# Patent

## Title
Gene products of Bacillus licheniformis which form odorous substances and improved biotechnological production methods based thereon

## Abstract

The present invention relates to 25 hitherto undescribed genes of B. licheniformis and gene products derived therefrom and all sufficiently homologous nucleic acids and proteins thereof. They occur in five different metabolic pathways for the formation of odorous substances. The metabolic pathways in question are for the synthesis of: 1) isovalerian acid (as part of the catabolism of leucine), 2) 2-methylbutyric acid and/or isobutyric acid (as part of the catabolism of valine and/or isoleucine), 3) butanol and/or butyric acid (as part of the metabolism of butyric acid), 4) propyl acid (as part of the metabolism of propionate) and/or 5) cadaverine and/or putrescine (as parts of the catabolism of lysine and/or arginine). The identification of these genes allows biotechnological production methods to be developed that are improved to the extent that, to assist these nucleic acids, the formation of the odorous substances synthesized via these metabolic pathways can be reduced by deactivating the corresponding genes in the micro-organism used for the biotechnological production. In addition, these gene products are thus available for preparing reactions or for methods according to their respective biochemical properties.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
The present application is a Section 365(c) continuation of International Application No. PCT/EP2005/006540 filed 17 Jun. 2005, which in turn claims the priority of DE Application 10 2004 031 177.3 filed Jun. 29, 2004, each of which is incorporated herein by reference.


FIELD OF THE INVENTION
The present invention relates to 25 not previously described genes of B. licheniformis and gene products derived therefrom which are involved in the formation of odorous substances in five different metabolic pathways, and to biotechnological production methods which are improved inasmuch as, on the basis of the identification of these genes, the formation of these odorous substances can be reduced.
The present invention is in the area of biotechnology, in particular the preparation of valuable products by fermentation of microorganisms able to form the valuable products of interest. This includes for example the preparation of low molecular weight compounds, for instance of dietary supplements or pharmaceutically relevant compounds, or of proteins for which, because of their diversity, there is in turn a large area of industrial use. In the first case, the metabolic properties of the relevant microorganisms are utilized and/or modified to prepare the valuable products; in the second case, cells which express the genes of the proteins of interest are employed. In both cases, genetically modified organisms (GMO) are mostly involved.
There is an extensive prior art on the fermentation of microorganisms, especially on the industrial scale; it extends from the optimization of the relevant strains in relation to the formation rate and the nutrient utilization via the technical design of the fermenters and up to the isolation of the valuable products from the relevant cells themselves and/or the fermentation medium. Both genetic and microbiological, and process engineering and biochemical approaches are applied thereto. The aim of the present invention is to improve this process in relation to a common property of the microorganisms employed, which impairs the actual fermentation step, specifically at the level of the genetic properties of the strains employed.
For industrial biotechnological production, the relevant microorganisms are cultured in fermenters which are designed appropriate for their metabolic properties. During the culturing, they metabolize the provided substrate and normally form, besides the actual product, a large number of other substances in which there is usually no interest and/or which may lead to unwanted side effects.
These include odorous and/or poisonous substances which are a nuisance and/or harmful and are discharged even during the fermentation via the exit air and/or are only incompletely removed during the subsequent working up of the valuable product and thus impair the quality of the product. The concomitant odorous and/or poisonous substances are thus deleterious firstly for the production process, meaning the staff involved and the surroundings of the plant. Secondly, failure to reach a desired quality (specification) of the product may lead to it being unavailable for the intended area of use (for example food production), which means a considerable economic disadvantage. Conversely, reducing the formation of odorous and/or poisonous substances could increase occupational and environmental safety and open up additional areas of use and markets for sales of the product.
Odors frequently found during fermentation of microorganisms are caused by small organic molecules from the classes of volatile, branched and unbranched fatty acids, alcohols and diamines. These include isovaleric acid, 2-methylbutyric acid, isobutyric acid from the class of branched fatty acids, butyric acid, propionic acid (unbranched fatty acids), butanol (alcohol), cadaverine and putrescine (diamines).
Some of these volatile substances are additionally toxic for humans and animals, for example cadaverine and putrescine, which are also known as ptomaines. They can therefore be defined not only as odorous substances but also, depending on the concentration and the exposure time for the relevant organism, as poisonous substances.
Efforts are being made even at present to remove such compounds subsequently from fermentation products. For this purpose, usual working up of the valuable products formed comprises, besides steps to remove cell detritus and high molecular weight compounds, also additional process steps which are referred to as deodorizing. To these are ordinarily added filtrations, precipitation steps and/or chromatography steps, each of which also contribute to a certain extent to the deodorizing. Nevertheless, all these steps carried out for removal lead to a purity which is only inadequate according to the above-mentioned criteria.
The exit air from the fermenter is likewise checked in order to minimize the pollution during the production process.
It would nevertheless be desirable to combat odors causally where possible, i.e. to prevent the relevant substances being produced at all. It would thus be possible firstly to keep the number of subsequent purification and working-up steps small, which appears to be advantageous because they represent in each case a physicochemical stress on the desired product, and reduce the yield. Overall, therefore, a better product quality would be obtained. Secondly, the production conditions would be improved per se, and the systems for filtering the fermenter exit air could be kept simpler. Such a combating of odors causally would, if the properties of the microorganism itself were to be changed thereby, also increase its tolerability for further operations on this microorganism.
SUMMARY OF THE INVENTION
The object was thus to reduce the formation of unpleasant odors and/or poisonous compounds which occurs during the fermentation of microorganisms, especially Gram-positive bacteria of the species Bacillus, and is attributable to the same. It was intended preferably that this take place at the genetic level in order to obtain odorous and/or poisonous substance-depleted microorganisms. In partial problems, this means identifying metabolic pathways relevant thereto, finding genes which code for proteins and/or enzymes which catalyze reactions lying on these pathways and are suitable as possible starting points for solving the problem, and, via identification of the relevant nucleotide sequences, acquiring tools for the desired genetic modification and providing corresponding applications.
To solve this problem, the following five metabolic pathways have been identified:

    (1) the metabolic pathway for synthesizing isovaleric acid (as part of leucine catabolism),
    (2) the metabolic pathway for synthesizing 2-methylbutyric acid and/or isobutyric acid (as part of valine and/or isoleucine catabolism),
    (3) the metabolic pathway for synthesizing butanol and/or butyric acid (as part of butyric acid metabolism),
    (4) the metabolic pathway for synthesizing propionic acid (as part of propionate metabolism) and
    (5) the metabolic pathway for synthesizing cadaverine and/or putrescine (as parts of lysine and/or arginine catabolism).


The following genes which code for proteins and/or enzymes which catalyze reactions lying on these pathways and are suitable as starting points for biotechnological production processes of the invention were then found; the non-consecutive numbering in some cases is based in each case on the complete description hereinafter of the respective metabolic pathways; in addition, some of them are involved in more than one of these pathways:

    
    
        on the metabolic pathway for synthesizing isovaleric acid and as part of leucine catabolism:
    
    
    (1) L-leucine dehydrogenase (E.C. 1.4.1.9),
    (2) 3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate dehydrogenase E1 (E.C. 1.2.4.2),
    (3) enzyme for hydrolyzing isovaleryl-CoA to isovaleric acid and coenzyme A,
    (4) acyl-CoA dehydrogenase (E.C. 1.3.99.-),
    (5) methylcrotonyl carboxylase,
    (6) 3-methylglutaconyl-CoA hydratase and
    (7) enoyl-CoA hydratase (E.C. 4.2.1.17);
    
        on the metabolic pathway for synthesizing 2-methylbutyric acid and/or isobutyric acid and as part of valine and/or isoleucine catabolism:
    
    
    (1) branched-chain amino acid aminotransferase (E.C. 2.6.1.42),
    (2) 3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate dehydrogenase E1 (E.C. 1.2.4.2),
    (3) enzyme for hydrolyzing 2-methylbutyryl-CoA to 2-methylbutyric acid or isobutyryl-CoA to isobutyric acid and coenzyme A,
    (4) acyl-CoA dehydrogenase (E.C. 1.3.99.-),
    (5) enoyl-CoA hydratase (protein) (E.C. 4.2.1.17),
    (6) 3-hydroxy-acyl-CoA dehydrogenase (E.C. 1.1.1.35),
    (7) acetyl-CoA acyltransferase,
    (8) enoyl-(3-hydroxyisobutyryl)-CoA hydrolase protein and
    (9) 3-hydroxyisobutyrate dehydrogenase (E.C. 1.1.1.31) or oxidoreductase
    (E.C. 1.1.-.-);
    
        on the metabolic pathway for synthesizing butanol and/or butyric acid and as part of butyric acid metabolism:
    
    
    (1) 3-hydroxybutyryl-CoA dehydrogenase (E.C. 1.1.1.157),
    (2) 3-hydroxybutyryl-CoA dehydratase (E.C. 4.2.1.55),
    (3) butyryl-CoA dehydrogenase (E.C. 1.3.99.25),
    (4) phosphate butyryltransferase (E.C. 2.3.1.19),
    (5) butyrate kinase (E.C. 2.7.2.7),
    (6) butyraldehyde dehydrogenase and
    (8) NADH-dependent butanol dehydrogenase A (E.C. 1.1.1.-);
    
        on the metabolic pathway for synthesizing propionic acid and as part of propionate metabolism:
    
    
    (1) succinate-propionate CoA-transferase,
    (2) acetate-CoA ligase or synthetase or propionate-CoA ligase or synthetase (E.C. 6.2.1.1) and
    (3) acetate-CoA ligase or synthetase or propionate-CoA ligase or synthetase (E.C. 6.2.1.1); and
    
        on the metabolic pathway for synthesizing cadaverine and/or putrescine and as parts of lysine and/or arginine catabolism:
    
    
    (1) lysine decarboxylase (E.C. 4.1.1.18) and/or arginine decarboxylase (E.C. 4.1.1.19),
    (2) agmatinase (E.C. 3.5.1.11) and
    (3) ornithine decarboxylase (E.C. 4.1.1.17).


Finally, nucleotide and amino acid sequences coding for these proteins/enzymes were completely determined by sequencing relevant genes in B. licheniformis DSM 13, and thus made available for the desired modification of the microorganisms of interest. They are compiled in the sequence listing for the present application. These involve the following nucleic acids (odd numbers) and amino acid sequences derived therefrom for enzymes or proteins as parts of those enzymes which consist of a plurality of subunits (even numbers below in each case):

    
    
        putative branched-chain amino acid aminotransferase (E.C. 2.6.1.42), defined by SEQ ID NO. 1 and 2,
        putative branched-chain amino acid aminotransferase (E.C. 2.6.1.42) defined by SEQ ID NO. 3 and 4,
        lysine and/or arginine decarboxylase (protein SpeA; E.C. 4.1.1.18 or E.C. 4.1.1.19) defined by SEQ ID NO. 5 (speA gene) and 6,
        NADH-dependent butanol dehydrogenase A (protein YugJ; E.C. 1.1.1.-) defined by SEQ ID NO. 7 (yugJ gene) and 8,
        butyryl-CoA dehydrogenase (E.C. 1.3.99.25) or acyl-CoA dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO. 9 and 10,
        butyryl-CoA dehydrogenase (E.C. 1.3.99.25) or acyl-CoA dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO. 11 and 12,
        3-hydroxybutyryl-CoA dehydrogenase (E.C. 1.1.1.157) defined by SEQ ID NO. 13 and 14,
        putative enoyl-CoA hydratase protein (E.C. 4.2.1.17) defined by SEQ ID NO. 15 and 16,
        probable enoyl-(3-hydroxyisobutyryl)-CoA hydrolase protein defined by SEQ ID NO. 17 and 18,
        probable enoyl-CoA hydratase (protein EchA8; E.C. 4.2.1.17) defined by SEQ ID NO. 19 (echA8 gene) and 20,
        acyl-CoA dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO. 21 and 22,
        acetate-CoA ligase or propionate-CoA ligase (or synthetase; protein AcsA; E.C. 6.2.1.1) defined by SEQ ID NO. 23 (acsA gene) and 24,
        3-hydroxybutyryl-CoA dehydratase (protein YngF; E.C. 4.2.1.55) defined by SEQ ID No. 25 (yngF gene) and 26,
        butyryl-CoA dehydrogenase (protein YusJ; E.C. 1.3.99.25) or acyl-CoA dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO. 27 (yusJ gene) and 28,
        3-hydroxyisobutyrate dehydrogenase (protein YkwC; E.C. 1.1.1.31) or oxidoreductase (E.C. 1.1.-.-) defined by SEQ ID NO. 29 (ykwC gene) and 30,
        probable phosphate butyryltransferase (E.C. 2.3.1.19) defined by SEQ ID NO. 31 and 32,
        probable butyrate kinase (E.C. 2.7.2.7) defined by SEQ ID NO. 33 and 34,
        acetate-CoA ligase or synthetase or propionate-CoA ligase or synthetase (protein AcsA; E.C. 6.2.1.1) defined by SEQ ID NO. 35 (acsA gene) and 36,
        acetate-CoA ligase or propionate-CoA ligase (protein Ytcl; E.C. 6.2.1.1) defined by SEQ ID NO. 37 (ytcl gene) and 38,
        lysine and/or arginine decarboxylase (protein speA; E.C. 4.1.1.18 or E.C. 4.1.1.19) defined by SEQ ID NO. 39 (speA gene) and 40,
        probable enoyl-CoA hydratase (E.C. 4.2.1.17) defined by SEQ ID NO. 41 (ysiB gene) and 42,
        similar to 3-hydroxy-acyl-CoA dehydrogenase (E.C. 1.1.1.35) defined by SEQ ID NO. 43 and 44,
        3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate dehydrogenase E1 (E.C. 1.2.4.2) defined by SEQ ID NO. 45 and 46,
        probable acetate-CoA ligase or propionate-CoA ligase (protein YhfL; E.C. 6.2.1.1) or acid-CoA ligase (E.C. 6.2.1.-) defined by SEQ ID NO. 47 (yhfL gene) and 48 or
        agmatinase (E.C. 3.5.1.11) defined by SEQ ID NO. 49 (ywhG gene) and 50.
    
