Method for Producing L-Amino Acids in Corynebacteria Using a Glycine Cleavage System

Abstract

It has been found, surprisingly, that the Corynebacterium humireducens strain comprises a very effective glycine cleavage system.

Claims

1-15. (canceled)

16. A glycine cleavage system comprising one or more of the enzymes GcvP, GcvT and GcvH, wherein: a) GcvP comprises a sequence at least 80% identical to the sequence of SEQ ID NO:40; b) GcvT comprises a sequence at least 80% identical to the sequence of SEQ ID NO:42; and c) GcvH comprises a sequence at least 80% identical to the sequence of SEQ ID NO:38.

17. The glycine cleavage system of claim 16, wherein said system comprises at least two of said enzymes.

18. The glycine cleavage system of claim 16, wherein said system comprises all three of said enzymes.

19. The glycine cleavage of claim 16, wherein: a) GcvP comprises a sequence at least 95% identical to the sequence of SEQ ID NO:40; b) GcvT comprises a sequence at least 95% identical to the sequence of SEQ ID NO:42; and c) GcvH comprises a sequence at least 95% identical to the sequence of SEQ ID NO:38.

20. The glycine cleavage system of claim 19, wherein said system comprises all three of said enzymes.

21. The glycine cleavage system of claim 16, wherein said system comprises at least one further polypeptide selected from the group consisting of: a) a LipA enzyme having a sequence at least 80% identical to the sequence of SEQ ID NO:48; b) a LipB enzyme having a sequence at least 80% identical to the sequence of SEQ ID NO:50; c) a Lpd enzyme having a sequence at least 80% identical to the sequence of SEQ ID NO:52; d) a LplA enzyme having a sequence at least 80% identical to the sequence of SEQ ID NO:94; e) a GcvL enzyme having a sequence at least 80% identical to the sequence of SEQ ID NO:96.

22. The glycine cleavage system of claim 21, wherein said system comprises all three of said enzymes and wherein: a) GcvP comprises a sequence at least 95% identical to the sequence of SEQ ID NO:40; b) GcvT comprises a sequence at least 95% identical to the sequence of SEQ ID NO:42; and c) GcvH comprises a sequence at least 95% identical to the sequence of SEQ ID NO:38.

23. The glycine cleavage system of claim 16, wherein said system comprises: a) GcvP comprising a sequence at least 96% identical to the sequence of SEQ ID NO:40; b) GcvT comprising a sequence at least 96% identical to the sequence of SEQ ID NO:42; c) GcvH comprising a sequence at least 96% identical to the sequence of SEQ ID NO:38; d) LipA comprising a sequence at least 96% identical to the sequence of SEQ ID NO:48; e) LipB comprising a sequence at least 96% identical to the sequence of SEQ ID NO:50; f) Lipd comprising a sequence at least 96% identical to the sequence of SEQ ID NO:52; g) LilA comprising a sequence at least 96% identical to the sequence of SEQ ID NO: 94; h) GcvL comprising a sequence at least 96% identical to the sequence of SEQ ID NO:96.

24. A recombinant microorganism, comprising the glycine cleavage system of claim 16.

25. The recombinant microorganism of claim 24, wherein one or more polynucleotides encoding the enzymes in the glycine cleavage system are overexpressed.

