TH35879A - Method for producing L-lysine - Google Patents

Abstract

DC60 (18/07/55) Recombinant autonomous DNA in Coliniform bacteria cells containing DNA sequences encoding aspartokinesase which is significantly reduced upon reversibility inhibition with L-lysine and L-threonine, and DNA sequences encoding diaminopimylate decarboxylase; Coliniform bacteria containing aspartokinesase which is significantly reduced upon reversibility inhibition with L-lysine and L-threonine, and containing DNA sequences amplified encoding diaminopimylate decarboxylase, and a method for L-lysine production consisting of steps of culturing Coliniform bacteria in suitable culture media to induce L-lysine production and accumulation in the bacterial culture medium, and collecting L-lysine from that culture medium. Recombinant autonomous DNA in Coliniform bacteria cells containing... The DNA sequences encoding aspartokines, which are reversibly inhibited by L-lysine and L-threonine, and the DNA sequences encoding diaminopimylate decarboxylase; coliniform bacteria containing aspartokines, which are reversibly inhibited by L-lysine and L-threonine, and containing the DNA sequences encoding diaminopimylate decarboxylase; and a method for L-lysine production consisting of steps for culturing coliniform bacteria in suitable culture media to induce L-lysine production and accumulation in the bacterial culture medium, and the collection of L-lysine from that culture medium.

Claims (22)

1. Autorecombined DNA in colineform bacterial cells consists of aspartokine-conducting DNA sequences, which are downregulated by reversible inhibition by L-lysine and L-threonine, and diaminopymylate decarboxylase-conducting DNA sequences. The aspartokine-conducting DNA sequence is a conventional aspartokine-conducting DNA sequence that is downregulated by reversible inhibition by L-lysine and L-threonine, and can be produced by reacting the conventional aspartokine-conducting DNA with mutagens. Diaminopimylate decarboxylase-induced DNA is either the original diaminopimylate decarboxylase-induced DNA or diaminopimylate kecarboxylase-induced DNA that has been mutated and retains diaminopimylate decarboxylase activity. 2. The reassembled DNA, according to claim 1, shows that the resulting aspacotine is a mutated aspacotine of colineform bacteria, where the amino acid sequence associated with alanine at position 279 from the nitrogenous end of the amino acid sequence shown in Table 5 is changed to an amino acid other than alanine, or an acidic amino acid, and the amino acid associated with alanine at position 30 from the nitrogenous end of the amino acid sequence shown in Table 7 is changed to an amino acid other than alanine, or an acidic amino acid. 3. The reassembled DNA, as per claim 1, in which the synthesized diaminopimylated decarboxylase has the amino acid sequence shown in the sequence table number 12. 4. DNA recombination according to claim 1, where the aspartokines-forming DNA sequence is hybridized as shown in Table 3 under specific conditions. 5. DNA reassembled according to claim 4, where the specific condition consists of 1.times SSC. 6. DNA reassembled according to claim 4, where the specific condition consists of 0.1 t.times SSC. 7. The recombined DNA according to claim 1, where the DNA sequence producing diaminopimylate decarboxylase consists of the sequences shown in sequence table number 11. 8. The reassembled DNA according to claim 1 also contains a sequence of phosphoenolpyruvate carboxylase-containing DNA used to construct classical phosphoenolpyruvate carboxylase, or is constructed by reacting phosphoenolpyruvate carboxylase-containing DNA with a mutagenic agent while retaining the activity of phosphoenolpyruvate carboxylase. 9. Colineform bacteria are altered by the introduction of the recombined DNA described in claim 1. 10. Colineform bacteria are altered by the introduction of the recombinant DNA specified in claim 8. 11. The L-lysine production method consists of culturing the colineform bacteria of patent 9 in a suitable culture medium for L-lysine production and accumulation in the culture medium, and collecting L-lysine from the culture medium. 12. The L-lysine production method consists of culturing the colineform bacteria of patent 10 in a suitable culture medium for L-lysine production and accumulation in the culture medium, and collecting L-lysine from the culture medium. 13. The L-lysine production method involves culturing altered colineform bacteria by introducing recombinant DNA into the culture medium. This recombinant DNA consists of the aspartokine-producing DNA sequence, which is reverse-inhibited by L-lysine, and the degraded L-threonine sequence, as well as the diaminopimylate carboxylase-producing DNA sequence. 14. Methods under Claim 13, where aspartokines-conducting DNA sequences are either classical aspartokines-conducting DNA sequences that have been reversibly degraded by L-lysine and L-threonine, or aspartokines-conducting DNA that has been reversibly degraded by L-lysine and L-threonine and can be produced by reacting classical aspartokines-conducting DNA with a mutagen, and diaminopimylate decarboxylase-conducting DNA sequences, classical diaminopimylate decarboxylase-conducting DNA, or diaminopimylate decarboxylase-conducting DNA that has been reacted with a mutagen and retains diaminopimylate decarboxylase activity. 15. The method pursuant to claim 13, where the constructed aspartokines is a mutated aspartokines of the Coliniform bacterium, where the amino acid sequence associated with alanine at position 279 counting from the nitrogenous end in the amino acid sequence shown in Table No. 5 is changed to threonine, and the amino acid sequence associated with alanine at position 30 counting from the nitrogenous end in the amino acid sequence shown in Table No. 7 is changed to threonine. 16. The method pursuant to claim 13, in which the synthesized diaminopimylate D-carboxylase has the amino acid sequence shown in the amino acid sequence table number 12. 17. The method under Claim 13, in which the DNA sequence that produces hybrid aspartokines is arranged as shown in Table 3 under specific conditions. 18. The procedure under Claim 17, the specific conditions of which include 1.times.SSC 19. The method under claim 18, the specific condition of which is 0.1 times.SSC. 20. The method under claim 13, in which the synthesized aspartokines have a subunit a with the amino acid sequence shown in Schedule No. 5, in which alanine at position 279 counting from the nitrogenous end of Schedule No. 5 is converted to an amino acid other than alanine or an acidic amino acid, and a subunit b with the amino acid sequence shown in Schedule No. 7, in which alanine at position 30 counting from the nitrogenous end of Schedule No. 7 is converted to an amino acid other than alanine or an acidic amino acid. 21. The method under claim 13, in which the DNA sequence producing diaminopimylate decarboxylase consists of the amino acid sequences shown in sequence table number 11. 22. The method under claim 13 also involves the sequencing of phosphoenolpyruvate carboxylase-containing DNA, which can be used to produce either the conventional form of phosphoenolpyruvate carboxylase or by reacting phosphoenolpyruvate carboxylase-containing DNA with a mutagenic agent while maintaining the activity of phosphoenolpyruvate carboxylase (Clause 22, page 4, figure 13).