    


All of them are made available by the present application.
The stated problem is thus solved in the same way in principle by all 25 nucleic acids of SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 49 which are indicated in the sequence listing and are obtainable from B. licheniformis DSM 13, including an in each case corresponding homology region which is defined hereinafter and which effects a delimitation from the sequences described in the prior art. It is likewise solved by the gene products derived therefrom of SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 and 50, once again including a corresponding homology region defined hereinafter. The respective most similar nucleic acid and amino acid sequences described in the prior art are compiled in Example 2 (Table 1) with reference to the relevant database entries. The homology regions claimed in each case have been defined on the basis of this information. Solutions according to the invention of the stated problem are preferably in each case those nucleic acids and proteins which actually originate from microorganisms.
Which of these genes is preferred must be ascertained experimentally taking account of the individual strain to be cultured (and possibly different gene activities) and the respective metabolic situation (for example (over)supply of certain C or N sources) in the individual case. For this it is necessary for a series of several mutants, which are to be produced in the same way in principle, of the various relevant genes to be generated in parallel and cultured under conditions which are otherwise identical.
Further solutions are represented by fermentation processes in which one or more of the metabolic pathways for synthesizing (1) isovaleric acid (as part of leucine catabolism), (2) 2-methylbutyric acid and/or isobutyric acid (as part of valine and/or isoleucine catabolism), (3) butanol and/or butyric acid (as part of butyric acid metabolism), (4) propionic acid (as part of propionate metabolism) and/or (5) cadaverine and/or putrescine (as parts of lysine and/or arginine catabolism) are functionally inactivated, preferably via the abovementioned enzymes/proteins which are active on these pathways, and particularly preferably via the nucleotide sequences provided according to the invention. The latter can be used in a manner known per se and established in the prior art, for example for producing knock-out constructs and for introducing them via vectors in the host cells so that gene disruption takes place.
Further solutions are represented by appropriately modified microorganisms in particular relevant to industrial production, all fermentation processes in which these are employed, and among these especially those used to produce valuable products.
In addition, these gene products are available on the basis of the present invention for reaction mixtures or processes according to their respective biochemical properties, by which is meant in particular the synthesis of (1) isovaleric acid, (2) 2-methylbutyric acid and/or isobutyric acid, (3) butanol and/or butyric acid, (4) propionic acid and/or (5) cadaverine and/or putrescine.
The present invention enables, at least as far as these important metabolic pathways are concerned, causal combating of odors. This is because it is possible by switching off the identified metabolic pathways via the proteins involved with the aid of the nucleic acids coding for these proteins to substantially prevent the relevant substances being produced at all. It is thus possible firstly to keep the number of subsequent purification and working-up steps small, which is advantageous because they represent in each case a physicochemical stress on the desired product and reduce the yield; the product quality is thus overall improved. Secondly, the production conditions are improved per se, and the systems for filtration of the fermenter exit air can be kept simpler. This causal combating of odors acts, because it operates at the genetic level, on the properties of the respective microorganism itself, thus increasing its tolerability of further operations on this microorganism.
In particular, industrial fermentation is improved thereby, which ought also to lead to a reduction of the costs of the fermentation products.
In addition, the identified genes and gene products are thus available for diverse applications, for example for chemical and/or at least partly biocatalyzed synthesis of the relevant compounds.
As described in the examples of the present application, it was possible by sequencing the genomic DNA of the B. licheniformis DSM 13, the reference strain obtainable from the Deutschen Sammlung von Mikroorganismen and Zellkulturen GmbH, Mascheroder Weg 1b, 38124 Brunswick, to identify said 25 novel genes for this species. These are ones which code for enzymes or enzyme subunits which are involved in the reactions described herein for synthesizing odorous substances.
The most similar genes and relevant proteins in each case which are known in this connection in the prior art show the sequence homologies indicated in Example 2 (Table 1) of the present application. The range of protection covered in each case by the present application is defined thereby. Accordingly, all the following nucleic acids and proteins represent in principle equivalent embodiments of the present invention:

    
    
        nucleic acid coding for a gene product (putative branched-chain amino acid aminotransferase; E.C. 2.6.1.42) involved in the synthesis of 2-methylbutyric acid and/or isobutyric acid and having a nucleotide sequence which shows at least 67% identity and with increasing preference at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the nucleotide sequence indicated in SEQ ID NO. 1,
        gene product (putative branched-chain amino acid aminotransferase; E.C. 2.6.1.42) involved in the synthesis of 2-methylbutyric acid and/or isobutyric acid and having an amino acid sequence which shows at least 73% identity and with increasing preference at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the amino acid sequence indicated in SEQ ID NO. 2;
        nucleic acid coding for a gene product (putative branched-chain amino acid aminotransferase; E.C. 2.6.1.42) involved in the synthesis of 2-methylbutyric acid and/or isobutyric acid and having a nucleotide sequence which shows at least 78% identity and with increasing preference at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the nucleotide sequence indicated in SEQ ID NO. 3;
        gene product (putative branched-chain amino acid aminotransferase; E.C. 2.6.1.42) involved in the synthesis of 2-methylbutyric acid and/or isobutyric acid and having an amino acid sequence which shows at least 83% identity and with increasing preference at least 85%, 87.5% 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the amino acid sequence indicated in SEQ ID NO. 4;
        nucleic acid speA coding for a gene product (lysine and/or arginine decarboxylase; E.C. 4.1.1.18 or 4.1.1.19) involved in the synthesis of cadaverine and/or putrescine and having a nucleotide sequence which shows at least 78% identity and with increasing preference at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the nucleotide sequence indicated in SEQ ID NO. 5;
        gene product SpeA (lysine and/or arginine decarboxylase; E.C. 4.1.1.18 or E.C. 4.1.1.19) involved in the synthesis of cadaverine and/or putrescine and having an amino acid sequence which shows at least 89% identity and with increasing preference at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the amino acid sequence indicated in SEQ ID NO. 6;
        nucleic acid yugJ coding for a gene product (NADH-dependent butanol dehydrogenase A; E.C. 1.1.1.-) involved in the synthesis of butanol and/or butyric acid and having a nucleotide sequence which shows at least 81% identity and with increasing preference at least 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the nucleotide sequence indicated in SEQ ID NO. 7;
        gene product YugJ (NADH-dependent butanol dehydrogenase A; E.C. 1.1.1.-) involved in the synthesis of butanol and/or butyric acid and having an amino acid sequence which shows at least 93% identity and with increasing preference at least 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5% and particularly preferably 100% identity to the amino acid sequence indicated in SEQ ID NO. 8;
        nucleic acid coding for a gene product (acyl-CoA dehydrogenase; E.C. 1.3.99.-) involved in the synthesis of isovaleric acid, 2-methylbutyric acid, isobutyric acid, butanol and/or butyric acid and having a nucleotide sequence which shows at least 79% identity and with increasing preference at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the nucleotide sequence indicated in SEQ ID NO. 9;
        gene product (acyl-CoA dehydrogenase; E.C. 1.3.99.-) involved in the synthesis of isovaleric acid, 2-methylbutyric acid, isobutyric acid or butanol and/or butyric acid and having an amino acid sequence which shows at least 86% identity and with increasing preference at least 87.5%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the amino acid sequence indicated in SEQ ID NO. 10;
        nucleic acid coding for a gene product (acyl-CoA dehydrogenase; E.C. 1.3.99.-) involved in the synthesis of isovaleric acid, 2-methylbutyric acid, isobutyric acid, butanol and/or butyric acid and having a nucleotide sequence which shows at least 64% identity and with increasing preference at least 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the nucleotide sequence indicated in SEQ ID NO. 11;
        gene product (acyl-CoA dehydrogenase; E.C. 1.3.99.-) involved in the synthesis of isovaleric acid, 2-methylbutyric acid, isobutyric acid, butanol and/or butyric acid and having an amino acid sequence which shows at least 67% identity and with increasing preference at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the amino acid sequence indicated in SEQ ID NO. 12;
        nucleic acid coding for a gene product (3-hydroxybutyryl-CoA dehydrogenase; E.C. 1.1.1.157) involved in the synthesis of butanol and/or butyric acid and having a nucleotide sequence which shows at least 67% identity and with increasing preference at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the nucleotide sequence indicated in SEQ ID NO. 13;
        gene product (3-hydroxybutyryl-CoA dehydrogenase; E.C. 1.1.1.157) involved in the synthesis of butanol and/or butyric acid and having an amino acid sequence which shows at least 69% identity and with increasing preference at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the amino acid sequence indicated in SEQ ID NO. 14;
        nucleic acid coding for a gene product (putative enoyl-CoA hydratase protein; E.C. 4.2.1.17) involved in the synthesis of isovaleric acid, 2-methylbutyric acid and/or isobutyric acid and having a nucleotide sequence which shows at least 65% identity and with increasing preference at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the nucleotide sequence indicated in SEQ ID NO. 15;
        gene product (putative enoyl-CoA hydratase protein; E.C. 4.2.1.17) involved in the synthesis of isovaleric acid, 2-methylbutyric acid and/or isobutyric acid and having an amino acid sequence which shows at least 62% identity and with increasing preference at least 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the amino acid sequence indicated in SEQ ID NO. 16;
        nucleic acid coding for a gene product (probable enoyl-(3-hydroxyisobutyryl)-coenzyme A hydrolase protein) involved in the synthesis of isovaleric acid, 2-methylbutyric acid and/or isobutyric acid and having a nucleotide sequence which shows at least 66% identity and with increasing preference at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the nucleotide sequence indicated in SEQ ID NO. 17;
        gene product (probable enoyl-(3-hydroxyisobutyryl)-coenzyme A hydrolase protein) involved in the synthesis of isovaleric acid, 2-methylbutyric acid and/or isobutyric acid and having an amino acid sequence which shows at least 66% identity and with increasing preference at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the amino acid sequence indicated in SEQ ID NO. 18;
        nucleic acid echA8 coding for a gene product (probable enoyl-CoA hydratase; E.C. 4.2.1.17) involved in the synthesis of isovaleric acid, 2-methylbutyric acid and/or isobutyric acid and having a nucleotide sequence which shows at least 48% identity and with increasing preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the nucleotide sequence indicated in SEQ ID NO. 19;
        gene product EchA8 (probable enoyl-CoA hydratase; E.C. 4.2.1.17) involved in the sythesis of isovaleric acid, 2-methylbutyric acid and/or isobutyric acid and having an amino acid sequence which shows at least 52% identity and with increasing preference at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the amino acid sequence indicated in SEQ ID NO. 20;
        nucleic acid coding for a gene product (acyl-CoA dehydrogenase; E.C. 1.3.99.-) involved in the synthesis of isovaleric acid, 2-methylbutyric acid and/or isobutyric acid and having a nucleotide sequence which shows at least 54% identity and with increasing preference at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the nucleotide sequence indicated in SEQ ID NO. 21;
        gene product (acyl-CoA dehydrogenase) involved in the synthesis of isovaleric acid, 2-methylbutyric acid and/or isobutyric acid and having an amino acid sequence which shows at least 65% identity and with increasing preference at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the amino acid sequence indicated in SEQ ID NO. 22;
        nucleic acid acsA coding for a gene product (acetyl-coenzyme A synthetase; E.C. 6.2.1.1) involved in the synthesis of propionic acid and having a nucleotide sequence which shows at least 67% identity and with increasing preference at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the nucleotide sequence indicated in SEQ ID NO. 23;
        gene product AscA (acetyl-coenzyme A synthetase; E.C. 6.2.1.1) involved in the synthesis of propionic acid and having an amino acid sequence which shows at least 65% identity and with increasing preference at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the amino acid sequence indicated in SEQ ID NO. 24;
        nucleic acid yngF coding for a gene product (3-hydroxybutyryl-CoA dehydratase; E.C. 4.2.1.55) involved in the synthesis of butanol and/or butyric acid and having a nucleotide sequence which shows at least 68% identity and with increasing preference at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the nucleotide sequence indicated in SEQ ID NO. 25;
        gene product YngF (3-hydroxybutyryl-CoA dehydratase; E.C. 4.2.1.55) involved in the synthesis of butanol and/or butyric acid and having an amino acid sequence which shows at least 69% identity and with increasing preference at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the amino acid sequence indicated in SEQ ID NO. 26;
        nucleic acid yusJ coding for a gene product (acyl-CoA dehydrogenase; E.C. 1.3.99.-) involved in the synthesis of isovaleric acid, 2-methylbutyric acid, isobutyric acid, butanol and/or butyric acid and having a nucleotide sequence which shows at least 77% identity and with increasing preference at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the nucleotide sequence indicated in SEQ ID NO. 27;
        gene product YusJ (acyl-CoA dehydrogenase; E.C. 1.3.99.-) involved in the synthesis of isovaleric acid, 2-methylbutyric acid, isobutyric acid, butanol and/or butyric acid and having an amino acid sequence which shows at least 86% identity and with increasing preference at least 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the amino acid sequence indicated in SEQ ID NO. 28;
        nucleic acid ykwC coding for a gene product (hypothetical oxidoreductase; E.C. 1.1.-.-) involved in the synthesis of 2-methylbutyric acid and/or isobutyric acid and having a nucleotide sequence which shows at least 77% identity and with increasing preference at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the nucleotide sequence indicated in SEQ ID NO. 29;
        gene product YkwC (hypothetical oxidoreductase; E.C. 1.1.-.-) involved in the synthesis of 2-methylbutyric acid and/or isobutyric acid and having an amino acid sequence which shows at least 85% identity and with increasing preference at least 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the amino acid sequence indicated in SEQ ID NO. 30;
        nucleic acid coding for a gene product (probable phosphate butyryltransferase; E.C. 2.3.1.19) involved in the synthesis of butanol and/or butyric acid and having a nucleotide sequence which shows at least 51% identity and with increasing preference at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the nucleotide sequence indicated in SEQ ID NO. 31;
        gene product (probable phosphate butyryltransferase; E.C. 2.3.1.19) involved in the synthesis of butanol and/or butyric acid and having an amino acid sequence which shows at least 69% identity and with increasing preference at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the amino acid sequence indicated in SEQ ID NO. 32;
        nucleic acid coding for a gene product (probable butyrate kinase; E.C. 2.7.2.7) involved in the synthesis of butanol and/or butyric acid and having a nucleotide sequence which shows at least 77% identity and with increasing preference at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the nucleotide sequence indicated in SEQ ID NO. 33;
        gene product (probable butyrate kinase; E.C. 2.7.2.7) involved in the synthesis of butanol and/or butyric acid and having an amino acid sequence which shows at least 84% identity and with increasing preference at least 85%, 87.5%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the amino acid sequence indicated in SEQ ID NO. 34;
        nucleic acid acsA coding for a gene product (acetyl-coenzyme A synthetase; E.C. 6.2.1.1) involved in the synthesis of propionic acid and having a nucleotide sequence which shows at least 79% identity and with increasing preference at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the nucleotide sequence indicated in SEQ ID NO. 35;
        gene product AcsA (acetyl-coenzyme A synthetase: E.C. 6.2.1.1) involved in the synthesis of propionic acid and having an amino acid sequence which shows at least 85% identity and with increasing preference at least 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the amino acid sequence indicated in SEQ ID NO. 36;
        nucleic acid ytcl coding for a gene product (acetate-CoA ligase; E.C. 6.2.1.1) involved in the synthesis of propionic acid and having a nucleotide sequence which shows at least 74% identity and with increasing preference at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the nucleotide sequence indicated in SEQ ID NO. 37;
        gene product Ytcl (acetate-CoA ligase; E.C. 6.2.1.1) involved in the synthesis of propionic acid and having an amino acid sequence which shows at least 77% identity and with increasing preference at least 80%, 85%, 87.5%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the amino acid sequence indicated in SEQ ID NO. 38;
        nucleic acid speA coding for a gene product (lysine and/or arginine decarboxylase; E.C. 4.1.1.18 or E.C. 4.1.1.19) involved in the synthesis of cadaverine and/or putrescine and having a nucleotide sequence which shows at least 68% identity and with increasing preference at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the nucleotide sequence indicated in SEQ ID NO. 39;
        gene product SpeA (lysine and/or arginine decarboxylase; E.C. 4.1.1.18 or E.C. 4.1.1.19) involved in the synthesis of cadaverine and/or putrescine and having an amino acid sequence which shows at least 66% identity and with increasing preference at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the amino acid sequence indicated in SEQ ID NO. 40;
        nucleic acid ysiB coding for a gene product (probable enoyl-CoA hydratrase; E.C. 4.2.1.17) involved in the synthesis of isovaleric acid, 2-methylbutyric acid and/or isobutyric acid and having a nucleotide sequence which shows at least 75% identity and with increasing preference at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the nucleotide sequence indicated in SEQ ID NO. 41;
        gene product YsiB (probable enoyl-CoA hydratrase; E.C. 4.2.1.17) involved in the synthesis of isovaleric acid, 2-methylbutyric acid and/or isobutyric acid and having an amino acid sequence which shows at least 77% identity and with increasing preference at least 80%, 85%, 87.5%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the amino acid sequence indicated in SEQ ID NO. 42;
        nucleic acid coding for a gene product (similar to 3-hydroxyacyl-CoA dehydrogenase; E.C. 1.1.1.35) involved in the synthesis of 2-methylbutyric acid and having a nucleotide sequence which shows at least 76% identity and with increasing preference at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the nucleotide sequence indicated in SEQ ID NO. 43;
        gene product (similar to 3-hydroxyacyl-CoA dehydrogenase) involved in the synthesis of 2-methylbutyric acid and having an amino acid sequence which shows at least 80% identity and with increasing preference at least 85%, 87.5%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the amino acid sequence indicated in SEQ ID NO. 44;
        nucleic acid coding for a gene product (2-oxoglutarate dehydrogenase E1 component; E.C. 1.2.4.2) involved in the synthesis of isovaleric acid, 2-methylbutyric acid and/or isobutyric acid and having a nucleotide sequence which shows at least 80% identity and with increasing preference at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the nucleotide sequence indicated in SEQ ID NO. 45;
        gene product (2-oxoglutarate dehydrogenase E1 component; E.C. 1.2.4.2) involved in the synthesis of isovaleric acid, 2-methylbutyric acid and/or isobutyric acid and having an amino acid sequence which shows at least 82% identity and with increasing preference at least 85%, 87.5%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the amino acid sequence indicated in SEQ ID NO. 46;
        nucleic acid yhfL coding for a gene product (probable acid-CoA ligase; E.C. 6.2.1.-) involved in the synthesis of propionic acid and having a nucleotide sequence which shows at least 67% identity and with increasing preference at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the nucleotide sequence indicated in SEQ ID NO. 47;
        gene product YhfL (probable acid-CoA ligase; E.C. 6.2.1.-) involved in the synthesis of propionic acid and having an amino acid sequence which shows at least 76% identity and with increasing preference at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the amino acid sequence indicated in SEQ ID NO. 48;
        nucleic acid ywhG coding for a gene product (agmatinase; E.C. 3.5.1.11) involved in the synthesis of cadaverine and/or putrescine and having a nucleotide sequence which shows at least 85% identity and with increasing preference at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and particularly preferably 100% identity to the nucleotide sequence indicated in SEQ ID NO. 49;
        gene product YwhG (agmatinase; E.C. 3.5.1.11) involved in the synthesis of cadaverine and/or putrescine and having an amino acid sequence which shows at least 97% identity and with increasing preference at least 97.5%, 98%, 98.5%, 99%, 99.5% and particularly preferably 100% identity to the amino acid sequence indicated in SEQ ID NO. 50.
    
