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 thereform 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 synthesised 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.

1 . A nucleic acid selected from the group consisting of:
 (a) nucleic acids 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 to the nucleotide sequence indicated in SEQ ID NO. 1;   (b) nucleic acids 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 to the nucleotide sequence indicated in SEQ ID NO. 3;   (c) nucleic acids 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 to the nucleotide sequence indicated in SEQ ID No. 5;   (d) nucleic acids 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 to the nucleotide sequence indicated in SEQ ID NO. 7;   (e) nucleic acids 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 75% identity to the nucleotide sequence indicated in SEQ ID NO. 9;   (f) nucleic acids 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 to the nucleotide sequence indicated in SEQ ID NO. 11;   (g) nucleic acids 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 to the nucleotide sequence indicated in SEQ ID NO. 13;   (h) nucleic acids 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 to the nucleotide sequence indicated in SEQ ID NO. 15;   (i) nucleic acids 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 to the nucleotide sequence indicated in SEQ ID NO. 17;   (j) nucleic acids 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 to the nucleotide sequence indicated in SEQ ID NO. 19;   (k) nucleic acids 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 to the nucleotide sequence indicated in SEQ ID NO. 21;   (l) nucleic acids 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 to the nucleotide sequence indicated in SEQ ID NO. 23;   (m) nucleic acids 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 to the nucleotide sequence indicated in SEQ ID NO. 25;   (n) nucleic acids 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 to the nucleotide sequence indicated in SEQ ID NO. 27;   (o) nucleic acids 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 to the nucleotide sequence indicated in SEQ ID NO. 29;   (p) nucleic acids 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 to the nucleotide sequence indicated in SEQ ID NO. 31;   (q) nucleic acids 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 to the nucleotide sequence indicated in SEQ ID NO. 33;   (r) nucleic acids 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 to the nucleotide sequence indicated in SEQ ID NO. 35;   (s) nucleic acids 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 to the nucleotide sequence indicated in SEQ ID NO. 37;   (t) nucleic acids 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 to the nucleotide sequence indicated in SEQ ID NO. 39;   (u) nucleic acids ysiB 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 75% identity to the nucleotide sequence indicated in SEQ ID NO. 41;   (v) nucleic acids 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 to the nucleotide sequence indicated in SEQ ID NO. 43;   (w) nucleic acids 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 to the nucleotide sequence indicated in SEQ ID NO. 45;   (x) nucleic acids 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 to the nucleotide sequence indicated in SEQ ID NO. 47; and   (y) nucleic acids 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 to the nucleotide sequence indicated in SEQ ID NO. 49.   
     
     
         2 . (canceled) 
     
     
         3 . A nucleic acid according to  claim 1  coding for a gene product involved in the synthesis of at least one compound selected from the group consisting of isovaleric acid, 2-methylbutyric acid, isobutyric acid, butanol, butyric acid, propionic acid, cadaverine and putrescine, the nucleic acid being naturally present in a microorganism. 
     
     
         4 .- 15 . (canceled) 
     
