Methods for producing sulphurous fine chemicals
AssigneeBASF SE
InventorsBurkhard Kröger, Oskar Zelder, Corinna Klopprogge, Hartwig Schröder, Stefan Häfner
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Abstract
The invention relates to methods for the fermentative production of sulfur-containing fine chemicals, in particular L-methionine, by using bacteria which express a nucleotide sequence coding for a methionine synthase (metH) gene.
Claims26
1-25. (canceled)
A method for the fermentative production of at least one sulfur-containing fine chemical selected from L-methionine and S-adenosylmethionine, which comprises the following steps:
- a) fermentation of a coryneform bacteria culture producing the desired sulfur-containing fine chemical, the coryneform bacteria expressing at least one heterologous nucleotide sequence which codes for a protein with methionine synthase (metH) activity, said sequence being less than 70% homologous to the meth-encoding sequence from Corynebacterium glutamicum ATCC 13032;
- b) concentration of the sulfur-containing fine chemical in the medium or in the bacterial cells, and
- c) isolation of the sulfur-containing fine chemical.
A method as claimed in any of the preceding claims, wherein the meth-encoding sequence comprises a coding sequence according to 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, 49 and 51 or a nucleotide sequence homologous thereto which is less than 70% homologous to the metH-encoding sequence from Corynebacterium glutamicum ATCC 13032 and codes for a protein with metH activity.
A method as claimed in any of the preceding claims, wherein the coding metH sequence is a DNA or RNA which can be replicated in coryneform bacteria or is stably integrated into the chromosome.
A method as claimed in any of the preceding claims, wherein the coding metH sequence is overexpressed.
A method as claimed in any of the preceding claims, wherein bacteria are fermented in which additionally at least one further gene of the biosynthetic pathway of the desired sulfur-containing fine chemical, or of a biosynthetic or metabolic pathway associated therewith, has been amplified or mutated such that its activity is not influenced by metabolic metabolites.
A method as claimed in any of the preceding claims, wherein bacteria are fermented in which at least one metabolic pathway, which reduces the production of the desired sulfur-containing fine chemical, is at least partially switched off.
A method as claimed in any of the preceding claims, wherein coryneform bacteria are fermented in which, at the same time, at least one of the genes selected from among
- a. the aspartate kinase-encoding gene lysC,
- b. the glyceraldehyde-3-phosphate dehydrogenase-encoding gene gap,
- c. the 3-phosphoglycerate kinase-encoding gene pgk,
- d. the pyruvate carboxylase-encoding gene pyc,
- e. the triose phosphate isomerase-encoding gene tpi,
- f. the homoserine O-acetyltransferase-encoding gene metA,
- g. the cystathionine gamma-synthase-encoding gene metB,
- h. the cystathionine gamma-lyase-encoding gene metC,
- i. the serine hydroxymethyltransferase-encoding gene glyA,
- j. the O-acetylhomoserine sulfhydrylase-encoding gene metY,
- k. the metH gene coding for vitamin B12-dependent methionine synthase,
- l. the serC gene coding for phophoserine aminotransferase,
- m. the serB gene coding for phosphoserine phosphatase,
- n. the cysE gene coding for serine acetyltransferase, and
- o. the hom gene coding for a homoserine dehydrogenase,
- is overexpressed or mutated in particular in such a way that the activity of the corresponding proteins is influenced by metabolic metabolites to a smaller extent, if at all, compared to nonmutated proteins and/or these proteins have a higher activity and/or substrate specificity.
A method as claimed in any of the preceding claims, wherein coryneform bacteria are fermented in which, at the same time, at least one of the genes selected from among
- a) the homoserine kinase-encoding gene thrB,
- b) the threonine dehydratase-encoding gene ilvA,
- c) the threonine synthase-encoding gene thrC,
- d) the meso-diaminopimelate D-dehydrogenase-encoding gene ddh,
- e) the phosphoenolpyruvate carboxykinase-encoding gene pck,
- f) the glucose-6-phosphate 6-isomerase-encoding gene pgi,
- g) the pyruvate oxidase-encoding gene poxB,
- h) the dihydrodipicolinate synthase-encoding gene dapA,
- i) the dihydrodipicolinate reductase-encoding gene dapB;
- and
- j) the diaminopicolinate decarboxylase-encoding gene,
- is attenuated by changing the rate of expression or by introducing a specific mutation or expresses a protein with reduced activity.
A method as claimed in one of the preceding claims, wherein microorganisms of the species Corynebacterium glutamicum are used.
A method as claimed in one of the preceding claims, wherein a microorganism with resistance to methionine biosynthesis inhibitors is employed.
Plasmid pCIS lysC thr311ile (SEQ ID NO:78), encoding lysC thr311ile.
Plasmid pC Phsdh metH_Sc (SEQ ID NO:79), encoding Streptomyces coelicolor metH.
A method for producing an L-methionine-containing animal feed additive from fermentation broths, which comprises the following steps:
- k) culturing and fermentation of an L-methionine-producing microorganism in a fermentation medium;
- l) removal of water from the L-methionine-containing fermentation broth;
- m) removal of from 0 to 100% by weight of the biomass formed during fermentation;
- and
- n) drying of the fermentation broth obtained according to b) and/or c), in order to obtain the animal feed additive in the desired powder or granule form.
Dependencies are inferred from the claim text.