    


In connection with the present application, an expression of the form “at least X %” means “X % to 100%, including the extreme values X and 100 and all integral and non-integral percentages in between”.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1: Metabolic pathway for the formation of isovaleric acid. Explanations: see text
FIG. 2: Metabolic pathway for the formation of 2-methylbutyric acid and/or isobutyric acid; aspect of the formation of 2-methylbutyric acid. Explanations: see text
FIG. 3: Metabolic pathway for the formation of 2-methylbutyric acid and/or isobutyric acid; aspect of the formation of isobutyric acid. Explanations: see text
FIG. 4: Metabolic pathway for the formation of butanol and/or butyric acid. Explanations: see text
FIG. 5: Metabolic pathway for the formation of propionic acid. Explanations: see text
FIG. 6: Metabolic pathway for the formation of cadaverine and/or putrescine; aspect of the formation of cadaverine. Explanations: see text
FIG. 7: Metabolic pathway for the formation of cadaverine and/or putrescine; aspect of the formation of putrescine. Explanations: see text



DETAILED DESCRIPTION
The designations of the respective enzymes are governed by the specific reactions catalyzed by them, as are depicted for example in FIGS. 1 to 7. (Detailed explanations of the figures and of the relevant metabolic pathways following hereinafter.) Thus, it is also possible for a single enzyme to be able to catalyze two reactions which are chemically virtually identical but are assigned to different pathways on the basis of the respective substrate. This may also be associated with a different enzyme classification (E.C. numbers) according to IUBMB. The enzyme designation is governed according to the invention according to the respective specific reaction. This is because the specific function which is implemented in the course of the present invention or is to be switched off where appropriate is also associated therewith.
For illustration, reference may be made by way of example to the enzyme which is indicated in SEQ ID NO. 18 and with which such a deviation is in fact located on the same metabolic pathway defined according to the invention. According to the relevant statement in SEQ ID NO. 17, this is a “probable enoyl-(3-hydroxyisobutyryl)-coenzyme A hydrolase protein”. At the time of the application, the IUBMB has not yet allocated an E.C. number for this reaction, which is why reference can be made for definition of the relevant enzymic activity only to reaction (6.) in FIG. 3. On the same metabolic pathway for synthesizing 2-methylbutyric acid and/or isobutyric acid (as part of valine and/or isoleucine catabolism) there is also a reaction which is catalyzed by an enoyl-CoA hydratase, reaction (3.) in FIG. 3; the situation is likewise for reaction (7.) in FIG. 1. A plurality of enzymes with E.C. class 4.2.1.17 are in each case suitable for this, for example those shown in SEQ ID NO. 16, 20 and 42 (see below), but also the enzyme according to SEQ ID NO. 18. In the course of this specific reaction, the enzyme according to SEQ ID NO. 18 is thus to be regarded as enoyl-CoA hydratase and assigned to E.C. class 4.2.1.17.
These genes and gene products can now be synthesized artificially by methods known per se, and without the need to reproduce the sequencing described in Example 1, in a targeted manner on the basis of these sequences.
As a further alternative thereto, it is possible to obtain the relevant genes from a Bacillus strain, in particular the strain B. licheniformis DSM 13 which is obtainable from the DSMZ, via PCR, it being possible to use the respective border sequences listed in the sequence listing for synthesizing primers. On use of other strains, the genes homologous thereto are obtained in each case, and the success of the PCR should increase with the closeness of the relationship of the selected strains to B. licheniformis DSM 13, because an increasing agreement in sequence also within the primer binding regions should be associated therewith.
As an alternative thereto, the nucleic acids indicated in the sequence listing can also be employed as DNA probes in order to detect the respective homologous genes in preparations of genomic DNA from other species. The procedure for this is known per se; as is the isolation of the genes obtained in this way, their cloning, their expression and obtaining of the relevant proteins. Consideration is given in this connection in particular to operating steps like those described for B. licheniformis itself in Example 1.
The existence of the relevant proteins in a strain of interest is detected in the first place by a chemical detection of whether the relevant odorous substances are formed. It is then possible for the enzymic activities presumed therefor to be ascertained in suitable detection reactions. This takes place for example by the starting compound relevant to the reaction in question being incubated with a cell extract. When the relevant enzymic activity is present, the products following in the relevant metabolic pathway should accumulate and, if all the subsequent enzymes are present, result in the odorous substance.
As detection at the level of molecular biology it is possible to synthesize proteins on the basis of the amino acid sequences shown in the present sequence listing, and to form antibodies against them. These can then be used for example in Western blots for detecting the homologous protein in cell extracts of the host cells of interest.
Among the nucleic acids mentioned herein and coding for a gene product of the invention involved in the synthesis of isovaleric acid, 2-methylbutyric acid, isobutyric acid, butanol, butyric acid, propionic acid, cadaverine and/or putrescine and defined as above on the basis of SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49, preference is given in each case to that present naturally in a microorganism, preferably a bacterium, particularly preferably a Gram-positive bacterium, among these preferably one of the genus Bacillus, among these particularly preferably one of the species B. licheniformis and among these very particularly preferably B. licheniformis DSM13.
It is thus possible as just described comparatively easy in relation to neosynthesis for the relevant nucleic acids to be obtained from natural species, especially microorganisms. Among these, increasing preference is given in view of the stated problem to those which can be fermented and which can in fact be employed in industrial fermentations. These include in particular representatives of the genera Staphylococcus, Corynebacterium and Bacillus. Mention should be made among these for example of S. carnosus and C. glutamicum, and B. subtilis, B. licheniformis, B. amyloliquefaciens, B. agaradherens, B. lentus, B. globigii and B. alkalophilus. Most preference is given to B. licheniformis DSM 13 because it was possible to obtain therefrom exactly the sequences listed in the sequence listing.
These explanations apply in the same way to the relevant proteins.
Thus, among the gene products mentioned herein and involved in the synthesis of isovaleric acid, 2-methylbutyric acid, isobutyric acid, butanol, butyric acid, propionic acid, cadaverine and/or putrescine and defined on the basis of SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50, preference is given in each case to those naturally formed by a microorganism, preferably by a bacterium, particularly preferably by a Gram-positive bacterium, among these preferably by one of the genus Bacillus, among these particularly preferably by one of the species B. licheniforms and among these very particularly preferably by B. licheniforms DSM 13.
The metabolic pathway utilized in Gram-positive bacteria of the genus Bacillus for synthesizing isovaleric acid as part of leucine catabolism is depicted in FIG. 1. It ultimately represents an interface between the citrate cycle and/or fatty acid metabolism and pyruvate metabolism as far as the synthesis of leucine.
The enzymes involved in the reactions shown in FIG. 1 are, as mentioned above, the following, where the relevant number designates the respective reaction step indicated in the figure:

    (1.) L-leucine dehydrogenase (E.C. 1.4.1.9),
    (2.) 3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate dehydrogenase E1 (E.C. 1.2.4.2),
    (3.) enzyme for hydrolyzing isovaleryl-CoA to isovaleric acid and coenzyme A (where non-enzymatic hydrolysis is also possible),
    (4.) acyl-CoA dehydrogenase (E.C. 1.3.99.-),
    (5.) methylcrotonyl carboxylase,
    (6.) 3-methylglutaconyl-CoA hydratase and
    (7.) enoyl-CoA hydratase (E.C. 4.2.1.17).


Solutions of the stated problem and thus independent embodiments of the present invention are thus represented by all processes for fermenting a microorganism in which at least one of the genes on a metabolic pathway for synthesizing isovaleric acid (as part of leucine catabolism) is functionally inactivated.
The advantages previously explained are associated with this solution.
Preference is given in this connection to any process of this type in which the microorganism now forms only 50% of the amount naturally formed under the same conditions, preferably now only 10%, particularly preferably no isovaleric acid.
These percentages (and all subsequent corresponding data for the further metabolic pathways) mean, in analogy to the statement made above for the sequence homology, once again all intermediate integral or fractional percentages in correspondingly preferred gradation. To determine these values, cells of an untreated strain and of a treated strain are fermented under conditions which are otherwise identical and, during the fermentation, the rate of formation of the unwanted odorous substance is suitably ascertained in a manner known per se. Since the strains are otherwise identical, the differences in the formation of this substance are attributable to the different gene activities. In this connection, any reduction in the formation of the odorous substance is desired according to the invention. Values comparable in percentage terms are obtained by taking samples (for instance from the exit air) from both fermentations and determining the content of the respective substance by analytical methods known per se. It is preferred to determine this value at the transition to the stationary phase of growth, because this time can usually be identified unambiguously and, at the same time, is normally associated with the highest metabolic rate.
Account is taken thereby of the generally high flexibility of microorganisms in relation to their metabolism. Thus, it is conceivable for inactivation of one gene to be partly compensated by enhancement of the activity of another gene and/or protein which is possibly not quite as effective in vivo. However, increasing preference is given to inactivation of the said pathway as extensively as possible. It is possible for this to test in the individual case the inactivation of various genes for the effectiveness according to the invention thereof and to select those with the strongest effect. It is additionally possible to combine a plurality of inactivations together.
Preference is given to a process according to the invention in which at least one of the following enzymes is functionally inactivated:

    (1.) L-leucine dehydrogenase (E.C. 1.4.1.9),
    (2.) 3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate dehydrogenase E1 (E.C. 1.2.4.2),
    (3.) enzyme for hydrolyzing isovaleryl-CoA to isovaleric acid and coenzyme A,
    (4.) acyl-CoA dehydrogenase (E.C. 1.3.99.-),
    (5.) methylcrotonyl carboxylase,
    (6.) 3-methylglutaconyl-CoA hydratase and
    (7.) enoyl-CoA hydratase (protein) (E.C. 4.2.1.17).