26. The recombinant microorganism of claim 24, wherein said microorganism overproduces L-methionine and comprises one or more of the of the following features: a) an attenuated polynucleotide (mcbR), which codes for a DNA binding domain having a sequence at least 95% identical to the sequence of SEQ ID NO:2; b) an attenuated polynucleotide (thrB gene), which codes for a homoserine kinase having a sequence at least 95%, identical to the sequence of SEQ ID NO:4; c) an attenuated polynucleotide (pgi), which codes for a glucose-6-phosphate isomerase having a sequence at least 95%, identical to the sequence of SEQ ID NO:6; d) an attenuated polynucleotide (pck), which codes for a phosphoenol-pyruvate carboxykinase having a sequence at least 95%, identical to the sequence of SEQ ID NO:8; e) an attenuated polynucleotide (metQ), which codes for a D-methionine-binding lipoprotein having a sequence at least 95%, identical to the sequence of SEQ ID NO:10; an attenuated polynucleotide (metP), which codes for a methionine transporter having a sequence at least 95%, identical to the sequence of SEQ ID NO:12; g) an attenuated polynucleotide (metN), which codes for an ATP-dependent methionine transporter having a sequence at least 95% identical to the sequence of SEQ ID NO:14; h) an attenuated polynucleotide (metK), which codes for an S-adenosyl-methionine synthase having a sequence at least 95% identical to the sequence of SEQ ID NO:16; i) an attenuated polynucleotide (metI), which codes for a methionine import system permease having a sequence at least 95% identical to the sequence of SEQ ID NO:18; j) an attenuated polynucleotide (dapA), which codes for a 4-hydroxy-tetrahydrodipicolinate synthase having a sequence at least 95% identical to the sequence of SEQ ID NO:20; k) an overexpressed polynucleotide (CBS), which codes for a cysteine synthase having a sequence at least 95% identical to the sequence of SEQ ID NO:22; l) an attenuated polynucleotide, which codes for a cg3031 homologue having a sequence at least 95% identical to the sequence of SEQ ID NO:24; m) an overexpressed polynucleotide (aecD), which codes for a cystathionine beta-lyase having a sequence at least 95% identical to the sequence of SEQ ID NO:26; n) an overexpressed polynucleotide (asd), which codes for an aspartate semialdehyde dehydrogenase having a sequence at least 95% identical to the sequence of SEQ ID NO:28; o) an overexpressed polynucleotide (metH), which codes for a 5-methyltetra-hydrofolate homocysteine methyltransferase (MetH, EC 2.1.1.13); P) an overexpressed polynucleotide (brnE), which codes for the smaller subunit of a transporter for branched-chain amino acids (BrnE) and having a sequence identity at least 95%, identical to the sequence of SEQ ID NO:30; q) an overexpressed polynucleotide (brnF), which codes for the larger subunit of a transporter for branched-chain amino acids (BrnF) having a sequence at least 95% identical to the sequence of SEQ ID NO:32; r) an overexpressed polynucleotide (cysE), which codes for a serine acetyl-transferase (CysE) having a sequence at least 95% identical to the sequence of SEQ ID NO:34; s) an overexpressed polynucleotide (cysK), which codes for a cysteine synthase (CysK) having a sequence at least 95% identical to the sequence of SEQ ID NO:36; t) an overexpressed polynucleotide (glyA), which codes for a serine hydroxymethyltransferase (GlyA) having a sequence at least 95% identical to the sequence of SEQ ID NO:44; u) an overexpressed polynucleotide (horn), which codes for an optionally feedback-resistant homoserine dehydrogenase (Horn) having a sequence at least 95% identical to the sequence of SEQ ID NO:46; v) an overexpressed polynucleotide (lysC), which codes for an optionally feedback-resistant aspartate kinase (LysC) having a sequence at least 95%, identical to the sequence of SEQ ID NO:54; w) an overexpressed polynucleotide (metB), which codes for a cystathionine gamma-synthase (MetB) having a sequence at least 95% identical to the sequence of SEQ ID NO:56; x) an overexpressed polynucleotide (metF), which codes for a 5,10-methylene-tetrahydrofolate reductase (MetF) having a sequence at least 95% identical to the sequence of SEQ ID NO:58; y) an overexpressed polynucleotide (metX), which codes for a homoserine O-acetyltransferase (MetX) having a sequence at least 95% identical to the sequence of SEQ ID NO:60; z) an overexpressed polynucleotide (metY), which codes for an O-acetylhomoserine lyase (MetY) having a sequence at least 95% identical to the sequence of SEQ ID NO:62; aa) an overexpressed polynucleotide (pyc), which codes for a pyruvate carboxylase (Pyc) having a sequence at least 95% identical to the sequence of SEQ ID NO:64; bb) an overexpressed polynucleotide (serA), which codes for an optionally feedback-resistant D-3-phosphoglycerate dehydrogenase (SerA) having a sequence at least 95% identical to the sequence of SEQ ID NO:66; cc) an overexpressed polynucleotide (serB), which codes for a phosphoserine phosphatase (SerB) having a sequence at least 95% identical to the sequence of SEQ ID NO:68; dd) an overexpressed polynucleotide (serC), which codes for a phosphoserine aminotransferase (SerC) having a sequence at least 95% identical to the sequence according of SEQ ID NO:70; ee) an overexpressed polynucleotide (ald), which codes for an alanine dehydrogenase (Ald) having a sequence at least 95% identical to the sequence of SEQ ID NO:72; ff) an overexpressed polynucleotide (cysD), which codes for the subunit of a sulphate adenylyltransferase (CysD) having a sequence at least 95% identical to the sequence of SEQ ID NO:74; gg) an overexpressed polynucleotide (cysH), which codes for an adenosine phosphosulphate reductase (CysH) having a sequence at least 95% identical to the sequence of SEQ ID NO:76; hh) an overexpressed polynucleotide (cysI), which codes for a sulphite reductase (CysI) having a sequence at least 95% identical to the sequence of SEQ ID NO:78; ii) an overexpressed polynucleotide (cysJ), which codes for (CysJ) having a sequence at least 95% identical to the sequence of SEQ ID NO:80; jj) an overexpressed polynucleotide (cysN), which codes for the subunit of a sulphate adenylyltransferase (CysN) having a sequence at least 95% identical to the sequence of SEQ ID NO:82; kk) an overexpressed polynucleotide (cysY), which codes for a cystathionine beta-synthase (CysY) having a sequence at least 95% identical to the sequence of SEQ ID NO:84; ll) an overexpressed polynucleotide (cysZ), which codes for a putative sulphate transporter (CysZ) having a sequence at least 95% identical to the sequence of SEQ ID NO:86; mm) an overexpressed polynucleotide (metE), which codes for a 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase (MetE) having a sequence at least 95% identical to the sequence of SEQ ID NO:88; nn) an overexpressed polynucleotide (ptH1), which codes for a peptidyl-tRNA hydrolase 1 (PtH1) having a sequence at least 95% identical to the sequence of SEQ ID NO:90; oo) an overexpressed polynucleotide (ptH2), which codes for a peptidyl-tRNA hydrolase 2 (PtH2) having a sequence at least 95% identical to the sequence of SEQ ID NO:92.

