TH71899B - Methods for the production of target substances by fermentation. - Google Patents

Abstract

DC60 (29/08/46) In the method for the production of target substances using microorganisms, which involves culturing gamma-proteobacterium in a medium to produce and accumulate target substances in the medium or cells, and the collection of those target substances, strains in which the ArcA protein does not function normally in cells are used by methods such as breaking the arcA gene on the chromosome.

Claims (10)

Revised October 1, 2018. Claim 1. A method for the production of target substances selected from a group comprising L-lysine, L-threonine, L-isoleucine, L-glutamic acid, L-glutamine, L-arginine, L-homoserine, and succinic acid, involving the culture of (formula)-proteobacterium in a medium under aerobic conditions to produce and accumulate the target substances in that medium or cells, and the accumulation of the target substances from the medium or cells where the (formula)-proteobacterium capable of producing the target substances has been modified so that the production of ArcA protein is reduced or eliminated, and where the ArcA protein is (A) or (B) of the following: (A) a protein with an amino acid sequence of SEQ ID NO: 32; (B) a protein with an amino acid sequence of SEQ ID NO: 32 that includes the substitution, deletion, addition, or addition of 2 to 20 amino acids and the enhancement of the ability (Formula)-Proteobacterium to produce the target substance when the production of that protein is reduced or eliminated in (Formula)-Proteobacterium compared to the original or unmodified strain. ++++++++++++++++++++++++++++++++++++++++ ------01/12/2017------(OCR) Page 1 of 2 pages. Claim 1. A method for the production of a target substance selected from a group consisting of L-lysine, L-threonine, L-isoleucine, L-glutamic acid, L-glutamine, L-arginine, L-homoserine, and succinic acid, which involves culturing (Formula)-Proteobacterium in a medium under aerobic conditions to produce and accumulate the target substance in that medium or cells, and the accumulation of the target substance from the medium or cells where the (Formula)-Proteobacterium capable of producing the target substance has been modified so that the production of ArcA protein is reduced or eliminated. And where ArcA protein is (A) or (B) as follows: (A) a protein with an amino acid sequence of SEQ ID NO: 32; (B) a protein with an amino acid sequence of SEQ ID NO: 32 that includes the substitution, deletion, addition or addition of 1 to 20 amino acids and the enhancement of the ability of (formula)-proteobacterium to produce the target substance when the production of that protein is reduced or depleted in (formula)-proteobacterium compared to the original or unmodified strain. 2. Method according to claim 1, where ArcA protein defined in (B) is a protein that has 90% or more similarity to the amino acid sequence of SEQ ID NO:32 and enhancement of the ability of (formula)-proteobacterium to produce the target substance when the production of that protein is reduced or depleted in (formula)-proteobacterium. (Formula)-Proteobacterium as opposed to the original or unmodified strain. 3. Methods according to claim 1 or 2, where the production of the ArcA protein is reduced or eliminated by the disruption pathway of the arcA gene on the chromosome, and where the arcA gene is DNA defined in (a) or (b) as follows: (a) DNA containing the nucleotide sequences of nucleotide numbers 101 to 817 of SEQ ID NO: 31; (b) DNA that can be paired with the nucleotide sequences of nucleotide numbers 101 to 817 of SEQ ID NO: 31 or a tracer that can be produced from the nucleotide sequences under strict conditions and transcription for a protein that enhances the ability of (Formula)-Proteobacterium to produce the target when the production of the protein is reduced or eliminated in (Formula)-Proteobacterium, page 2 of 2, is a bacterial strain compared to the original or unmodified strain under the following stringent conditions: 1 X SSC, 0.1% SDS at 60°C. 4. The method according to any one of the claims 1 through 3, where (Formula)-Proteobacterium is a bacterial strain of the genus Escherichia or Pantoea. 1. Gamma-proteobacterium, which has the ability to produce target substances and is modified so that the ArcA protein does not function normally. 2. Gamma-proteobacterium pursuant to claim 1, where a normally functioning ArcA protein is a protein defined in (A) or (B) of the following: (A) a protein with an amino acid sequence of: 32; (B) a protein with an amino acid sequence of: 32, which includes a substitution, deletion, addition or addition of one or more amino acids and improves the ability to produce the target substance when the protein is not normally functioning in gamma-proteobacterium compared to the case where the protein is normally functioning. 3. Gamma-proteobacterium under claim 1, whereby a normally functioning ArcA protein is a protein that is 70% or greater similar to the amino acid sequence of 32 and thus exhibits improved target production ability when the protein is not normally functioning in gamma-proteobacterium compared to the case where the protein is normally functioning. 4. Gamma-proteobacterium under claim 1, whereby a normally functioning ArcA protein is a protein with a 32-amino acid sequence, which includes the substitution, deletion, addition, or addition of 2 to 20 amino acids, and which improves its ability to produce target substances when the protein is not normally functioning in gamma-proteobacterium compared to the case where the protein is normally functioning. 5. Gamma-proteobacterium, according to any of the claims 1 through 4, where the ArcA protein does not function normally by means of chromosomal arcA gene fragmentation. 6. Gamma-Proteobacterium pursuant to claim 5, where the arcA gene is DNA defined in (a) or (b) the following; (a) DNA containing the nucleotide sequence of nucleotides 101 to 817 of sequence 31; (b) DNA capable of recombination with the nucleotide sequence of nucleotides 101 to 817 of sequence 31, or a test that can be produced from that nucleotide under strict conditions and transcribed for a protein that improves the ability to produce the target substance when the protein is not functioning normally in gamma-proteobacterium, compared to the case where the protein is functioning normally. 7. Gamma-proteobacterium under any of the claims 1 through 6 where the bacteria belong to the Escherichia genus. 8. Gamma-proteobacterium as defined in any of the claims 1 through 7, where the target substance is an L-amino acid. 9. Gamma-proteobacterium pursuant to claim clause 8, where L-amino acids are selected from a group comprising L-lysine, L-glutamic acid, and L-arginine. 10. A method for the production of the target substance, consisting of the cultivation of gamma-proteobacterium according to any of the claims 1 through 9 in a medium for the production and accumulation of the target substance in the medium or cells, and the collection of the target substance from that medium or cells.