This is because, as depicted in FIG. 1, these activities may be connected with the metabolic pathway under consideration here.
As already stated above and described in the examples of the present application, it was possible by sequencing the genomic DNA of B. licheniformis DSM 13 to identify several of the genes coding for enzymes located on this pathway, or for subunits thereof. The genes involved are the following (the preceding number designates in each case the reaction in which the relevant enzyme is involved):

    (2.) 3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate dehydrogenase E1 (E.C. 1.2.4.2) defined by SEQ ID NO. 45,
    (4.) a subunit of acyl-CoA dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO. 9, 11, 21 or 27 (yusJ gene), and
    (7.) enoyl-CoA hydratase (protein) (E.C. 4.2.1.17) defined by SEQ ID NO. 15, 17, 19 (echA8 gene) or 41 (ysiB gene).


The amino acid sequences derived therefrom are indicated in SEQ ID NO. 46, 10, 12, 22, 28, 16, 18, 20 and 42, respectively. It was thus possible to identify these specific gene products in the course of the present invention as involved in this metabolic pathway for synthesizing isovaleric acid

    (as part of leucine catabolism).


A process of the invention which is therefore preferred is one where the functionally inactivated enzyme is the homolog, which is naturally active in the relevant microorganism, to one of the following proteins from B. licheniformis DSM13:

    (2.) 3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate dehydrogenase E1 (E.C. 1.2.4.2) defined by SEQ ID NO. 46,
    (4.) a subunit of acyl-CoA dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO. 10, 12, 22 or 28, and
    (7.) enoyl-CoA hydratase (protein) (E.C. 4.2.1.17) defined by SEQ ID NO. 16, 18, 20 or 42.


A preferred process of the invention is one where the enzyme is functionally inactivated at the genetic level, preferably by inactivation of a gene which corresponds to the nucleic acid which codes for one of the following proteins from B. licheniformis DSM 13:

    (2.) 3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate dehydrogenase E1 (E.C. 1.2.4.2) defined by SEQ ID NO. 45,
    (4.) a subunit of acyl-CoA dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO. 9, 11, 21 or 27 (yusJ gene), and
    (7.) enoyl-CoA hydratase (protein) (E.C. 4.2.1.17) defined by SEQ ID NO. 15, 17, 19 (echA8 gene) or 41 (ysiB gene).


This is because, in accordance with the stated problem, it was preferably intended to find a causal solution, meaning one applying at the level of molecular biology. This is available with the stated nucleotide sequences. Example 3 explains how corresponding deletions can be undertaken; further statements concerning this are given hereinafter because they apply in principle to all described metabolic pathways.
A preferred process of the invention is thus one where, for inactivation at the genetic level, one of the nucleic acids of the invention within the region designated above homologous to

    (2.) SEQ ID NO. 45,
    (4.) 9, 11, 21 or 27 and
    (7.) 15, 17, 19 or 41

has been used, preferably one, particularly preferably two parts in each case one of these sequences which in each case comprise at least 70 connected positions.



This can be detected for example by a molecular biological investigation (such as, for example, restriction, sequencing) of the gene region modified by the mutagenesis.
A further embodiment of the present invention is represented by the use of a gene which corresponds to the nucleic acid which codes for one of the following proteins of B. licheniformis DSM 13 for functional inactivation of a metabolic pathway for synthesizing isovaleric acid (as part of leucine catabolism) at the genetic level in a microorganism:

    (2.) 3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate dehydrogenase E1 (E.C. 1.2.4.2) defined by SEQ ID NO. 45,
    (4.) a subunit of acyl-CoA dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO. 9, 11, 21 or 27 (yusJ gene), and
    (7.) enoyl-CoA hydratase (protein) (E.C. 4.2.1.17) defined by SEQ ID NO. 15, 17, 19 (echA8 gene) or 41 (ysiB gene).


The same statements as previously made about the corresponding processes apply in principle to such uses.
Accordingly, a preferred use according to the invention of nucleic acids of the invention is within the region of homology designated above to

    (2.) SEQ ID NO. 45,
    (4.) 9, 11, 21 or 27 and
    (7.) 15, 17, 19 or 41 for functional inactivation, preferably of one, particularly preferably of two parts in each case of one of these sequences, where these parts in each case comprise at least 70 connected positions.


Further embodiments based on these fermentation processes and uses are detailed hereinafter because they can be applied in principle to all the metabolic pathways described within the scope of the present invention.
The metabolic pathway utilized in Gram-positive bacteria of the genus Bacillus for synthesizing 2-methylbutyric acid as part of isoleucine catabolism is depicted in FIG. 2; the corresponding pathway proceeding via the same enzymes in principle for synthesizing isobutyric acid as part of valine catabolism is evident from FIG. 3. This aspect, which is regarded in connection with the present application as a single pathway, of bacterial metabolism ultimately represents, like the pathway considered previously too, an interface between the citrate cycle and/or fatty acid metabolism and pyruvate metabolism as far as the synthesis of the two amino acids isoleucine and valine.
As already mentioned, the following enzymes are involved in the reactions shown in FIGS. 2 and 3, in each case the relevant numbers of the reaction steps indicated in the figures being indicated:

    (1.) branched-chain amino acid aminotransferase (E.C. 2.6.1.42; reaction 1 in FIGS. 2 and 3),
    (2.) 3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate dehydrogenase E1 (E.C. 1.2.4.2; reaction 2 in FIGS. 2 and 3),
    (3.) enzyme for hydrolyzing 2-methylbutyryl-CoA to 2-methylbutyric acid (reaction 3 in FIG. 2) or isobutyryl-CoA to isobutyric acid and coenzyme A (reaction 3 in FIG. 3; a non-enzymatic hydrolysis also being possible in both cases),
    (4.) acyl-CoA dehydrogenase (E.C. 1.3.99.-; reaction 4 in FIGS. 2 and 3),
    (5.) enoyl-CoA hydratase (protein) (E.C. 4.2.1.17; reaction 5 in FIGS. 2 and 3),
    (6.) 3-hydroxy-acyl-CoA dehydrogenase (E.C. 1.1.1.35) (reaction 6 in FIG. 2),
    (7.) acetyl-CoA acyltransferase (reaction step 7 in FIG. 2),
    (8.) enoyl-(3-hydroxyisobutyryl)-CoA hydrolase protein (step 6 in FIG. 3) and
    (9.) 3-hydroxyisobutyrate dehydrogenase (E.C. 1.1.1.31) or oxidoreductase
    (E.C. 1.1.-.-; step 7 in FIG. 3).


Solutions of the stated problem and thus independent embodiments of the present invention are thus represented by all processes for fermenting a microorganism in which at least one of the genes on a metabolic pathway for synthesizing 2-methylbutyric acid and/or isobutyric acid (as part of valine and/or isoleucine catabolism) is functionally inactivated.
The advantages already explained are associated with this solution.
Preference is given in this connection to any process of this type in which the microorganism now forms only 50% of the amount formed naturally under the same conditions, preferably now only 10%, particularly preferably no 2-methylbutyric acid and/or isobutyric acid.
Account is thereby taken, as explained above for the first metabolic pathway described, of the generally high flexibility of microorganisms in relation to their metabolism.
A preferred process of the invention is one in which at least one of the following enzymes is functionally inactivated:

    (1.) branched-chain amino acid aminotransferase (E.C. 2.6.1.42),
    (2.) 3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate dehydrogenase E1 (E.C. 1.2.4.2),
    (3.) enzyme for hydrolyzing 2-methylbutyryl-CoA to 2-methylbutyric acid or isobutyryl-CoA to isobutyric acid and coenzyme A,
    (4.) acyl-CoA dehydrogenase (E.C. 1.3.99.-),
    (5.) enoyl-CoA hydratase (protein) (E.C. 4.2.1.17),
    (6.) 3-hydroxy-acyl-CoA dehydrogenase (E.C. 1.1.1.35),
    (7.) acetyl-CoA acyltransferase,
    (8.) enoyl-(3-hydroxyisobutyryl)-CoA hydrolase protein and
    (9.) 3-hydroxyisobutyrate dehydrogenase (E.C. 1.1.1.31) or oxidoreductase (E.C. 1.1.-.-).


This is because, as depicted in FIGS. 2 and 3, these activities may be associated with the metabolic pathway considered.
As stated previously and described in the examples of the present application, it was possible by sequencing the genomic DNA of B. licheniformis DSM 13 to identify several of the genes which code for enzymes located on this pathway, or for subunits thereof. These involve the following genes (the preceding number designates in each case the reaction in which the relevant enzyme is involved):

    (1.) branched-chain amino acid aminotransferase (E.C. 2.6.1.42) defined by SEQ ID NO. 1 or 3,
    (2.) 3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate dehydrogenase E1 (E.C. 1.2.4.2) defined by SEQ ID NO. 45,
    (4.) acyl-CoA dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO. 9, 11, 21 or 27 (yusJ gene),
    (5.) enoyl-CoA hydratase (protein) (E.C. 4.2.1.17) defined by SEQ ID NO. 15, 17, 19 (echA8 gene) or 41 (ysiB gene),
    (6.) 3-hydroxy-acyl-CoA dehydrogenase (E.C. 1.1.1.35) defined by SEQ ID NO. 43,
    (8.) enoyl-(3-hydroxyisobutyryl)-CoA hydrolase protein defined by SEQ ID NO. 17 and
    (9.) 3-hydroxyisobutyrate dehydrogenase (E.C. 1.1.1.31) or oxidoreductase
    (E.C. 1.1.-.-) defined by SEQ ID NO. 29 (ykwC gene).


The amino acid sequences derived therefrom are indicated in SEQ ID NO. 2, 4, 46, 10, 12, 22, 28, 16, 18, 20, 42, 44, 18 and 30. It was thus possible in the course of the present invention to identify the specific gene products as involved in this metabolic pathway for synthesizing 2-methylbutyric acid and/or isobutyric acid (as part of valine and/or isoleucine catabolism).
A preferred process of the invention is therefore one where the functionally inactivated enzyme is the homolog, naturally active in the relevant microorganism, to one of the following proteins from B. licheniformis DSM 13:

    (1.) branched-chain amino acid aminotransferase (E.C. 2.6.1.42) defined by SEQ ID NO. 2 or 4,
    (2.) 3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate dehydrogenase E1 (E.C. 1.2.4.2) defined by SEQ ID NO. 46,
    (4.) acyl-CoA dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO. 10, 12, 22 or 28,
    (5.) enoyl-CoA hydratase (protein) (E.C. 4.2.1.17) defined by SEQ ID NO. 16, 18, 20 or 42,
    (6.) 3-hydroxy-acyl-CoA dehydrogenase (E.C. 1.1.1.35) defined by SEQ ID NO. 44,
    (8.) enoyl-(3-hydroxyisobutyryl)-CoA hydrolase protein defined by SEQ ID NO. 18 and
    (9.) 3-hydroxyisobutyrate dehydrogenase (E.C. 1.1.1.31) or oxidoreductase (E.C. 1.1.-.-) defined by SEQ ID NO. 30.


A preferred process of the invention is one where the enzyme is functionally inactivated at the genetic level, preferably by inactivation of a gene which corresponds to the nucleic acid which codes for one of the following proteins of B. licheniformis DSM 13:

    (1.) branched-chain amino acid aminotransferase (E.C. 2.6.1.42) defined by SEQ ID NO. 1 or 3,
    (2.) 3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate dehydrogenase E1 (E.C. 1.2.4.2) defined by SEQ ID NO. 45,
    (4.) acyl-CoA dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO. 9, 11, 21 or 27 (yusJ gene),
    (5.) enoyl-CoA hydratase (protein) (E.C. 4.2.1.17) defined by SEQ ID NO. 15, 17, 19 (echA8 gene) or 41 (ysiB gene),
    (6.) 3-hydroxy-acyl-CoA dehydrogenase (E.C. 1.1.1.35) defined by SEQ ID NO. 43,
    (8.) enoyl-(3-hydroxyisobutyryl)-CoA hydrolase protein defined by SEQ ID NO. 17 and
    (9.) 3-hydroxyisobutyrate dehydrogenase (E.C. 1.1.1.31) or oxidoreductase (E.C. 1.1.-.-) defined by SEQ ID NO. 29 (ykwC gene).


This is because, in accordance with the stated problem, the intention was preferably to find a causal solution, meaning one applying at the level of molecular biology. Example 3 explains how corresponding deletions can be undertaken; further statements concerning this are given hereinafter.
A preferred process of the invention is thus one where, for inactivation at the genetic level, one of the nucleic acids of the invention within the region designated above and homologous to

    (1.) SEQ ID NO. 1 or 3,
    (2.) 45,
    (4.) 9, 11, 21 or 27,
    (5.) 15, 17, 19 or 41,
    (6.) 43,
    (8.) 17 and
    (9.) 29

has been used, preferably one, particularly preferably two parts in each case of one of these sequences which in each case comprise at least 70 connected positions.



A further embodiment of the present invention is represented by the use of a gene which corresponds to the nucleic acid which codes for one of the following proteins of B. licheniformis DSM 13 for functional inactivation of a metabolic pathway for synthesizing isovaleric acid (as part of leucine catabolism) at the genetic level in a microorganism:

    (1.) branched-chain amino acid aminotransferase (E.C. 2.6.1.42) defined by SEQ ID NO. 1 or 3,
    (2.) 3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate dehydrogenase E1 (E.C. 1.2.4.2) defined by SEQ ID NO. 45,
    (4.) acyl-CoA dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO. 9, 11, 21 or 27 (yusJ gene),
    (5.) enoyl-CoA hydratase (protein) (E.C. 4.2.1.17) defined by SEQ ID NO. 15, 17, 19 (echA8 gene) or 41 (ysiB gene),
    (6.) 3-hydroxy-acyl-CoA dehydrogenase (E.C. 1.1.1.35) defined by SEQ ID NO. 43,
    (8.) enoyl-(3-hydroxyisobutyryl)-CoA hydrolase protein defined by SEQ ID NO. 17 and
    (9.) 3-hydroxyisobutyrate dehydrogenase (E.C. 1.1.1.31) or oxidoreductase (E.C. 1.1.-.-) defined by SEQ ID NO. 29 (ykwC gene).


