKKj (g / l) Rt-ptkt (g / l) Rt-pTal (g / l) 24 2,3-BDL 46.4 45.56 38.3 acetoin 8.0 7.3 8.2 acetate 11.9 7.3 10.3 ethanol 3.2 1.4 0.0 48 2,3-BDL 59.7 53.8 52.7 acetoin 17.1 21.4 12.4 acetate 22.1 11.7 12.0 ethanol 1.4 1.3 1.0 72 2,3-BDL 60.6 61.2 55.1 acetoin 18.9 17.6 13.9 acetate 29.9 16.3 15.2 ethanol 1.9 2.1 1.5

As already observed in the shake flask experiments (Example 4), the sole overexpression of the transketolase or tansaldolase gene did not lead to a significant change in the product spectrum. By sole overexpression of the transketolase or transaldolase, the yield of 2,3-butanediol or the other products could not be significantly improved. On the contrary, the product yield in the transaldolase overexpressing strain R.t. pTal was rather reduced.

7. Example:

Production of C4 and C2 compounds with transketolase and transaldolase overexpressing recombinant R. terrigena strains R.t. pTkt-pTal and R.t. pTkt valley by fed-batch fermentation with xylose as C source

Fed-batch fermentation of strains R.t. pTkt-pTal and R.t.-pTkt valley with xylose as C source was performed as described in Example 6. The control strain used was R.t. pKKj. The preparation of the recombinant strains used was described in Example 3. Precult (FM2-G-amp) and Batch (FM2-X-amp) strains of the strains R.t. pKKj and R.t. pTkt-Tal contained ampicillin (see Example 3). Precult culture (FM2-G-tet + amp) and batch medium (FM2-X-tet + amp) in the fermentation of the strain Rt-pTkt-pTal transformed with the two plasmids pTkt and pTal-tet contained both ampicillin and tetracycline (see FIG. Example).

Table 8 shows the time dependent formation of C2 and C4 products in the R. terrigena control strain Rt-pKKj and in the recombinant, transketolase and transaldolase overproducing Raoultella terrigena strains Rt-pTkt-pTal and Rt-pTkt-Tal. Table 8 Production history of the recombinant R. terrigena strains Rt-pTkt-pTal and Rt-pTkt-valley in fed-batch fermentation with xylose as C source Time (h) product Rt-pKKj (g / l) Rt-pTkt-pTal (g / l) Rt-ptkt valley (g / l) 24 2,3-BDL 44.6 43.8 58.8 acetoin 13.1 6.9 4.5 acetate 9.1 7.0 6.8 ethanol 2.2 2.4 2.9 48 2,3-BDL 56.7 69.7 68.8 acetoin 14.2 16.0 13.3 acetate 13.2 3.2 10.2 ethanol 1.9 2.2 2.6 72 2,3-BDL 57.5 104.1 89 acetoin 21.3 10.1 15.9 acetate 28.3 4.7 19.5 ethanol 2.0 2.2 1.6

In contrast to the recombinant strains Rt-pTkt and Rt-pTal, in each of which only one of the two genes was overexpressed (Example 6), the strains according to the invention characterized Rt-pTkt-pTal and Rt-pTkt-Tal, in which both the transketolase and the transaldolase gene were overexpressed in the fermentation with xylose as C source by a significantly improved C4 production. The increase compared to the control strain Rt-pKKj for the strain Rt-pTkt-Tal was 54% and for the strain Rt-pTkt-pTal 80%, especially with respect to 2,3-butanediol. Based on the sum of the C4 products acetoin and 2,3-butanediol, the increase compared with the control strain Rt-pKKj was 33% for the strain Rt-pTkt-Tal and 45% for the strain Rt-pTkt-pTal. Diacetyl could not be detected in any of the strains. In addition to the absolute yield, the space-time yield for the C4 products (sum of 2,3-butanediol and acetoin) was significantly improved (after 72 h fermentation period) of 1.09 gl ^-1 h ^-1 for the control strain Rt-pKKj to 1.49 gl ^-1 h ^-1 for strain Rt-pTkt-pTal and 1.46 gl ^-1 h ^-1 for strain Rt-pTkt-Tal. Accordingly, the strains according to the invention are distinguished not only by a markedly improved C4 productivity over the control strain Rt-pkkj, but are also significantly improved over that at Yu and Saddler, Appl. Environ. Microbiol. (1983) 46: 630-635 disclosed prior art (about 0.6 gl ^-1 h ^-1 , as out there 3 and 4 removed).

