TH77083A - A new strain of rumen bacteria And the process for preparing succinic acid using this - Google Patents

Abstract

DC60 (01/07/48) The proposed invention relates to a new strain of bacterial rumen that results from gene destruction. Lactate dehydrogenase (ldhA) and pyruvate formales (pfl) (which are involved in the production of lactic, formic and acetic acid) from holes. Maine bacteria; New mutant bacteria (Mannheimia succinicproducens LPK7) with destruction of the lactate dehydrogenase (ldhA) genes, pyruvate formates (pfl) genes, trans phosphate genes. Acetylase (pta) and acetate kinase (ackA) gene; New mutant bacteria (Mannheimia succinicproducens LPK4) with destruction of the lactate dehydrogenase (ldhA) gene, pyruvate formularase (pfl) gene, and the gene formate. Phoenol pyruvate carboxylase (ppc) involved in fixation Carbon dioxide in the metabolic pathway of the production of succinic acid; And methods for producing succinic acid Which is characterized by the use of the above mutated strains in anaerobic conditions Bacterial species This invention mutant has properties capable of producing highly concentrated succinic acid. While producing acid Other types of organic matter in small quantities or not produced at all. When compared with the previous generic lineage used in production Different types of organic acids, so the artificial mutant bacterial strains are useful as pathogens For industrial production of succinic acid

Claims (9)

All rights reserved which will not appear on the advertisement page :------03/08/2018------(OCR) Page 1 of 3 pages of rights reserved. 1. Mutant rumen bacteria in which the lactate dehydrogenase-encoding (gene-/dhA) and pyruvate formate-lyase-encoding (pfl) genes are destroyed and capable of producing succinic acid in the absence of oxygen were selected from a group comprising Mannheimia, Actinobacillus and Anaerobiospirillum. 2. Mutant rumen bacteria in which the lactate dehydrogenase-encoding gene (IDHA), pyruvate formate-lyase-encoding gene (PFL), phosphotransacetylase-encoding gene (PTA) and acetate kinase-encoding gene (AckA) were destroyed and had the ability to produce succinic acid in the absence of oxygen. The rumen bacteria were selected from a group consisting of the genera Mannheimia, Actinobacillus and Anaerobiospirillum. 3. Mutant rumen bacteria in which the lactate dehydrogenase-encoding gene (IDHA), pyruvate formate-lyase-encoding gene (PFL) and phosphoenolpyruvate-encoding gene (PPC) are lost and have the ability to produce succinic acid in the absence of oxygen. These rumen bacteria were selected from a group consisting of the genera Mannheimia, Actinobacillus and Anaerobiospirillum. 4. Any of the patented rumen bacteria in paragraphs 1-3, where the said rumen bacteria are the only bacteria that produce the main product of fermentation (homofermentative bacteria). 5. The mutant rumen bacteria claimed under Claim 1, wherein the mutant rumen bacteria are the Mannheimia s p. LPK strains, all three rumen bacteria strains. 6. The rumen bacterium mutant claimed under claim 1, wherein the said Mannheimia s p.LPK strain is KCTC 10558BP. 7. The mutant rumen bacteria claimed under claim no. 2, where the mutant rumen bacteria are the strain Mannheimia s p. LPK7. All three rumen bacteria strains 8. The rumen bacterium mutant under patent no. 7, namely Mannheimia s p.LPK7, is KCTC 10626BP. 9. Mutant rumen bacteria claimed under claim no. 3, where the mutant rumen bacteria are Mannheimia s p. LPK4 strains. All three rumen bacteria strains1. Production method of mutant rumen bacteria that have the ability to produce succinic acid in the absence of oxygen. The method consists of inactivating the genes encoding lactate dehydrogenase {IdhA) and the genes encoding pyruvate kinase {pfl} from selected rumen bacteria from the group consisting of the genera Mannheimia, Actinobacillus and Anaerobiospirillum.1Method for production of mutant rumen bacteria that have the ability to produce 1-chaksinic acid in the presence of oxygen.The method consists of inactivating the genes encoding phosphotransacetylase (pta) and acetate kinase (aackA) in additional from selected rumen bacteria from the group consisting of the genera Mannheimia, Actinobacillus and Anaerobiospirillum. The genes encoding lactate dehydrogenase {IdhA) and pyruvate formate lyase (pfl) were knocked out.1 A method for producing mutant rumen bacteria that are capable of producing succinic acid in the absence of oxygen. The method consists of knocking out the phosphoenolpyruvate (IPPC) genes from selected rumen bacteria from the group consisting of the genus Mannheimia, genus Actinobacillus and genus Anaerobiospirillum. The lactate dehydrogenase {IdhA) and pyruvate formate lyase {pfl) genes are destroyed1. The method of producing mutant rumen bacteria claimed in claim 11 or 12, wherein the mutant rumen bacteria in which the lactate