The same as previously stated concerning the corresponding processes applies in principle to such uses.
Accordingly, a preferred use according to the invention is of nucleic acids of the invention within the regions designated above and homologous to

    (1.) SEQ ID NO. 1 or 3,
    (2.) 45,
    (4.) 9, 11, 21 or 27,
    (5.) 15, 17, 19 r 41,
    (6.) 43,
    (8.) 17 and
    (9.) 29

for functional inactivation, preferably of one, particularly preferably of two parts in each case of one of these sequences, where these parts comprise in each case at least 70 connected positions.



Further embodiments based on these fermentation processes and uses are detailed hereinafter.
The metabolic pathway utilized in Gram-positive bacteria of the genus Bacillus for synthesizing butanol and/or butyric acid as part of butyric acid metabolism is depicted in FIG. 4. This metabolic pathway is ultimately derived from fatty acid metabolism.
As previously mentioned, the following enzymes are involved in the reactions shown in FIG. 4, the relevant number designating the respective reaction step indicated in the figure:

    (1.) 3-hydroxybutyryl-CoA dehydrogenase (E.C. 1.1.1.157),
    (2.) 3-hydroxybutyryl-CoA dehydrogenase (E.C. 4.2.1.55),
    (3.) butyryl-CoA dehydrogenase (E.C. 1.3.99.25),
    (4.) phosphate butyryltransferase (E.C. 2.3.1.19),
    (5.) butyrate kinase (E.C. 2.7.2.7),
    (6.) butyraldehyde dehydrogenase and
    (8.) NADH-dependent butanol dehydrogenase A (E.C. 1.1.1.-).


Reaction (7.) normally takes place by non-enzymatic oxidation by atmospheric oxygen.
Solutions of the stated problem and thus independent embodiments of the present invention are thus represented by all processes for fermenting a microorganism in which at least one of the genes on a metabolic pathway for synthesizing butanol and/or butyric acid (as part of butyric acid metabolism) is functionally inactivated.
The advantages already explained are associated with this solution.
Preference is given in this connection to any process of this type in which the microorganism now forms only 50% of the amount naturally formed under the same conditions, preferably now only 10%, particularly preferably no butanol or no butyric acid.
This takes account, as explained above for the first metabolic pathway described, of the generally high flexibility of microorganisms in relation to their metabolism.
A preferred process of the invention is one in which at least one of the following enzymes is functionally inactivated:

    (1.) 3-hydroxybutyryl-CoA dehydrogenase (E.C. 1.1.1.157),
    (2.) 3-hydroxybutyryl-CoA dehydratase (E.C. 4.2.1.55),
    (3.) butyryl-CoA dehydrogenase (E.C. 1.3.99.25),
    (4.) phosphate butyryltransferase (E.C. 2.3.1.19),
    (5.) butyrate kinase (E.C. 2.7.2.7),
    (6.) butyraldehyde dehydrogenase and
    (8.) NADH-dependent butanol dehydrogenase A (E.C. 1.1.1.-).


This is because, as depicted in FIG. 4, these activities can be associated with the metabolic pathway under consideration here.
As stated above and described in the examples of the present application, it was possible by sequencing the genomic DNA of B. licheniformis DSM 13 to identify several of the genes which code for enzymes located on this pathway, or for subunits thereof. These are the following genes (the preceding number designates in each case the reaction in which the relevant enzyme is involved):

    (1.) 3-hydroxybutyryl-CoA dehydrogenase (E.C. 1.1.1.157) defined by SEQ ID NO. 13,
    (2.) 3-hydroxybutyryl-CoA dehydratase (E.C. 4.2.1.55) defined by SEQ ID NO. 25 (yngF gene),
    (3.) butyryl-CoA dehydrogenase (E.C. 1.3.99.25) defined by SEQ ID NO. 9, 11 or 27 (yusJ gene),
    (4.) phosphate butyryltransferase (E.C. 2.3.1.19) defined by SEQ ID NO. 31,
    (5.) butyrate kinase (E.C. 2.7.2.7) defined by SEQ ID NO. 33 and
    (8.) NADH-dependent butanol dehydrogenase A (E.C. 1.1.1.-) defined by SEQ ID NO. 7 (yugJ gene).


The amino acid sequences derived therefrom are indicated in SEQ ID NO. 14, 26, 10, 12, 28, 32, 34 and 8. It was thus possible in the course of the present invention to identify these specific gene products as involved in this metabolic pathway for synthesizing butanol and/or butyric acid (as part of butyric acid metabolism).
A preferred process of the invention is therefore one where the functionally inactivated enzyme is the homolog, which is naturally active in the relevant microorganism, to one of the following proteins from B. licheniformis DSM 13:

    (1.) 3-hydroxybutyryl-CoA dehydrogenase (E.C. 1.1.1.157) defined by SEQ ID NO. 14,
    (2.) 3-hydroxybutyryl-CoA dehydratase (E.C. 4.2.1.55) defined by SEQ ID NO. 26,
    (3.) butyryl-CoA dehydrogenase (E.C. 1.3.99.25) defined by SEQ ID NO. 10, 12 or 28,
    (4.) phosphate butyryltransferase (E.C. 2.3.1.19) defined by SEQ ID NO. 32,
    (5.) butyrate kinase (E.C. 2.7.2.7) defined by SEQ ID NO. 34 and
    (8.) NADH-dependent butanol dehydrogenase A (E.C. 1.1.1.-) defined by SEQ ID NO. 8.


The preferred process according to the invention is one where the enzyme is functionally inactivated at the genetic level, preferably by inactivation of a gene which corresponds to the nucleic acid which codes for one of the following proteins of B. licheniformis DSM 13:

    (1.) 3-hydroxybutyryl-CoA dehydrogenase (E.C. 1.1.1.157) defined by SEQ ID NO. 13,
    (2.) 3-hydroxybutyryl-CoA dehydratase (E.C. 4.2.1.55) defined by SEQ ID NO. 25 (yngF gene),
    (3.) butyryl-CoA dehydrogenase (E.C. 1.3.99.25) defined by SEQ ID NO. 9, 11 or 27 (yusJ gene),
    (4.) phosphate butyryltransferase (E.C. 2.3.1.19) defined by SEQ ID NO. 31,
    (5.) butyrate kinase (E.C. 2.7.2.7) defined by SEQ ID NO. 33 and
    (8.) NADH-dependent butanol dehydrogenase A (E.C. 1.1.1.-) defined by SEQ ID NO. 7 (yugJ gene).


This is because, in accordance with the stated problem, it was intended preferably to find a causal solution, meaning one applying at the level of molecular biology. Example 3 explains how corresponding deletions can be undertaken; further statements concerning this are given hereinafter.
Thus, a preferred process of the invention is one where, for inactivation at the genetic level, one of the nucleic acids of the invention within the region designated above and homologous to

    (1.) SEQ ID NO. 13,
    (2.) 25,
    (3.) 9, 11 or 27,
    (4.) 31,
    (5.) 33 and
    (6.) 7

has been used, preferably one, particularly preferably two parts in each case of one of these sequences, which in each case comprise at least 70 connected positions.



A further embodiment of the present invention is represented by the use of a gene which corresponds to the nucleic acid which codes for one of the following proteins of B. licheniformis DSM 13 for functional inactivation of a metabolic pathway for synthesizing butanol and/or butyric acid (as part of butyric acid metabolism) at the genetic level in a microorganism:

    (1.) 3-hydroxybutyryl-CoA dehydrogenase (E.C. 1.1.1.157) defined by SEQ ID NO. 13,
    (2.) 3-hydroxybutyryl-CoA dehydratase (E.C. 4.2.1.55) defined by SEQ ID NO. 25 (yngF gene),
    (3.) butyryl-CoA dehydrogenase (E.C. 1.3.99.25) defined by SEQ ID NO. 9, 11 or 27 (yusJ gene),
    (4.) phosphate butyryltransferase (E.C. 2.3.1.19) defined by SEQ ID NO. 31,
    (5.) butyrate kinase (E.C. 2.7.2.7) defined by SEQ ID NO. 33 and
    (8.) NADH-dependent butanol dehydrogenase A (E.C. 1.1.1.-) defined by SEQ ID NO. 7 (yugJ gene).


The same as has previously been stated concerning the corresponding processes applies in principle to such uses.
Accordingly, a preferred use according to the invention is of nucleic acids of the invention within the region designated above and homologous to

    (1.) SEQ ID NO. 13,
    (2.) 25,
    (3.) 9, 11 or 27,
    (4.) 31,
    (5.) 33 and
    (8.) 7

for functional inactivation, preferably of one, particularly preferably of two parts in each case of one of these sequences, where these parts comprise in each case at least 70 connected positions.



Further embodiments based on these fermentation processes and uses are detailed hereinafter.
The metabolic pathway utilized in Gram-positive bacteria of the genus Bacillus for synthesizing propionic acid (as part of propionate metabolism) is depicted in FIG. 5. This metabolic pathway ultimately represents an interface between the citrate cycle and fatty acid metabolism.
As already mentioned, the following enzymes are involved in the reactions shown in FIG. 5, where the relevant number designates the respective reaction step indicated in the figure:

    (1.) succinate-propionate CoA-transferase,
    (2.) acetate-CoA ligase or synthetase or propionate-CoA ligase or synthetase (E.C. 6.2.1.1) and
    (3.) acetate-CoA ligase or synthetase or propionate-CoA ligase or synthetase (E.C. 6.2.1.1).


Solutions of the stated problem and thus independent embodiments of the present invention are thus represented by all processes for fermenting a microorganism in which at least one of the genes on a metabolic pathway for synthesizing propionic acids (as part of propionate metabolism) is functionally inactivated.
The previously explained advantages are associated with this solution.
Preference is given in this connection to any process of this type in which the microorganism now forms only 50% of the amount naturally formed under the same conditions, preferably now only 10%, particularly preferably no propionic acid.
This takes account, as explained above for the first metabolic pathway described, of the generally high flexibility of microorganisms in relation to their metabolism.
A preferred process of the invention is one in which at least one of the following enzymes is functionally inactivated:

    (1.) succinate-propionate CoA-transferase,
    (2.) acetate-CoA ligase or synthetase or propionate-CoA ligase or synthetase (E.C. 6.2.1.1) and
    (3.) acetate-CoA ligase or synthetase or propionate-CoA ligase or synthetase (E.C. 6.2.1.1).


This is because, as depicted in FIG. 5, these activities can be connected with the metabolic pathway under consideration herein.
As already stated above and described in the examples in the present application, it was possible to identify by sequencing the genomic DNA of B. licheniformis DSM 13 several of the genes which code for enzymes located on this pathway, or for subunits thereof. These are the following genes (the preceding number designates in each case the reaction in which the relevant enzyme is involved):

    acetate-CoA ligase or synthetase or propionate-CoA ligase or synthetase (E.C. 6.2.1.1) defined by SEQ ID NO. 35 (acsA gene), 37 (ytcl gene), 47 (yhfL gene) or 23 (acsA gene).


The amino acid sequences derived therefrom are indicated in SEQ ID NO. 36, 38, 48 and 24. It was thus possible to identify the specific gene products in the course of the present invention as involved in this metabolic pathway for synthesizing propionic acid (as part of propionate metabolism).
A preferred process of the invention is therefore one where the functionally inactivated enzyme is the homolog, which is naturally active in the relevant microorganism, to one of the following proteins from B. licheniformis DSM 13: acetate-CoA ligase or synthetase or propionate-CoA ligase or synthetase (E.C. 6.2.1.1) defined by SEQ ID NO. 36, 38, 48 or 24.
A preferred process of the invention is one where the enzyme is functionally inactivated at the genetic level, preferably by inactivation of a gene which corresponds to the nucleic acid which codes for one of the following proteins of B. licheniformis DSM 13: acetate-CoA ligase or synthetase or propionate-CoA ligase or synthetase (E.C. 6.2.1.1) defined by SEQ ID NO. 35 (acsA gene), 37 (ytcl gene), 47 (yhfL gene) or 23 (acsA gene).
This is because, in accordance with the stated problem, the intention was preferably to find a causal solution, meaning one applying at the level of molecular biology. Example 3 explains how corresponding deletions can be undertaken; further statements concerning this are given hereinafter.
A preferred process of the invention is thus one where, for the inactivation at the genetic level, one of the nucleic acids of the invention has been used within the region designated above and homologous to SEQ ID NO. 35, 37, 47 or 23, preferably one, particularly preferably two parts in each case of one of these sequences which comprise in each case at least 70 connected positions.
A further embodiment of the present invention is represented by the use of a gene which corresponds to the nucleic acid which codes for one of the following proteins of B. licheniformis DSM 13 for the functional inactivation of a metabolic pathway for synthesizing propionic acid (as part of propionate metabolism) at the genetic level in a microorganism: acetate-CoA ligase or synthetase or propionate-CoA ligase or synthetase (E.C. 6.2.1.1) defined by SEQ ID NO. 35 (acsA gene), 37 (ytcl gene), 47 (yhfL gene) or 23 (acsA gene).
The same as previously stated concerning the corresponding processes applies in principle to uses of this type.
Accordingly, preference is given to such a use according to the invention of nucleic acids of the invention within the region designated above and homologous to SEQ ID NO. 35, 37, 47 or 23 for functional inactivation, preferably of one, particularly preferably of two parts in each case of one of these sequences, where these parts comprise in each case at least 70 connected positions.
Further embodiments based on these fermentation processes and uses are detailed hereinafter.
The metabolic pathway utilized in Gram-positive bacteria of the genus Bacillus for synthesizing cadaverine and/or putrescine (as parts of lysine and/or arginine catabolism) is depicted in FIGS. 6 (for lysine and the cadaverine derived therefrom) and 7 (for arginine and the putrescine derived therefrom). This aspect, which is designated as a single pathway in the present application, of the bacterial metabolism is ultimately derived as side pathway from amino acid metabolism and in the second case additionally from the urea cycle.
As already mentioned, the following enzymes are involved in the reactions shown in FIGS. 6 and 7, where the relevant number designates the respective reaction step indicated in the figures:

    (1.) lysine decarboxylase (E.C. 4.1.1.18) and/or arginine decarboxylase (E.C. 4.1.1.19) (single demonstrated reaction in FIG. 6; step 1 in FIG. 7; the case where the same enzyme is able to catalyze both reactions also applies here),
    (2.) agmatinase (E.C. 3.5.1.11); step 2 in FIG. 7) and
    (3.) ornithine decarboxylase (E.C. 4.1.1.17; step 3 in FIG. 7).