Furthermore, in the fermentation, the production of the by-product acetate was significantly reduced by 31% for the strain R.t. pTkt-Tal and by 83% for the strain R.t.-pTkt-pTal.

Table 8 thus shows that the spectrum of C4 and C2 production is altered by the simultaneous overexpression of the transketolase and transaldolase genes in the strains Rt-pTkt-pTal and Rt-pTkt-Tal, whereby the proportion of 2, 3-butanediols in total production (sum of C2 and C4 products) from 52.7% for the control strain Rt-pKKj to 86% for the strain Rt-pTkt-pTal, and 70.6% for the strain Rt- ptkt valley increased. The production strains according to the invention are thus distinguished not only by an overall higher C4 production, but also by a higher proportion of 2,3-butanediol in the total amount of C2 and C4 compounds produced (selectivity).

This surprising shift of the product spectrum in favor of 2,3-butanediol in the fermentation of xylose is unexpected, since on the one hand the respective overexpression of tansketolase and transaldolase alone had no influence on the 2,3-butanediol production (6th example) and the state the technique for other microorganisms ( US 7998722 and US 8034591 ) at least two additional genes had to be overexpressed in order to improve the xylose utilization in these strains and to increase the production of ethanol.

Furthermore, it was surprising that the transketolase and transaldolase are two enzymes of the pentose phosphate pathway, which are involved in the metabolism of the C source xylose and of which a direct effect on the biosynthesis of 2,3-butanediol was not expected. The starting point of 2,3-butanediol biosynthesis is pyruvate, the end product of glycolysis. Pyruvate is also the starting material for the other products acetoin, acetate and ethanol, so any conceivable direct influence of transketolase and transaldolase overexpression on the availability of pyruvate would also have affected the production of these compounds. This was not observed, so that the concomitant overexpression of the transketolase and transaldolase genes in the Rt-pTkt-pTal and Rt-pTkt-Tal production strains in fed-batch fermentation with the C-source xylose selectively inhibits the production of 2,3-butanediol improved.

Example 8: Fermentation with the C source xylose in 330 l scale

The strain Raoultella terrigena R.t. pTkt-pTal, which contained both the transketolase expression vector pTkt and the transaldolase expression vector pTal-tet, was fermented (see Examples 3 and 7).

Preparation of inoculum for the pre-fermenter: An inoculum of Rt-pTkt-pTal in LBtet + amp medium (LB medium containing additionally 15 mg / l tetracycline and 200 mg / l ampicillin) was prepared by adding 2x100 ml LBtet + amp Medium, in each case in a 1 l Erlenmeyer flask, each with 0.25 ml of a glycerol culture (overnight culture of the strain in LBtet + amp medium, mixed with glycerol in a final concentration of 20% v / v and stored at -20 ° C) were inoculated. Cultivation was carried out for 7 h at 30 ° C. and 120 rpm on an infus orbital shaker (cell density OD ₆₀₀ / ml of 0.5-2.5). 100 ml of the preculture were used to inoculate 8 liters of fermentation medium. Two pre-fermenters were inoculated with 8 l fermentation medium each.

Prefermenters: The fermentation was performed three fermenters the company Sartorius BBI Systems GmbH in two Biostat ^® C-DCU. Fermentation medium was FM2-G-tet + amp (see 3rd example). The fermentation took place in the so-called batch mode.

2 × 8 l FM2-G-tet + amp were inoculated with 100 ml of inoculum. Fermentation temperature was 30 ° C. pH of the fermentation was 6.3 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 pO ₂ was adjusted to 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 a fermentation time of 18 hours (cell density OD ₆₀₀ / ml of 30-40), the two pre-fermenters were used as the inoculum for the main fermenter.

