RU2001125710A - Method for the enzymatic production of non-proteinogenic L-amino acids - Google Patents

Claims (19)

1. A method of producing non-proteinogenic L-amino acids by direct fermentation using a well-known microorganism strain characterized by an impaired regulation of cysteine metabolism, which is carried out according to the well-known method, characterized in that in the fermentation process a nucleophilic compound is added in such quantities that this leads to the production of non-proteinogenic L-amino acids by the microorganism strain. 2. The method according to claim 1, characterized in that after the end of the fermentation non-proteinogenic L-amino acids are isolated from the medium for fermentation using widely known methods. 3. The method according to claim 1 or 2, characterized in that add a nucleophilic compound containing a fragment selected from the group including

4. The method according to claim 1 or 2, characterized in that the nucleophilic compound is selected from the group of compounds comprising a thiol of the general formula (1): HSR ₁ (1) where R ¹ denotes a monovalent substituted or unsubstituted alkyl, alkoxygroup, aryl or heteroaryl having a maximum of 15 carbon atoms; azole of the general formula (2) or (3):

as well as its esters and ethers or salts, where X and Y have the same or different meanings and are CR ⁴ or N, where R ⁴ is —H, —COOH, —OH, —NH ₂ , —NO ₂ ^- , —SH, —SO ₃ ^- , —F, - C1, -Br, -I, C ₁ -C ₅ alkylcarbonyl- or R ¹ , where R ¹ has the meanings indicated for formula (1), and wherein R ² and R ³ have the same or different meanings and are designated R ⁴ , or C ¹ and C ² in the formula (3) instead of the substituents R ² and R ^{3 are} attached to the ring using the bridge [-CR ⁵ -R ⁶ ] _a , where a is 1, 2, 3 or 4, with R ⁵ and R ⁶ are identical or different and denote R ⁴ and one or more groups [-CR ⁵ R ⁶ -], which are not adjacent, may be life substituted with oxygen, sulfur or optionally iminofragmentom substituted C ₁ -C ₅ -alkyl, and two adjacent groups [-CR ⁵ R6-] may be replaced by a group [-CR5 = R6-] or a group [-CR ⁵ = N-] , Z-isoxazolinone of the general formula (4) or (5):

as well as its esters and ethers or salts, where X, R ¹ , R ² and R are as defined above, and C ¹ and C ² in the formula (5) instead of the substituents R ² and R ³ can be attached to the ring using specified for the formula (3) of the bridge. 5. The method according to any one of claims 1 to 4, characterized in that the method according to the invention uses a strain of microorganisms selected from the group including the following strains: strains with modified cysE alleles; strains containing export genes; strains with altered CysB activity; strains that are obtained using methods of nonspecific mutagenesis in combination with screening methods for the purpose of overproduction of cysteine or to reduce the decomposition of cysteine. 6. The method according to any one of claims 1 to 5, characterized in that a strain of Escherichia coli is used as a strain of microorganisms. 7. The method according to PP.1,5 or 6, characterized in that the cultivation of the microorganism strain in the fermenter is carried out continuously, periodically (party cultivation), or using a batch process with water. 8. The method according to any one of claims 1 to 7, characterized in that the carbon source and nucleophilic compound are added continuously during the fermentation process. 9. The method according to any one of claims 1 to 8, characterized in that the addition of the nucleophilic compound begins 6-8 hours after the start of fermentation and continues until the end of the fermentation. 10. The method according to any one of claims 1 to 9, characterized in that the amount of nucleophilic compound added is from 10 to 1000 mmol per 1 liter of the initial volume of the fermentation medium. 11. The method according to any one of claims 1 to 10, characterized in that sugar, sugar alcohol or organic acids are used as a carbon source for fermentation. 12. The method according to claim 11, characterized in that the carbon sources are added in a form that ensures that the glucose content in the fermenter is 0.1-50 g / l. 13. The method according to any one of claims 1 to 12, characterized in that ammonia, ammonium salts or protein hydrolysates are used as a nitrogen source. 14. The method according to any one of claims 1 to 13, characterized in that the pH of the fermentation medium is 4-10 and the incubation temperature is 15-45 ° C. 15. The method according to any one of claims 1 to 14, characterized in that it is carried out under aerobic conditions for growth. 16. The method according to any one of claims 1 to 15, characterized in that after removal of the biomass from the fermentation medium by filtration or centrifugation, non-proteinogenic L-amino acids are isolated from the culture supernatant by extraction, adsorption, ion exchange chromatography, precipitation or crystallization. 17. The method according to any one of claims 1 to 16, characterized in that the non-proteinogenic L-amino acid is an amino acid of General formula (6) having the L-configuration:

where Z is a monovalent radical selected from the group consisting of radicals of formulas (7) - (13)

as well as their esters, ethers or salts, where R ¹ , R ² , R ³ , R ⁴ , X and Y have the meanings indicated for formulas (1) to (5). 18. A compound selected from the group consisting of 1, 2, 3, 4-tetrazolyl-L-alanine and its derivatives, 1, 2, 3-triazolyl-L-alanine and its derivatives and S-heteroaryl-L-cysteine. 19. The compound according to p. 18, selected from the group including: 1, 2, 3, 4-tetrazol-1-yl-L-alanine, 1, 2, 3, 4-tetrazol-2-yl-L-alanine, 1, 2, 3-benzotriazol-1-yl-L-alanine, 1, 2, 3-benzotriazol-2-yl-L-alanine, 5-carboxy-1, 2, 3-benzotriazol-1-yl-L- alanine, 5-carboxy-1, 2, 3-benzotriazol-2-yl-L-alanine, 1, 2, 4-triazol-3-yl-L-cysteine, thiazol-2-yl-l-cysteine, imidazole- 2-yl-L-cysteine, thien-2-yl-L-cysteine, pyridin-2-yl-L-cysteine, pyrimidin-2-yl-L-cysteine, benzothiazol-2-yl-L-cysteine and benzoxazole- 2-yl-L-cysteine.