Solutions of the stated problem and thus independent embodiments of the present invention are thus represented by all processes for fermenting a microorganism in which at least one of the genes on a metabolic pathway for synthesizing cadaverine and/or putrescine (as parts of lysine and/or arginine catabolism) is functionally inactivated.
The previously explained advantages are associated with this solution.
Preference is given in this connection to any process of this type in which the microorganism now forms only 50% of the amount naturally formed under the same conditions, preferably now only 10%, particularly preferably no cadaverine and/or no putrescine.
This takes account, as explained above for the first metabolic pathway described, of the generally high flexibility of microorganisms in relation to their metabolism.
A preferred process of the invention is one where at least one of the following enzymes is functionally inactivated:

    (1.) lysine decarboxylase (E.C. 4.1.1.18) and/or arginine decarboxylase (E.C. 4.1.1.19),
    (2.) agmatinase (E.C. 3.5.1.11) and
    (3.) ornithine decarboxylase (E.C. 4.1.1.17).


This is because, as depicted in FIGS. 6 and 7, these activities can be associated with the metabolic pathway under consideration here.
As stated above and described in the examples of the present application, it was possible by sequencing the genomic DNA of B. licheniformis DSM 13 to identify several of the genes coding for enzymes located on this pathway, or for subunits thereof. These are the following genes

    (the preceding number designates in each case the reaction in which the relevant enzyme is involved):
    (1.) lysine and/or arginine decarboxylase (E.C. 4.1.1.18 or E.C. 4.1.1.19) defined by SEQ ID NO. 5 (speA gene) or 39 (speA gene) and
    (2.) agmatinase (E.C. 3.5.1.11) defined by SEQ ID NO. 49 (ywhG gene).


The amino acid sequences derived therefrom are indicated in SEQ ID NO. 6, 40 and 50. It was thus possible in the course of the present invention to identify these specific gene products as involved in this metabolic pathway for synthesizing cadaverine and/or putrescine (as parts of lysine and/or arginine catabolism).
A preferred process of the invention is therefore one where the functionally inactivated enzyme is the homolog, which is naturally active in the relevant microorganism, to one of the following proteins from licheniformis DSM 13:

    (1.) lysine and/or arginine decarboxylase (E.C. 4.1.1.18 or E.C. 4.1.1.19) defined by SEQ ID NO. 6 or 40 and
    (2.) agmatinase (E.C. 3.5.1.11) defined by SEQ ID NO. 50.


A preferred process of the invention is one where the enzyme is functionally inactivated at the genetic level, preferably by inactivation of a gene which corresponds to the nucleic acid which codes for one of the following proteins of B. licheniformis DSM 13:

    (1.) lysine and/or arginine decarboxylase (E.C. 4.1.1.18 or E.C. 4.1.1.19) defined by SEQ ID NO. 5 (speA gene) or 39 (speA gene) and
    (2.) agmatinase (E.C. 3.5.1.11) defined by SEQ ID NO. 49 (ywhG gene).


This is because, in accordance with the stated problem, the intention was preferably to find a causal solution, meaning one applying at the level of molecular biology. Example 3 explains how corresponding deletions can be undertaken; further statements concerning this are given hereinafter.
Thus, preference is given to a process of the invention where for the inactivation at the genetic level one of the nucleic acids of the invention within the region designated above and homologous to

    (1.) SEQ ID NO. 5 or 39 and
    (2.) 49

has been used, preferably one, particularly preferably two parts in each case of one of these sequences which in each case comprise at least 70 connected positions.



A further embodiment of the present invention is represented by the use of a gene which corresponds to the nucleic acid which codes for one of the following proteins of B. licheniformis DSM 13 for functional inactivation of a metabolic pathway for synthesizing cadaverine and/or putrescine (as parts of lysine and/or arginine catabolism) at the genetic level in a microorganism:

    (1.) lysine and/or arginine decarboxylase (E.C. 4.1.1.18 or E.C. 4.1.1.19) defined by SEQ ID NO. 5 (speA gene) or 39 (speA gene) and
    (2.) agmatinase (E.C. 3.5.1.11) defined by SEQ ID NO. 49 (ywhG gene).


The same as has previously been stated concerning corresponding processes applies in principle to such uses.
Accordingly, preference is given to a use according to the invention of nucleic acids of the invention within the region designated above and homologous to

    (1.) SEQ ID NO. 5 or 39 and
    (2.) 49

for functional inactivation, preferably of one, particularly preferably of two parts in each case of one of these sequences, where these parts comprise in each case at least 70 connected positions.



Further embodiments based on these fermentation processes and uses are detailed hereinafter.
Embodiments which are preferred in each case of the uses described above according to the invention of the genes and/or nucleic acids on each of the described five metabolic pathways are those where the functional inactivation takes place during the fermentation of the microorganism.
This is because in accordance with the stated problem the intention was to improve the fermentation at the genetic level. On fermentation of microorganisms which have been correspondingly modified via these genes and/or nucleic acids is to be expected that the amount of the odorous and/or poisonous substances is less than with unmodified strains. This advantage, which emerges during the fermentation, is preferred according to the invention because it has advantageous effects both on the production process, meaning the fermentation process, and on the subsequent working up.
Among these, preference is given to any use of this type where (if present) with increasing preference 2, 3 or 4 of the genes mentioned for each metabolic pathway ((1.) for synthesizing isovaleric acid, (2.) for synthesizing 2-methylbutyric acid and/or isobutyric acid, (3.) for synthesizing butanol and/or butyric acid, (4) for synthesizing propionic acid and/or (5.) for synthesizing cadaverine and/or putrescine) are inactivated.
This is because, as already explained, microorganisms may in individual cases escape inactivation by activating an alternative pathway or at least enzymes with comparable reactions and thus continuing to form the relevant odorous and/or poisonous substance. This problem can be solved in particular by blocking a plurality of single reactions.
Preference is further given to any use of this type where (if present in the relevant microorganism) with increasing preference 2, 3, 4 or 5 of the metabolic pathways (1.) for synthesizing isovaleric acid, (2.) for synthesizing 2-methylbutyric acid and/or isobutyric acid, (3.) for synthesizing butanol and/or butyric acid, (4.) for synthesizing propionic acid and/or (5.) for synthesizing cadaverine and/or putrescine are blocked at least in part.
This is because firstly the inactivation of a single reaction may block a plurality of said pathways. This applies for example to butyryl-CoA dehydrogenase (E.C. 1.3.99.25) defined by SEQ ID NO. 9, 11 or 27 (yusJ gene) which occurs on the first three metabolic pathways mentioned; or to the three following enzymes or groups of enzymes which are equally involved in the two pathways mentioned first: 3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate dehydrogenase E1 (E.C. 1.2.4.2) defined by SEQ ID NO. 46, a subunit of acyl-CoA dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO. 10, 12, 22 or 28, and enoyl-CoA hydratase (protein) (E.C. 4.2.1.17) defined by SEQ ID NO. 16, 18, 20 or 42. In these cases too, the enzymic activities are defined with reference to the reactions described above and indicated in the figures.
Secondly, it is possible by generally known methods of molecular biology to inactivate a plurality of genes in parallel, so that in principle all these pathways can be switched off and thus correspondingly favorable fermentation processes can be obtained.
In one alternative, all these uses of genes and/or of the described nucleic acids of the invention are ones where in each case a nucleic acid coding for an inactive protein and having a point mutation is employed.
Nucleic acids of this type can be generated by methods of point mutagenesis known per se. Such methods are described for example in relevant handbooks such as that of Fritsch, Sambrook and Maniatis “Molecular cloning: a laboratory manual”, Cold Spring Harbour Laboratory Press, New York, 1989. In addition, numerous commercial construction kits are now available therefor, for instance the QuickChange® kit from Stratagene, La Jolla, USA. The principle thereof is for oligonucleotides having single exchanges (mismatch primers) to be synthesized and hybridized with the gene in single-stranded form; subsequent DNA polymerization then affords corresponding point mutants. It is possible to use for this purpose the respective species-specific sequences of these genes. Owing to the high homologies, it is possible and particularly advantageous according to the invention to carry out this reaction on the basis of the nucleotide sequences provided in the sequence listing. These sequences can also serve to design appropriate mismatch primers for related species.
In one alternative, all these uses of genes and/or of the described nucleic acids of the invention are ones where in each case a nucleic acid with a deletion mutation or insertion mutation is employed, preferably comprising the border sequences, in each case comprising at least 70 to 150 nucleic acid positions, of the region coding for the protein.
These methods are also familiar per se to the skilled worker. It is thus possible to prevent the formation of one or more of the described gene products by the host cell by cutting out part of the relevant gene on an appropriate transformation vector via restriction endonucleases, and subsequently transforming the vector into the host of interest, where the active gene is replaced by the inactive copy via the homologous recombination which is still possible until then. In the embodiment of insertion mutation it is possible merely to introduce the intact gene interruptingly or, instead of a gene portion, another gene, for example a selection marker. Phenotypical checking of the mutation event is possible thereby in a manner known per se.
In order to enable these recombination events which are necessary in each case between the defective gene introduced into the cell and the intact gene copy which is endogenously present for example on the chromosome, it is necessary according to the current state of knowledge that in each case there is agreement in at least 70 to 150 connected nucleic acid positions, in each case in the two border sequences to the non-agreeing part, with the part lying between being immaterial. Accordingly, preferred embodiments are those including only two flanking regions with at least one of these sizes.
In an alternative embodiment of this use, nucleic acids having a total of two nucleic acid segments which in each case comprise at least 70 to 150 nucleic acid positions, and thus flank at least partly, preferably completely, the region coding for the protein, are employed. The flanking regions can in this connection be ascertained starting from the known sequences by methods known per se, for example with the aid of outwardly directed PCR primers and a preparation of genomic DNA as template (anchored PCR). This is because it is not obligatory for the segments to be protein-encoding in order to make it possible to exchange the two gene copies by homologous recombination. According to the present invention it is possible to design the primers required for this on the basis of the nucleotide sequences indicated in the sequence listing also for other species of Gram-positive bacteria and, among these, in particular for those of the genus Bacillus. As an alternative to this experimental approach it is possible to take such regions which are at least in part non-coding for many of the genes from related species, for example from B. subtilis database entries, for example the SubtiList database of the Institute Pasteur, Paris, France (http://genolist.pasteur.fr/SubtiList/genome.cgi) or the databases specified in Example 2.
The present invention is aimed in particular at providing genetically improved microorganisms for biotechnological production. Thus, every microorganism in which at least one of the genes which corresponds to the nucleic acid which codes for one of the following proteins of B. licheniformis DSM 13 is functionally inactivated represents an embodiment of the present invention:

    
    
        putative branched-chain amino acid aminotransferase (E.C. 2.6.1.42) defined by SEQ ID NO. 1,
        putative branched-chain amino acid aminotransferase (E.C. 2.6.1.42) defined by SEQ ID NO. 3,
        lysine and/or arginine decarboxylase (protein SpeA; E.C. 4.1.1.18 or E.C. 4.1.1.19) defined by SEQ ID NO. 5 (speA gene),
        NADH-dependent butanol dehydrogenase A (protein YugJ; E.C. 1.1.1.-) defined by SEQ ID NO. 7 (yugJ gene),
        butyryl-CoA dehydrogenase (E.C. 1.3.99.25) or acyl-CoA dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO. 9,
        butyryl-CoA dehydrogenase (E.C. 1.3.99.25) or acyl-CoA dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO. 11,
        3-hydroxybutyryl-CoA dehydrogenase (E.C. 1.1.1.157) defined by SEQ ID NO. 13,
        putative enoyl-CoA hydratase protein (E.C. 4.2.1.17) defined by SEQ ID NO. 15,
        probable enoyl-(3-hydroxyisobutyryl)-CoA hydrolase protein defined by SEQ ID NO. 17,
        probable enoyl-CoA hydratase (protein EchA8; E.C. 4.2.1.17) defined by SEQ ID NO. 19 (echA8 gene),
        acyl-CoA dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO. 21,
        acetate-CoA ligase or propionate-CoA ligase (or synthetase; protein AcsA; E.C. 6.2.1.1) defined by SEQ ID NO. 23 (acsA gene),
        3-hydroxybutyryl-CoA dehydratase (protein YngF; E.C. 4.2.1.55) defined by SEQ ID NO. 25 (yngF gene),
        butyryl-CoA dehydrogenase (protein YusJ; E.C. 1.3.99.25) or acyl-CoA dehydrogenase (E.C. 1.3.99.-) defined by SEQ ID NO. 27 (yusJ gene),
        3-hydroxyisobutyrate dehydrogenase (protein YkwC; E.C. 1.1.1.31) or oxidoreductase (E.C. 1.1.-.-) defined by SEQ ID NO. 29 (ykwC gene),
        probable phosphate butyryltransferase (E.C. 2.3.1.19) defined by SEQ ID NO. 31,
        probable butyrate kinase (E.C. 2.7.2.7) defined by SEQ ID NO. 33,
        acetate-CoA ligase or synthetase or propionate-CoA ligase or synthetase (protein AcsA; E.C. 6.2.1.1) defined by SEQ ID NO. 35 (acsA gene),
        acetate-CoA ligase or propionate-CoA ligase (protein Ytcl; E.C. 6.2.1.1) defined by SEQ ID NO. 37 (ytcl gene),
        lysine and/or arginine decarboxylase (protein speA; E.C. 4.1.1.18 or E.C. 4.1.1.19) defined by SEQ ID NO. 39 (speA gene),
        probable enoyl-CoA hydratase (E.C. 4.2.1.17) defined by SEQ ID NO. 41 (ysiB gene),
        similar to 3-hydroxyacyl-CoA dehydrogenase (E.C. 1.1.1.35) defined by SEQ ID NO. 43,
        3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate dehydrogenase E1 (E.C. 1.2.4.2) defined by SEQ ID NO. 45,
        probable acetate-CoA ligase or propionate-CoA ligase (protein YhfL; E.C. 6.2.1.1) or acid-CoA ligase (E.C. 6.2.1.-) defined by SEQ ID NO. 47 (yhfL gene) or
        agmatinase (E.C. 3.5.1.11) defined by SEQ ID NO. 49 (ywhG gene).
    