Main fermentor: The fermentation was ^® in a Biostat D 500 fermenter (working volume 330 l, vessel volume 500 l) of the company Sartorius BBI Systems GmbH performed. Fermentation medium was FM2-X-tet + amp with xylose as C source (see 3rd example). The fermentation took place in the so-called fed-batch mode.

180 l of FM2-X-tet + amp were inoculated with 16 l of inoculum. Fermentation temperature was 30 ° C. pH of the fermentation was 6.3 and was kept constant with the correction agents 25% NH ₄ OH, or 6 NH ₃ PO ₄ . The aeration was carried out with compressed air at a constant flow of 1 vvm, based on the initial volume. The oxygen partial pressure pO ₂ was adjusted to 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 the fermentation, xylose consumption was measured by off-line xylose measurement with an Fe analyzer. YSI determined (see 2nd example). As soon as the xylose concentration of the fermentation mixtures was about 20 g / l (8-10 h after inoculation), the metered addition of a 75% w / v xylose feed solution was started. The flow rate of the feed was chosen so that a xylose concentration of 10-20 g / l could be maintained during the production phase. After completion of the fermentation, the volume in the fermenter was 300 l.

The analysis of the fermentation parameters was carried out as described in Example 2. The production process is shown in Table 9. Table 9 Production of 2,3-butanediol by fed-batch fermentation in 330 l scale Time (h) product Rt-pTkt-pTal (g / l) 24 2,3-BDL 59.9 acetoin 8.4 acetate 3.5 ethanol 3.4 48 2,3-BDL 79.5 acetoin 17.7 acetate 3.8 ethanol 2.2 72 2,3-BDL 91.5 acetoin 20.7 acetate 9.9 ethanol 2.2

This is followed by a sequence protocol according to WIPO St. 25. This can be downloaded from the official publication platform of the DPMA.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• US 2012288908 AA [0006]
• WO 12104234 A1 [0008]
• US 7998722 [0021, 0021, 0146]
• DE 102011004915 [0077]
• DE 102011003394 [0094, 0112, 0117]
• DE 102011003386 [0098]
• US8034591 [0146]

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Claims (9)

Production strain for the fermentative production of C4 products which is able to utilize as C source in the fermentation of lignocellulosic biomass and the hemicellulose isolable therefrom, producible from a parent strain, characterized in that the parent strain is a naturally 2 , 3-butanediol production is capable of wild-type strain and the production strain at the same time an increased compared to the parent strain expression of a gene encoding a transketolase (EC 2.2.1.1) and a gene encoding a transaldolase (EC 2.2.1.2). Production strain according to claim 1, characterized in that it has been produced from an initial strain of the genus Raoultella, Klebsiella, Bacillus, Paenibacillus, Serratia or Lactobacillus. Production strain according to Claim 1 or 2, characterized in that the C4 products are 2,3-butanediol, acetoin or diacetyl. Production strain according to one of Claims 1 to 3, characterized in that the sugar obtained from lignocellulose-containing biomass and the hemicellulose isolable therefrom are pentoses (C5-sugars), particularly preferably xylose. Production strain according to one of claims 1 to 4, characterized in that the overexpression of the transketolase and transaldolase genes is enhanced such that the enzyme activity of the transketolase and transaldolase in each case by at least a factor of 2, preferably by at least a factor of 5 and more preferably by at least the Factor 10 is increased compared to the enzyme activity in the parent strain. Production strain according to one of claims 1 to 5, characterized in that when using xylose as the C source in the fermentation or the culture in the shaking flask, the yield of C4 products 2,3-butanediol and acetoin, by more than 20% in Comparison to the parent strain is increased. A process for the preparation of C4 products from C5 sugars, characterized in that a production strain according to claim 1 to 6 is cultivated in a cultivation medium. A 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 takes place and a Cultivation period for C4 production between 10 h and 200 h is present. A method according to claim 7, or 8, characterized in that a cultivation takes place in a fermentation volume greater than 300 liters.

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