dehydrogenase {IdhA) and pyruvate formate lyase {pfl) genes are destroyed is the Mannheimia s p. LPK KCTC 10558BP1. The method of producing mutant rumen bacteria claimed in claim 10, wherein the lactate dehydrogenase {IdhA) and pyruvate formate lyase {pfl) genes are destroyed by homologous recombination1. The method of producing mutant rumen bacteria claimed in claim 14 Whereas a similar integration method is performed by using a gene exchange vector comprising a disrupted lactate dehydrogenase {IdhA) gene and a gene exchange vector comprising a disrupted pyruvate formate lyase {pfl) gene.1 A method for producing mutant rumen bacteria claimed in claim 15, wherein the gene exchange vector comprising a disrupted lactate dehydrogenase {IdhA) gene is pMLKO-sacB as described in Figure 1, and the gene exchange vector comprising a disrupted pyruvate formate lyase {pfl) gene is pMPKO-sacB as described in Figure 2.1 A method for producing mutant rumen bacteria claimed in claim 11, wherein the disruption of the phosphotraacetylase {pta) gene and the acetate kinase {iackA) gene are A method of producing mutant rumen bacteria claimed in claim 17, wherein the method of producing mutant rumen bacteria is carried out by using a gene exchange vector comprising a gene encoding the enzyme phosphotransacetylase {pta) and a gene encoding the enzyme acetate\'kinase (ackA) that has been inactivated. Page 3 of 31 A method of producing mutant rumen bacteria claimed in claim 18, wherein the gene exchange vector comprising a gene encoding the enzyme phosphotransacetylase (pta) and a gene encoding the enzyme acetate\'kinase (ackA) that has been inactivated, namely pPTA-sacB, as described in Figure 62. A method of producing mutant rumen bacteria claimed in claim 12, wherein the gene encoding The phosphoenol pyruvate (ppc) enzyme is produced by a similar combination method. The method for producing mutant rumen bacteria claimed in claim 20, wherein the similar combination method is performed by using a gene exchange vector containing a gene encoding the enzyme phosphoenol pyruvate {ppc) inactivated. The method for producing mutant rumen bacteria claimed in claim 21, wherein the gene exchange vector contains a gene encoding the enzyme phosphoenol pyruvate (ppc) inactivated, the inactivated being pPPC-sacB. The process or method for producing mutant bacteria includes the steps of cultivating the mutant rumen bacteria claimed in claims 1-3, any one of which comprises the steps of extracting succinic acid from a liquid medium. The method for producing succinic acid claimed in claim 23, wherein the cultivating step is a fermentation step that yields only a small fraction of the primary product. (homofermentation)2. A method for producing succinic acid claimed in claim 23, wherein the rumen bacteria mutants are Mannheimia s p. LPKKCTC 10558BP, LPK7 KCTC 10626BP, or a method that produces the LPK4 mutants in a manner that is specific to them. And the bacteria became the LPK4 strain obtained from the said process were cultured for use in succinic acid production. ------------ ------26/01/2018------(OCR) Page 1 of 3 pages Claim A mutant rumen bacteria in which the lactate dehydrogenase? encoding gene (IdhA) and the pyruvate formate-lyase? encoding gene (pjอ) are destroyed, and have the ability to produce succinic acid in the absence of oxygen, whereby the said rumen bacteria were selected from a group consisting of the genera Mannheimia, the genera Actinobacillus and the genus Anaerobiospirillum. The mutant rumen bacteria in which the lactate dehydrogenase? encoding gene (IdhA), the pyruvate formate-lyase-encoding gene (pjอ) are destroyed, and have the ability to produce succinic acid in the absence of oxygen, whereby the said rumen bacteria were selected from a group consisting of the genera Mannheimia, the genera Actinobacillus and the genus Anaerobiospirillum. The phosphotransacetylase-encoding gene (pta) and the acetate kinase-encoding gene (ackA) were depleted, and the rumen bacteria were selected from a group consisting of the Mannheimia, Actinobacillus and Anaerobiospirillum gene families. The rumen bacteria were mutants in which the lactate dehydrogenase (IdhA) encoding gene, the pyruvate formate-lyase-encoding gene (pfl) and the phosphoenolpyruvate-encoding gene (ppc) were depleted, and the rumen bacteria were Selected from the group consisting of the genus Mannheimia, genus Aetinobacillus and genus Anaerobiospirillum, the rumen bacterium mutant claimed in any one of claims 1-3, wherein said rumen bacterium is a homofermentative bacterium, the rumen bacterium mutant claimed in claim 1, wherein said rumen bacterium is the Mannheimia s p. LPK strain as shown in Figure 3, the rumen bacterium mutant claimed in claim 1, wherein said Mannheimia s p. LPK strain is KCTC 10558BP, the rumen bacterium mutant claimed in claim 2, wherein said rumen bacterium is a homofermentative bacterium, Namely, the Mannheimia s p. LPK7 strain as shown in Figure 8, the mutant rumen bacteria claimed in Claim 7, wherein the Mannheimia s p. LPK7 strain is KCTC 10626BP, the mutant rumen bacteria claimed in Claim 3, wherein the mutant rumen bacteria is Mannheimia s p. LPK4 as shown in Figure 9 1, a method for producing mutant rumen bacteria capable of producing succinic acid in the absence of oxygen, comprising the deletion of the gene encoding lactate dehydrogenase (IdhA) and the gene encoding pyruvate formylatease (pfl) from rumen bacteria selected from the group consisting of the genus Mannheimia, the genus Actinobacilliis and the genus Anaerobiospirillum. 