    


“Corresponds” means in this connection in each case a gene of the organism under consideration which codes for a gene product having the same biochemical activity as defined above in connection with the respective metabolic pathways. This is generally at the same time the gene of all those of this organism which are translated in vivo which shows the greatest homology in each case to the stated gene from B. licheniformis (usually more than 40% identity, as can be found by an alignment of the two sequences as carried out in Example 2).
Among these, in accordance with the above statements, there is increasing preference in each case for a microorganism in which 2, 3 or 4 of the genes mentioned for each metabolic pathway ((1.) for synthesizing isovaleric acid, (2.) for synthesizing 2-methylbutyric acid and/or isobutyric acid, (3.) for synthesizing butanol and/or butyric acid, (4.) for synthesizing propionic acid and/or (5.) for synthesizing cadaverine and/or putrescine) are inactivated.
In addition, in accordance with the above statements, there is increasing preference in each case for a microorganism in which 2, 3, 4 or 5 of the metabolic pathways (1.) for synthesizing isovaleric acid, (2.) for synthesizing 2-methylbutyric acid and/or isobutyric acid, (3.) for synthesizing butanol and/or butyric acid, (4.) for synthesizing propionic acid and/or (5.) for synthesizing cadaverine and/or putrescine are blocked at least in part.
In addition, among these in each case a microorganism which is a bacterium is preferred.
This is because they have particular importance for biotechnological production. On the other hand, the relevant pathways have been described for microorganisms of the genus Bacillus. 
A microorganism which is preferred among these is in each case a Gram-negative bacterium, in particular one of the genera Escherichia Coli, Klebsiella, Pseudomonas or Xanthomonas, in particular strains of E. coli K12, E. coli B or Klebsiella planticola, and very especially derivatives of the strains Escherichia coli BL21 (DE3), E. coli RV308, E. coli DH5α, E. coli JM109, E. coli XL-1 or Klebsiella planticola (Rf).
This is because these are important strains for molecular biological operations on genes, for instance for cloning (see examples), and additionally important producer strains.
As alternative thereto, in each case a microorganism which is a Gram-positive bacterium is preferred, in particular one of the genera Bacillus, Staphylococcus or Corynebacterium, very especially of the species Bacillus lentus, B. licheniformis, B. amyloliquefaciens, B. subtilis, B. globigii or B. alcalophilus, Staphylococcus carnosus or Corynebacterium glutamicum, and among these very particularly preferably B. licheniformis DSM 13.
This is because these are particularly important for the biotechnological production of valuable products and proteins because they are naturally able to secrete them into the surrounding medium. On the other hand, they are increasingly related to the B. licheniformis employed for the present application, so that the working steps described and derived from the sequences disclosed in each case should proceed more successfully as the extent of relationship to B. licheniformis DSM 13 increases. It is thus to be assumed for example that a gene indicated in the sequence listing can, after point mutation, be used in a related species directly for a deletion mutation without the need to isolate the homologous gene from the strain itself for this purpose.
The present invention is aimed in particular at improving fermentation processes. Thus, every process for fermenting a microorganism of the invention described above represents an embodiment of the present invention.
These processes and the processes described above in each case in connection with an influence on one of the five metabolic pathways described are in particular processes where a valuable product is produced, in particular a low molecular weight compound or a protein.
This is because these are the essential areas of use of biotechnological production by fermentation of microorganisms.
Among these, preference is given in each case to a process where the low molecular weight compound is a natural product, a dietary supplement or a pharmaceutically relevant compound.
This is because they are important product groups for biotechnological production by fermentation of microorganisms.
Among such biotechnological processes for producing proteins by fermentation of microorganisms, preference is given in each case to a process where the protein is an enzyme, in particular one from the group of α-amylases, proteases, cellulases, lipases, oxidoreductases, peroxidases, laccases, oxidases and hemicellulases.
This is because these are important enzymes produced on the industrial scale, for example for incorporation in detergent or cleaning compositions.
In addition, the gene products provided according to the invention are available for further applications. Thus, the present invention is also implemented by any use of any gene product of the invention in a reaction mixture or process appropriate for its biochemical properties, which is defined as described above with reference to SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50.
Among these are preferably included uses (1.) for synthesizing isovaleric acid, (2.) for synthesizing 2-methylbutyric acid and/or isobutyric acid, (3.) for synthesizing butanol and/or butyric acid, (4.) for synthesizing propionic acid and/or (5.) for synthesizing cadaverine and/or putrescine, where appropriate in suitable combination with further enzymes.
Thus, the products of the metabolic pathways described are simple organic chemical compounds for which there is certainly a need in chemistry, for example to employ them as starting materials for more complex syntheses. Preparation thereof can be considerably simplified, especially when stereochemical reactions are involved, by the use of appropriate enzymes, because they in most cases specifically form one enantiomer. The term used when such synthetic routes are undertaken in at least one reaction step by biological catalysts is biotransformation. All gene products of the invention are suitable in principle therefor.
The following examples illustrate the present invention further.
EXAMPLES
All molecular biological working steps follow standard methods as indicated for example in the handbook by Fritsch, Sambrook and Maniatis “Molecular cloning: a laboratory manual”, Cold Spring Harbour Laboratory Press, New York, 1989, or comparable relevant works. Enzymes and construction kits are employed in accordance with the respective manufacturer's instructions.
Example 1
Identification of the Nucleic Acids Shown in SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 49 from B. licheniformis DSM 13
The genomic DNA was prepared by standard methods from the strain B. licheniformis DSM 13, which is available to anyone from the Deutsche Sammiung von Mikroorganismen and Zelikulturen GmbH, Mascheroder Weg 1b, 38124 Brunswick, mechanically fractionated and fractionated by electrophoresis in a 0.8% agarose gel. For a shotgun cloning of the smaller fragments, the fragments 2 to 2.5 kb in size were eluted from the agarose gel, dephosphorylated and ligated as blunt-ended fragments into the Smal restriction cleavage site of the vector pTZ19R-Cm. This is a derivative which confers chloramphenicol resistance of the plasmid pTZ19R which is obtainable from Fermentas (St. Leon-Rot). A gene library of the smaller fragments was obtained thereby. As second shotgun cloning, the genomic fragments obtained by a partial restriction with the enzyme Saulllal were ligated into the SuperCos 1 vector system (“Cosmid Vector Kit”) from Stratagene, La Jolla, USA, resulting in a gene library over the predominantly larger fragments.
The relevant recombinant plasmids were isolated and sequenced from the bacteria E. Coli DH5α (D. Hannahan (1983): “Studies on transformation on Escherichia coli”; J. Mol. Microbiol., volume 166, pages 557-580) obtainable by transformation with the relevant gene libraries. The dye termination method (dye terminator chemistry) was employed in this case, carried out by the automatic sequencers MegaBACE 1000/4000 (Amersham Bioscience, Piscataway, USA) and ABI Prism 377 (Applied Biosystems, Foster City, USA).
In this way, inter alia, the sequences SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 49 indicated in the sequence listing of the present application were obtained. The amino acid sequences derived therefrom are indicated—the relevant ones under the higher number in each case—under SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 and 50.
Example 2
Sequence Homologies
After ascertaining the DNA and amino acid sequences as in Example 1, in each case the most similar homologs disclosed to date were ascertained by searching the databases GenBank (National Center for Biotechnology Information NCBI, National Institute of Health, Bethesda, Md., USA), EMBL European Bioinformatics Institute (EBI) in Cambridge, Great Britain (http://www.ebi.ac.uk), Swiss-Prot (Geneva Bioinformatics (GeneBio) S.A., Geneva, Switzerland; http://www.genebio.com/sprot.html) and PIR (Protein Information Resource, National Biomedical Research Foundation, Georgetown University Medical Center, Washington, D.C., USA; http://www.pir.georgetown.edu). The nr (nonredundant) option was chosen in this connection.
The ascertained DNA and amino acid sequences were compared with one another via alignments in order to determine the degree of homology; the computer program used for this was Vector NTI® Suite Version 7, which is obtainable from Informax Inc., Bethesda, USA. In this case, the standard parameters of this program were used, meaning for comparison of the DNA sequences: K-tuple size: 2; Number of best Diagonals: 4; Window size: 4; Gap penalty: 5; Gap opening penalty: 15 and Gap extension penalty: 6.66. The following standard parameters applied to the comparison of the amino acid sequences: K-tuple size: 1; Number of best Diagonals: 5; Window size: 5; Gap penalty: 3; Gap opening penalty: 10 and Gap extension penalty: 0.1. The results of these sequence comparisons are compiled in Table 1 below, together with an indication of the respective enzyme names, meaning functions, E.C. numbers and the relevant metabolic pathways. The numbering of enzymes known in the prior art is the consistent nomenclature of the abovementioned databases.







TABLE 1







Genes and proteins of most similarity to the genes and proteins


respectively ascertained in Example 1.


The meanings therein are:


ID the SEQ ID NO. indicated in the sequence listing in the present application;


E.C. No. the number according to the international enzyme classification


(Enzyme Nomenclature of the IUBMB).













Name of the enzyme







(where possible of







the gene) and


Identity to the
Identity to the



additional


most closely
most closely



information where

Metabolic
related at the
related at the


ID
appropriate
E.C. No.
pathway
DNA level %
protein level %





1, 2
putative branched-
2.6.1.42
valine/isoleucine
62.40% to gb|AE017003.1|,
69% to



chain amino acid

catabolism
B. cereus ATCC
aminotransferase



aminotransferase


14579, section 6
IV from B.






of 18 of the
anthracis Ames






complete genome
(NP_655296.1)


3, 4
putative branched-
2.6.1.42
valine/isoleucine
73.80% to
79% to



chain amino acid

catabolism
emb|Z49992.1|BS
branched-chain



aminotransferase


CELABCD,
amino acid






B. subtilis genes
aminotransferase






celA, celB, celC,
from B. subtilis






celD and ywaA
168







(NP_391734.1)


5, 6
lysine and/or
4.1.1.18
cadaverine
74.00% to
85% to lysine



arginine
or
and/or
emb|X58433.1|BS
decarboxylase



decarboxylase
4.1.1.19
putrescine
CADDNA
from B. subtilis



(speA)

synthesis (lysine
B. subtilis, cad
(NP_389346;





and/or arginine
gene for lysine
A54546)





catabolism)
decarboxylase



7, 8
NADH-dependent
1.1.1.-
butyric acid
76.30% to
89% to NADH-



butanol

metabolism
emb|Z93934.1|BS
dependent



dehydrogenase A


Z93934,
butanol



(yugJ)


B. subtilis,
dehydrogenase






genomic DNA
from B. subtilis






fragment from
168






patB to yugK
(NP_391015.1)


 9, 10
acyl-CoA
1.3.99.-/
leucine
74.70% to
82% to short-



dehydrogenase
1.3.99.25
catabolism
emb|Z49782.1|BS
chain specific



(sic, i.e. more

valine/
DNA320D,
acyl-CoA



generally indicated

isoleucine
B. subtilis,
dehydrogenase



in the sequence

catabolism,
chromosomal
from B. cereus



listing)/butyryl-

butyric acid
DNA (region 320-
ATCC 14579



CoA dehydrogenase

metabolism
321 degrees)
(NP_835003.1)


11, 12
acyl-CoA
1.3.99.-
leucine
59.30% to
63% to C-



dehydrogenase

catabolism
emb|Z49782.1|BS
terminal domain



(sic, i.e. more


DNA320D,
of acyl-CoA



generally indicated


B. subtilis,
dehydrogenase



in the sequence


chromosomal
from B. anthracis



listing)/butyryl-


DNA (region 320-
Ames



CoA dehydrogenase.


