Method for producing L-leucine, L-valine, L-isoleucine, α-ketoisovalerate, α-keto-beta-methylvalerate, or α-ketoisocaproate using recombinant Corynebacteria that contain the ilvBN operon which can be induced by propionate
The invention relates to a process for the production of amino acids and keto acids using microorganisms, in which a promoter inducible by propionate makes possible the regulated expression of certain genes.
1. A process for the production of an L-amino acid selected from the group consisting of L-leucine, L-valine and L-isoleucine or of an α-keto acid selected from the group consisting of α-ketoisovalerate, α-keto-methylvalerate and α-ketoisocaproate, said process comprising:
a) fermenting microorganisms of the genus Corynebacterium , wherein:
i) said microorganisms comprise, in replicable form, a polynucleotide with operator activity, the sequence of which is at least 85% identical to the sequence of position 1 to 121 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 and to which the activator PrpR binds; and
ii) functionally downstream of the polynucleotide with operator activity, at the 3′-end, are a second polynucleotide having propionate- or 2-methylcitrate-inducible promoter activity; as well as genes ilvB and ilvN encoding for the subunits of an acetolactate synthase, and which regulates the transcription of the genes ilvBN as a function of the addition of the activator PrpR, in a medium;
b) during the fermenting of said microorganisms, there is a first phase (growth phase), which takes place without inducer, and a second phase during which propionate or 2-methylcitrate is added as an inducer, whereupon the desired L-amino acid or α-keto acid is synthesized under conditions in which the desired L-amino acid or α-keto acid is enriched in the medium and/or in the cells.
2. The process of claim 1 , wherein said polynucleotide with operator activity comprises a polynucleotide (“IR 1”), the sequence of which is at least 90% identical to the sequence of position 22 to position 49 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, and also a polynucleotide (“IR 2”), the sequence of which is at least 90% identical to the sequence of position 77 to position 105 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.
3. The process of claim 2 , wherein the polynucleotide with operator activity comprises a sequence that is at least 90% identical to the sequence of position 1 to 121 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.
4. The process of claim 2 , wherein:
a) said IR 1 polynucleotide is at least 96% identical to the sequence of position 22 to position 49 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3;
b) said IR 2 the sequence is at least 96% identical to the sequence of position 77 to position 105 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; and
c) said polynucleotide with operator activity comprises a sequence that is at least 96% identical to the sequence of position 1 to 121 of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.
5. The process of claim 1 , wherein the polynucleotide with promoter activity comprises a sequence that is at least 90% identical to the sequence of position 122 to 206 of SEQ ID NO:4.
6. The process of claim 1 , wherein the polynucleotide with promoter activity comprises a sequence that is at least 98% identical to the sequence of position 122 to 206 of SEQ ID NO:4.
7. The process of claim 1 , wherein the gene ilvB codes for a polypeptide, the sequence of which is at least 90% identical to the sequence of SEQ ID NO:9 and the gene ilvN is a polynucleotide codes for a polypeptide, the sequence of which is identical at least 90% identical to the sequence of SEQ ID NO:10.
8. The process of claim 1 , wherein the gene ilvB codes for a polypeptide, the sequence of which is at least 98% identical to the sequence of SEQ ID NO:9 and the gene ilvN is a polynucleotide codes for a polypeptide, the sequence of which is identical at least 98% identical to the sequence of SEQ ID NO:10.
9. The process of claim 1 , wherein said microorganisms further comprise enzymes of the biosynthetic pathway of the desired L-amino acid or α-keto acid that are amplified and/or metabolic pathways that decrease the formation of the desired L-amino acid or α-keto acid that are at least partially attenuated.
10. The process of claim 1 , wherein L-valine is synthesized.
11. The process of claim 1 , wherein the Corynebacteria employed in the culturing phase go through at least 16 generations and the fermentation comprises at least four stages.
12. The process of claim 11 wherein the Corynebacteria employed in the culturing phase go through at least 24 generations and the fermentation comprises at least a shaker flask stage, a PreSeed fermenter stage, a seed fermenter stage and a production fermenter stage.