     
         16 . A process for fermenting a substrate using a microorganism, the process comprising:
 (a) providing a microorganism, the microorganism having at least one gene on a metabolic pathway for synthesizing a predetermined product being functionally inactivated, the functionally inactivated enzyme being selected from the group consisting of:   (1) functionally inactivated enzymes of the metabolic pathway for synthesizing isovaleric acid (as part of leucine catabolism) selected from the group consisting of:   (i) L-leucine dehydrogenase (E.C. 1.4.1.9),   (ii) 3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate dehydrogenase E1 (E.C. 1.2.4.2),   (iii) enzyme for hydrolyzing isovaleryl-CoA to isovaleric acid and coenzyme A,   (iv) acyl-CoA dehydrogenase (E.C. 1.3.99.-),   (v) methylcrotonyl carboxylase,   (vi) 3-methylglutaconyl-CoA hydratase, and   (vii) enoyl-CoA hydratase (protein) (E.C. 4.2.1.17);   (2) functionally inactivated enzymes of the metabolic pathway for synthesizing 2-methylbutyric acid and/or isobutyric acid (as part of valine and/or isoleucine catabolism) from the group consisting of:   (i) branched-chain amino acid aminotransferase (E.C. 2.6.1.42),   (ii) 3-methyl-2-oxobutanoate dehydrogenase or 2-oxoglutarate dehydrogenase E1 (E.C. 1.2.4.2),   (iii) enzyme for hydrolyzing 2-methylbutyryl-CoA to 2-methylbutyric acid or isobutyryl-CoA to isobutyric acid and coenzyme A,   (iv) acyl-CoA dehydrogenase (E.C. 1.3.99.-),   (v) enoyl-CoA hydratase (protein) (E.C. 4.2.1.17),   (vi) 3-hydroxy-acyl-CoA dehydrogenase (E.C. 1.1.1.35),   (vii) acetyl-CoA acyltransferase,   (viii) enoyl-(3-hydroxyisobutyryl)-CoA hydrolase protein and   (ix) 3-hydroxyisobutyrate dehydrogenase (E.C. 1.1.1.31) or oxidoreductase (E.C.1.1.-.-);   (3) functionally inactivated enzymes of the metabolic pathway for synthesizing butanol and/or butyric acid (as part of butyric acid metabolism) from the group consisting of:   (i) 3-hydroxybutyryl-CoA dehydrogenase (E.C. 1.1.1.157),   (ii) 3-hydroxybutyryl-CoA dehydratase (E.C. 4.2.1.55),   (iii) butyryl-CoA dehydrogenase (E.C. 1.3.99.25),   (iv) phosphate butyryltransferase (E.C. 2.3.1.19),   (v) butyrate kinase (E.C. 2.7.2.7),   (vi) butyraldehyde dehydrogenase and   (vii) NADH-dependent butanol dehydrogenase A (E.C. 1.1.1.-)   (4) functionally inactivated enzymes of the metabolic pathway for synthesizing propionic acid (as part of propionate metabolism) from the group consisting of:   (i) 3-hydroxybutyryl-CoA dehydrogenase (E.C. 1.1.1.157),   (ii) 3-hydroxybutyryl-CoA dehydratase (E.C. 4.2.1.55),   (iii) butyryl-CoA dehydrogenase (E.C. 1.3.99.25),   (iv) phosphate butyryltransferase (E.C. 2.3.1.19),   (v) butyrate kinase (E.C. 2.7.2.7),   (vi) butyraldehyde dehydrogenase, and   (viii) NADH-dependent butanol dehydrogenase A (E.C. 1.1.1.-) and   (5) functionally inactivated enzymes of metabolic pathway for synthesizing cadaverine and/or putrescine (as parts of lysine and/or arginine catabolism) from the group consisting of:   (i) lysine decarboxylase (E.C. 4.1.1.18) and/or arginine decarboxylase (E.C. 4.1.1.19),   (ii) agmatinase (E.C. 3.5.1.11) and   (iii) ornithine decarboxylase (E.C. 4.1.1.17);   (b) providing a fermentable substrate; and   (c) permitting the microorganism to ferment the substrate.   
     
     
         17 .- 21 . (canceled) 
     
     
         22 . A microorganism in which at least one of the genes which corresponds to the nucleic acid which codes for one predetermined protein of  B. licheniformis  DSM 13 is functionally inactivated, the predetermined protein being selected from the group consisting of:
 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 (E.C.4.2.1.17) 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).   
     
     
         23 . A microorganism according to  claim 22  in which at least two of the genes are functionally inactivated. 
     
     
         24 . A microorganism according to  claim 22 , in which at least one of the metabolic pathways selected from the group consisting of the 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 (5.) for synthesizing cadaverine and/or putrescine, are blocked at least in part. 
     
     
         25 . A microorganism according to  claim 22 , wherein the microorganism is is a bacterium. 
     
     
         26 . A microorganism according to  claim 25 , the bacterium being selected from the group of Gram-negative bacteria consisting of the genera  Escherichia coli, Klebsiella, Pseudomonas  and  Xanthomonas.    
     
     
         27 . A microorganism according to  claim 25 , the bacterium being selected from the group of Gram-negative bacteria strains  Escherichia coli  BL21 (DE3),  E. coli  RV308,  E. coli  DH5 α, E. coli  JM109,  E. coli  XL-1 and  Klebsiella planticola  (Rf). 
     
     
         28 . A microorganism according to  claim 25 , the bacterium being selected from the group of Gram-positive genera  Bacillus, Staphylococcus  and  Corynebacterium.    
     
     
         29 . A microorganism according to  claim 25 , the bacterium being selected from the group of Gram-positive species  Bacillus lentus, B. licheniformis, B. amyloliquefaciens, B. subtilis, B. globigii  or  B. alcalophilus, Staphylococcus carnosus  and  Corynebacterium glutamicum    
     
     
         30 . A microorganism according to  claim 29  wherein the bacterium is  B. licheniformis  DSM 13.