321 degrees)
(NP_653803.1)



The first codon







ought to be







translated in vivo







as methionine






13, 14
3-hydroxyburyryl-
1.1.1.157
butyric cid
62.40% to
65% to the NAD-



CoA dehydrogenase

metabolism
gb|AE017015.1|,
binding domain






B. cereus ATCC
of 3-hydroxyacyl-






14579, section 18
CoA






of 18 of the
dehydrogenase,






complete genome
from B. anthracis







Ames







(NP_653804.1)


15, 16
putative enoyl-
4.2.1.17
leucine
61.00% to
58% to YhaR



CoA hydratase

catabolism,
emb|Y14078.1|BS
from B. subtilis



protein

valine/
Y14078,
168





isoleucine
B. subtilis, 8.7 Kb
(CAB12828.2)





catabolism
chromosomal







DNA:







downstream of







the glyB-prsA







region



17, 18
probable enoyl-
not yet allo-
leucine
61.90% to
62% to 3-



(3-hydroxy-
cated
catabolism,
gb|AE017031.1|,
hydroxy-



isobutyryl)-

valine/
B. anthracis
isobutyryl-



coenzyme A

isoleucine
Ames, section 8
coenzyme A



hydrolase protein

catabolism
of 18 of the
hydrolase from






complete genome
B. cereus ATCC







14579







(NP_832055.1;







AAP09256)


19, 20
probable enoyl-CoA
4.2.1.17
leucine
43.50% to
48% to 3-hydroxy-



hydratase (echA8).

catabolism,
gb|AC084761.2|,
butyryl-CoA



The first codon

valine/
Gallus gallus,
dehydratase from



ought to be

isoleucine
clone WAG-69H2,
B subtilis 168



translated in vivo

catabolism
complete
(NP_390732.1)



as methionine


sequence



21, 22
actyl-CoA
1.3.99.-
leucine
49.90% to
61% to acyl-CoA



dehydrogenase

catabolism,
gb|AE015940.1|,
dehydrogenase





valine/
Clostridium tetani
from B. cereus





isoleucine
E88, section 5 of
ATCC 14579





catabolism
10 of the
(NP_832051.1)






complete genome



23, 24
acetyl-coenzyme
6.2.1.1
propionate
63.00% to
61% to acetyl-



A synthetase

metabolism
dbj|AP001511.1|,
CoA synthetase



(indicated thus in


B. halodurans,
from B.



the sequence


genomic DNA,
halodurans



listing) or


section 5/14
(NP_242003.1)



propionate-CoA







ligase (acsA)






25, 26
3-hydroxybutyryl-
4.2.1.55
butyric acid
63.80% to
65% to



CoA dehydratase

metabolism
emb|Y13917.1|BS
hydroxybutyryl



(yngF)


Y13917,
dehydratase






B. subtilis, genes
from






ppsE, yngL,
B. subtilis






yngK, yotB, yngJ,
(AAF32340.1)






yngI, yngH, yngG







and yngF and partial genes







ppsD and yngE



27, 28
acyl-CoA
1.3.99./
leucine
72.90% to
82% of butyryl-



dehydrogenase
1.3.99.25
catabolism,
emb|Y13917.1|BS
CoA



(sic, i.e. more

valine/
Y13917,
dehydrogenase



generally,

isoleucine
B. subtilis, genes
from B. subtilis



indicated in the

catabolism,
ppsE, yngL,
168



sequence listing)/

butyric acid
yngK, yotB, yngJ,
(NP_389708.1)



butyryl-CoA

metabolism
yngI, yngH, yngG




dehydrogenase


and yngF and




(yusJ)


partial genes







ppsD and yngE



29, 30
3-hydroxy-
1.1.1.31 or 1.1.-.-
valine
72.80% to
81% to 3-



isobutyrate

catabolism
emb|AJ222587.1|
hydroxy



dehydrogenase/


BS16829KB,
isobutyrate



hypothetical


B. subtilis, 29 kB
dehydrogenase



oxidoreductase


DNA fragment
from B. subtilis



(sic, i.e. more


from the gene
168



generally,


ykwC to the gene
(NP_389279.1)



indicated in the


cse15




sequence listing)







(ykwC)






31, 32
probable phosphate
2.3.1.19
butyric acid
46.30% to
65% to



butyryl-transferase

metabolism
gb|S81735.1|S81
phosphate






735, leucine
butyryl-






dehydrogenase
transferase







from B. subtilis 168







(NP_390289.1)


33, 34
probable butyrate
2.7.2.7
butyric acid
72.50% to
80% to



kinase

metabolism
emb|Z99116.2|BS
branched-chain






UB0013,
fatty acid kinase






B. subtilis,
from B. subtilis






complete genome
168






(section 13 of 21):
(NP_390287.1)






from 2409151 to







2613687



35, 36
acetyl-coenzyme A
6.2.1.1
propionate
74.90% to
81% to acetyl-



synthetase

metabolism
emb|Z99119.2|BS
CoA synthetase



(indicated thus in


UB0016,
from B. subtilis



the sequence


B. subtilis,
168



listing) or


complete genome
(NP_390846.1)



propionate-CoA


(section 16 of 21):
and to acetate-



ligase (acsA)


from 3013458 to
CoA ligase from






3213379
B. subtilis







(P39062,







S39646)


37, 38
acetate-CoA ligase
6.2.1.1
propionate
70% to
73% to acetate-



(indicated thus in

metabolism
emb|Z99119.2|BS
CoA ligase from



the sequence


UB0016,
B. subtilis 168



listing) or


B. subtilis,
(NP_390834.1,



propionate-CoA


complete genome
E69989)



ligase (ytcI).


(section 16 of 21):




The first codon


from 3013458 to




ought to be


3213379




translated in vivo







as methionine






39, 40
lysine and/or
4.1.1.18
cadaverine
63.40% to
62% to lysine



arginine
or
and/or
emb|Z99104.2|BS
decarboxylase



decarboxylase
4.1.1.19
putrescine
UB0001,
from B. subtilis



(speA)

synthesis
B. subtilis,
168





(lysine and/or
complete genome
(NP_387908.1)





arginine
(section 1 of 21):
and B. perfrigens





catabolism)
from 1 to 213080
(NP_976355)


41, 42
probable enoyl-CoA
4.2.1.17
leucine
70.30% to
73% to 3-



hydratase (ysiB)

catabolism,
emb|Z75208.1|BS
hydroxybutyryl-





valine/
Z75208,
CoA dehydratase





isoleucine
B. subtilis, genomic
from B. subtilis





catabolism
sequence,
168






89009 bp
(NP_390732.1)


43, 44
similar to
1.1.1.35
isoleucine
71.60% to
76% to 3-



3-hydroxyacyl-CoA

catabolism
emb|Z99120.2|BS
hydroxyacyl-CoA



dehydrogenase


UB0017,
dehydrogenase






B. subtilis,
from B. subtilis






complete genome
168






(section 17 of 21):
(NP_391163.1)






from 3213330 to







3414388



45, 46
3-methyl-2-
1.2.4.2
leucine
75.70% to
78% to E1



oxobutanoate

catabolism,
emb|X54805.1|BS
subunit of



dehydrogenase/

valine/
ODHA, B. subtilis,
2-oxoglutarate



2-oxoglutarate

isoleucine
odhA gene for
dehydrogenase



dehydrogenase E1

catabolism
2-oxoglutarate
from B. subtilis



component (sic,


dehydrogenase
(CAB13829.2)



i.e. indicated







more generally







in the sequence







listing)






47, 48
probable acid-CoA
6.2.1.-
propionate
62.20% to
72% to long-



ligase (yhfL)

metabolism
gb|AE017001.1|,
chain fatty acid-






B. cereus ATCC
CoA ligase from






14579, section 4
B. subtilis 168






of 18 of the
(NP_388908.1)






complete genome



49, 50
agmatinase (ywhG)
3.5.1.11
cadaverine
80.9% to
95% to





and/or
B. subtilis, gene
agmatinase





putrescine
BSUB0020
(agmatine





synthesis (lysine
(Genebank,
ureohydrolase)





and/or arginine
complete
from B. subtilis





catabolism)
genome)
168 (P70999)









It is evident that the genes found and the gene products derived therefrom are respectively novel genes and proteins with a clear distance from the prior art disclosed to date.
Example 3
Functional Inactivation of One or More of the Genes Shown in SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 49 in B. licheniformis 
Principle of the Preparation of a Deletion Vector
Each of these genes can be functionally inactivated for example by means of a so-called deletion vector. This procedure is described per se for example by J. Vehmaanperä et al. (1991) in the publication “Genetic manipulation of Bacillus amyloliquefaciens”; J. Biotechnol., volume 19, pages 221-240.
A suitable vector for this is pE194 which is characterized in the publication “Replication and incompatibility properties of plasmid pE194 in Bacillus subtilis” by T. J. Gryczan et al. (1982), J. Bacteriol., volume 152, pages 722-735. The advantage of this deletion vector is that it possesses a temperature-dependent origin of replication. pE194 is able to replicate in the transformed cell at 33° C., so that initial selection for successful transformation takes place at this temperature. Subsequently, the cells comprising the vector are incubated at 42° C. The deletion vector no longer replicates at this temperature, and a selection pressure is exerted on the integration of the plasmid via a previously selected homologous region into the chromosome. A second homologous recombination via a second homologous region then leads to excision of the vector together with the intact gene copy from the chromosome and thus to deletion of the gene which is located in the chromosome in vivo. Another possibility as second recombination would be the reverse reaction to integration, meaning recombination of the vector out of the chromosome, so that the chromosomal gene would remain intact. The gene deletion must therefore be detected by methods known per se, for instance in a southern blot after restriction of the chromosomal DNA with suitable enzymes or with the aid of the PCR technique on the basis of the size of the amplified region.
It is thus necessary to select two homologous regions of the gene to be deleted, each of which should include at least 70 base pairs in each case, for example the 5′ region and the 3′ region of the selected gene. These are cloned into the vector in such a way that they flank a part coding for an inactive protein, or are in direct succession, omitting the region in between. The deletion vector is obtained thereby.
Deletion of the Genes Considered Here
A deletion vector of the invention is constructed by PCR amplification of the 5′ and 3′ regions of one of these genes of interest in each case. The sequences SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 49 indicated in the sequence listing are available for designing suitable primers and originate from B. licheniformis, but ought also to be suitable, because of the homologies to be expected, for other species, especially of the genus Bacillus. 
The two amplified regions suitably undergo intermediate cloning in direct succession on a vector useful for these operations, for example on the vector pUC18 which is suitable for cloning steps in E. coli. 
The next step is a subcloning into the vector pE194 selected for deletion, and transformation thereof into B. subtilis DB104, for instance by the method of protoplast transformation according to Chang & Cohen (1979; “High Frequency Transformation of Bacillus subtilis Protoplasts by Plasmid DNA”; Molec. Gen. Genet. (1979), volume 168, pages 111-115). All working steps must be carried out at 33° C. in order to ensure replication of the vector.
In a next step, the vector which has undergone intermediate cloning is likewise transformed by the method of protoplast transformation into the desired host strain, in this case B. licheniformis. The transformants obtained in this way and identified as positive by conventional methods (selection via the resistance marker of the plasmid; check by plasmid preparation and PCR for the insert) are subsequently cultured at 42° C. under selection pressure for presence of the plasmid through addition of erythromycin. The deletion vector is unable to replicate at this temperature, and the only cells to survive are those in which the vector is integrated into the chromosome, and this integration most probably takes place in homologous or identical regions. Excision of the deletion vector can then be induced subsequently by culturing at 33° C. without erythromycin selection pressure, the chromosomally encoded gene being completely deleted from the chromosome. The success of the deletion is subsequently checked by southern blotting after restriction of the chromosomal DNA with suitable
Such transformants in which the relevant gene is deleted are normally additionally distinguished by a limitation on the formation of the odorous or poisonous substance resulting from the relevant metabolic pathway. In the cases where the cell has no substitute pathway for synthesizing the relevant compound, the relevant metabolic pathway is completely blocked so that this compound is no longer formed at all, and the strain modified in this way no longer has the relevant odorous component.

## Claims

1. A process for fermenting a substrate using a microorganism, the process comprising:
(a) providing the microorganism having (1) a butyryl-CoA dehydrogenase gene and (2) a lysine and/or arginine decarboxylase gene involved in metabolic pathways for synthesizing odorous substances;
(b) functionally inactivating (1) the butyryl-CoA dehydrogenase gene and (2) the lysine and/or arginine decarboxylase gene to form functionally inactivated genes;
(c) providing a fermentable substrate; and
(d) permitting the microorganism to ferment the substrate,
wherein the microorganism is of a genus Bacillus, and
wherein the metabolic pathways for synthesizing the odorous substances are at least partially blocked, thereby reducing odor produced during fermentation.

2. The process according to claim 1, wherein the microorganism having the functionally inactivated genes forms less than 50% of the amount of the odorous substances formed in the microorganism in which (1) the butyryl-CoA dehydrogenase gene and (2) the lysine and/or arginine decarboxylase gene on the metabolic pathways for synthesizing the odorous substance are not functionally inactivated, wherein the odorous substances are (1) butanol and/or butyric acid and (2) cadaverine and/or putrescine, respectively.

3. The process according to claim 2, wherein the microorganism having the functionally inactivated genes forms less than 10% of the amount of the odorous substances in the microorganism in which the butyryl-CoA dehydrogenase gene and the lysine and/or arginine decarboxylase gene on the metabolic pathways for synthesizing the odorous substances are not functionally inactivated.

4. The process according to claim 3, wherein the microorganism having the functionally inactivated genes forms none of the odorous substances in the microorganism in which the butyryl-CoA dehydrogenase gene and the lysine and/or arginine decarboxylase gene on the metabolic pathways for synthesizing the odorous substances are not functionally inactivated.

5. The process according to claim 1, wherein the butyryl-CoA dehydrogenase gene encodes an amino acid sequence as defined by SEQ ID NO. 10 and wherein the lysine and/or arginine decarboxylase gene encodes an amino acid sequence selected from SEQ ID NOS: 6 or 40.

6. The process according to claim 1 wherein the functionally inactivated genes are functionally inactivated by a point mutation.

7. The process according to claim 1 wherein the functionally inactivated genes are functionally inactivated by a deletion mutation.

8. The process according to claim 1 wherein the functionally inactivated genes are functionally inactivated by an insertion mutation.

9. The process according to claim 1 wherein a low molecular weight compound is produced selected from the group consisting of natural products, dietary supplements and pharmaceutically relevant compounds, and enzymes.

10. The process according to claim 9, wherein the enzymes are selected from the group consisting of a-amylases, proteases, cellulases, lipases, oxidoreductases, peroxidases, laccases, oxidases and hemicellulases.

11. The process according to claim 1,
wherein (1) the functionally inactivated butyryl-CoA dehydrogenase gene and (2) the lysine and/or arginine decarboxylase gene are on the metabolic pathways for synthesizing (1) butanol and/or butyric acid and (2) cadaverine and/or putrescine, respectively.

12. A process for fermenting a substrate using a microorganism, the process comprising:
(a) providing the microorganism having (1) a butyryl-CoA dehydrogenase gene and (2) a lysine and/or arginine decarboxylase gene involved in metabolic pathways for synthesizing predetermined products, wherein the butyryl-CoA dehydrogenase gene and the lysine and/or arginine decarboxylase gene are functionally inactivated to at least partially block the metabolic pathways for synthesizing (1) butanol and/or butyric acid and (2) cadaverine and/or putrescine, respectively,
(b) providing a fermentable substrate; and
(c) permitting the microorganism to ferment the substrate,
wherein the microorganism is a genus Bacillus.

13. A process of preparing a microorganism of a genus Bacillus, the process comprising:
(a) providing the microorganism having (1) a butyryl-CoA dehydrogenase gene and (2) a lysine and/or arginine decarboxylase gene involved in metabolic pathways for synthesizing odorous substances; and
(b) functionally inactivating the butyryl-CoA dehydrogenase gene and the lysine and/or arginine decarboxylase gene effective to at least partially block the metabolic pathways for synthesizing the odorous substances.