TH20135A - Mutant phosphoenol pyruvate Carboxylase, genes of these compounds and methods for producing amino acids. - Google Patents

Abstract

????????????????? ???????????? ??? ???????????????????????? ???????????????? ???? ??? ??????????????????????? 625 ??????? N-?????????????????????????????????? ????????? ????????????????????? ???????????? 438 ??????? N- ???????????? ?????????? ???????? ????????????????????? Escherichia coli ???? ?????????? ????????? ???????? ????????????????????? ???????????? ??????????????????????????????????????????? ?????????????????????????????????????????????????

Claims (13)

1. The amino acid production method involves selecting Escherichia microorganisms with DNA sequences transcribing phosphoenolpyruvate carboxylase mutants that have been rendered insensitive to reinhibition by aspartic acid. This is achieved by culturing the Escherichia microorganisms in a suitable medium containing a selected inhibitor of phosphoenolpyruvate carboxylase, chosen from groups including 3-bromopyruvate, aspartic acid-beta-hydrazide, and DL-trio-beta-hydroxyaspartic acid. Amino acids are then isolated from this medium, selected from groups including L-lysine, L-threonine, L-methionine, L-isoleucine, L-glutamic acid, L-arginine, and L-proline. 2. Method of Claim 1, where the mutated phosphoenolpyruvate carboxylate has a glutamic acid sequence at position 625 from the N- end that has been mutated to a sequence of amino acids other than glutamic acid. 3. Method of Claim 1, where the phosphoenolpyruvate carboxylate mutant has a set of glutamic acid compounds at position 625 from the N- end replaced by lysine. 4. Method of Claim 1, where the phosphoenol pyruvate carboxylase mutant has an arginine compound at position 222 mutated into an amino acid other than arginine, and the glutamic acid compound at position 223 is mutated into an amino acid other than glutamic, with the amino acid position measured from the N- terminal of the phosphoenol pyruvate carboxylase. 5. Method according to claim 1, where the phosphoenolpyruvate carboxylase mutant in which the arginine at position 222 is replaced by histidine and the glutamic acid at position 223 is replaced by lysine, with the amino acid position measured from the N- terminal of the phosphoenolpyruvate carboxylase. 6. Method of Claim 1, where the phosphoenolpyruvate carboxylase mutant contains a serine compound at position 288 mutated to an amino acid other than serine; a glutamic acid compound at position 289 mutated to an amino acid other than glutamic acid; a methionine compound at position 551 mutated to an amino acid other than methionine; and a glutamic acid compound at position 804 mutated to an amino acid other than glutamic acid, where the amino acid positions are measured from the N- terminal of the phosphoenolpyruvate carboxylase. 7. Method of Claim 1, where the phosphoenolpyruvate carboxylase mutant has the serine compound at position 288 replaced by phenylalanine, the glutamyl compound at position 289 replaced by lysine, the methionine compound at position 551 replaced by isoleucine, and the glutamic acid compound at position 804 replaced by lysine, with the amino acid position measured from the N- terminal of the phosphoenolpyruvate carboxylase. 8. Method of Claim 1, where the phosphoenolpyruvate carboxylate mutant has an alanine sequence at position 867, measured from the N- end, that has been mutated into an amino acid other than alanine. 9. Method of Claim 1, where the phosphoenolpyruvate carboxylate mutant has an alanine compound at position 867, measured from the N- end, replaced by threonine. 10. Method for producing amino acids consisting of: culturing Escherichia or Coryneform bacteria in a suitable medium and isolating amino acids from the medium, selected from a group containing L-lysine, L-threonine, L-methionine, L-isoleucine, L-glutamic acid, L-arginine, and L-proline. The microorganisms are transduced by allowing DNA fragments to be incorporated into chromosomal DNA or by recombinant DNA created by binding the DNA fragments to autocoupling DNA vectors in Escherichia or Coryneform bacteria cells. The DNA fragments encode a phosphoenolpyruvate carboxylase mutant originating from the Escherichia microbial remains; a phosphoenolpyruvate carboxylase mutant that is inactivated by aspartic acid. And the phosphoenolpyruvate carboxylase mutant, which has an arginine compound at position 438 from the N- end, is mutated into an amino acid other than arginine. 11. Method pursuant to claim 10, where a phosphoenolpyruvate carboxylase mutant with an arginine compound at position 438 is replaced by cysteine. 12. The amino acid production method involves culturing Escherichia or Coryneform bacteria in a suitable medium and isolating selected amino acid components from a group including L-lysine, L-threonine, L-methionine, L-isoleucine, L-glutamic acid, L-arginine, and L-proline. The microorganisms are transduced by allowing DNA fragments to be incorporated into chromosomal DNA or by recombinant DNA generated by binding the DNA fragments to autocapitatively paired DNA vectors in Escherichia or Coryneform bacteria cells. These DNA fragments encode a phosphoenolpyruvate carboxylase mutant originating from the remnants of Escherichia microorganisms. This mutant phosphoenolpyruvate carboxylase has been mutated to become insensitive to aspartic acid's reactivation inhibitory action. There is a lysine compound at position 620 from the N- end that has been mutated into an amino acid other than lysine. 13. The method under claim 12, where the phosphoenolpyruvate carboxylase mutant has the lysine compound at position 620 replaced by serine.