27. The recombinant microorganism of claim 24, wherein said microorganism is a Corynebacterium.

28. A method for overproducing an L-amino acid using a recombinant microorganism comprising the glycine cleavage system of claim 16.

29. The method of claim 28, wherein the recombinant microorganism overproduces an L-amino acid selected from the group consisting of: L-alanine, L-valine, L-amino acids of the glutamate family, L-glutamate, L-glutamine, L-proline and L-arginine, L-aspartate, L-asparagine, L-methionine, L-lysine, L-isoleucine and L-threonine.

30. The method of claim 29, wherein said L-amino acid is L-methionine.

31. The method of claim 29, wherein only low amounts of glycine occur as a by-product.

Description

WORKING EXAMPLES

Example 1

Alanine and Valine Production by C. Humireducens

[0350] To assess alanine and valine production, the strain C. humireducens (DSM 45392) was cultured in a shaking flask batch. For this purpose, the C. humireducens strain was incubated in 10 ml of BHI liquid medium (Brain Heart Infusion; Merck) (37 g/l H.sub.2O) at 37 C. at 200 rpm for 24 h as preculture. 10 ml of shaking flask medium were then inoculated to an OD.sub.660 of 0.2 and cultured at 37 C. at 200 rpm for 48 h. To prepare said medium, 20 g of ammonium sulphate, 0.4 g of MgSO.sub.4*7H.sub.2O, 0.6 g of KH.sub.2PO.sub.4 and 10 g of yeast extract were dissolved in 750 ml of H.sub.2O. The pH of the solution was adjusted to 7.8 with 20% NH.sub.4OH and the solution was then autoclaved. 4 ml of a vitamin solution (pH 7 with NH.sub.4OH), consisting of 0.25 g/l of thiamine, 50 mg/l of cyanocobalamin, 25 mg/l of biotin and 1.25 g/l of pyridoxine, were then added. In addition, 140 ml of a sterile-filtered 50% glucose solution and 50 g of dry autoclaved CaCO.sub.3 were added and the medium subsequently made up to one litre. After culturing, the supernatant of four parallel cultures was in each case analysed by HPLC to determine the alanine, glycine and valine content with a detection limit of 0.01 g/l.

[0351] The strain C. humireducens after culturing for 48 h in shaking flask medium at 37 C., 200 rpm at a shaking flask scale produces around 0.81 g/l of alanine (net yield: 0.011 g.sub.alanine/g.sub.glucose) and 1.6 g/l of valine (net yield: 0.022 g.sub.valine/g.sub.glucose) (Tab. 1). Glycine was only produced in small amounts as by-product.

TABLE-US-00001 TABLE 1 Analytical data from a shaking flask experiment with the strain C. humireducens. The values measured after culturing with cells and with the blank medium are shown. Alanine Valine (g/l) (g/l) C. humireducens 1.27 1.9 Blank medium without cells 0.46 0.3

Example 3

Glutamate Performance Assay

[0352] For the L-glutamate performance assay, the strain C. humireducens (DSM 45392) was cultured in a shaking flask batch. For this purpose, the C. humireducens strain was incubated in 10 ml of BHI liquid medium (Brain Heart Infusion; Merck) (37 g/l H.sub.2O) at 37 C. at 200 rpm for 24 h as preculture. 10 ml of shaking flask medium were then inoculated to an OD.sub.660 of 0.2 and cultured at 37 C. at 200 rpm for 48 h. To prepare said medium, 20 g of ammonium sulphate, 0.4 g of MgSO.sub.4*7H.sub.2O, 0.6 g of KH.sub.2PO.sub.4 and 10 g of yeast extract were dissolved in 750 ml of H.sub.2O. The pH of the solution was adjusted to 7.8 with 20% NH.sub.4OH and the solution was then autoclaved. 4 ml of a vitamin solution (pH 7 with NH.sub.4OH), consisting of 0.25 g/l of thiamine, 50 mg/l of cyanocobalamin, 25 mg/l of biotin and 1.25 g/l of pyridoxine, were then added. In addition, 140 ml of a sterile-filtered 50% glucose solution and 50 g of dry autoclaved CaCO.sub.3 were added. 5 ml of a 400 mM sterile-filtered threonine stock solution were then added and the medium was subsequently made up to one litre.

[0353] After culturing, the supernatant of four parallel cultures was in each case analysed by HPLC to determine the glutamate content with a detection limit of 0.01 g/l.

[0354] The strain C. humireducens after culturing for 48 h in shaking flask medium at 37 C., 200 rpm at a shaking flask scale produced 1.8 (+/0.6) g/l of L-glutamate. The initial concentration of L-glutamate in the medium was 0.78 (+/0.1) g/l. Glycine was only produced in small amounts as by-product.