1Method for the production of mutant rumen bacteria capable of producing succinic acid in the absence of oxygen. The method consists of additionally inactivating the genes encoding the enzymes phosphotransacetylase (IPTA) and acetyl kinase (AckA) from selected rumen bacteria from the group consisting of G. BUS Mannheimia, G. BUS Actinobacillus and G. BUS Anaerobiospirillum. The genes encoding lactate dehydrogenase (IdhA) and pyruvate formate lyase (PFL) were inactivated. 1Method for the production of mutant rumen bacteria capable of producing succinic acid in the absence of oxygen. The method consists of inactivating the genes encoding the enzymes phosphoenolpyruvate (PPC) from selected rumen bacteria from the group consisting of G. BUS Mannheimia, G. BUS Actinobacillus and G. BUS Anaerobiospirillum. The method for producing mutant rumen bacteria claimed in claim 11 or 12, wherein the mutant rumen bacteria in which the lactate dehydrogenase (IdhA) and pyruvate formate lyase (pfl) genes are destroyed is the Mannheimia s p. LPK KCTC 10558BP strain. The method for producing mutant rumen bacteria claimed in claim 10, wherein the genes are destroyed. The method of producing the mutant rumen bacteria claimed in claim 14, wherein the homologous recombination method is performed by using a gene exchange vector comprising a disrupted lactate dehydrogenase (IdhA) gene, and a gene exchange vector comprising a disrupted pyruvate formymethyl ester (pfl) gene. The method of producing the mutant rumen bacteria claimed in claim 15, wherein the homologous recombination method is performed by using a gene exchange vector comprising a disrupted lactate dehydrogenase (IdhA) gene, and a gene exchange vector comprising a disrupted pyruvate formymethyl ester (pfl) gene. The gene exchange vector containing a disrupted lactate dehydrogenase (IdhA) gene is pMLKO-SacB as described in Figure 1, and the gene exchange vector containing a disrupted pyruvate phosphate lyase (pfl) gene is pMPKO-sacB as described in Figure 2. 1. The method for producing mutant rumen bacteria claimed in claim 11, wherein the disruption of the phosphotransacetylase (pta) gene and the acetate kinase (ackA) gene is carried out by a similar recombination method as claim 1. 2. The method for producing mutant rumen bacteria claimed in claim 17, wherein the recombination method is carried out by a similar recombination method as claim 1. A similar method is performed by using a gene exchange vector consisting of a phosphotransacetylase (pta) gene and a ketone kinase (ackA) gene inactivated. Page 3 of 3 1. A method for producing mutant rumen bacteria claimed in claim 18, wherein the gene exchange vector consisting of a phosphotransacetylase (pta) gene and a ketone kinase (ackA) gene inactivated is pPTA-sacB, as described in Figure 6. 2. A method for producing mutant rumen bacteria claimed in claim 12, wherein the ketone kinase (ppc) gene inactivation is performed by a similar combination method. 2. A method for producing mutant rumen bacteria claimed in claim 20, wherein the combination method is A similar method is performed by using a gene exchange vector containing a disrupted phosphoenol pyruvate (ppc) enzyme-encoding gene. 2 A method for producing mutant rumen bacteria claimed in claim 21, wherein the gene exchange vector contains a disrupted phosphoenol pyruvate (ppc) enzyme-encoding gene, pPPC-sacB, as described in Figure 7. 2 A method for producing succinic acid, wherein said method comprises the steps of cultivating any one of the mutant rumen bacteria claimed in claims 1-3, and extracting succinic acid from the culture medium in a liquid slurry. 2 A method for producing succinic acid claimed in claim 23, wherein the cultivating step involves homofermentation, yielding only a fraction of the primary product. 2 A method for producing succinic acid claimed in claim 23, wherein the mutant rumen bacteria claimed in claim 23 The claimed strains are Mamheimia s p. LPK KCTC 10558BP, LPK7 KCTC 10626BP or LPK4 as shown in Figure 9. ------------ Edited 11 Aug 2017 The claimed strains are mutant rumen bacteria in which the lactate dehydrogenase-encoding gene (IdhA) and pyruvate formate-1yase-encoding gene (pf1) are destroyed and have the ability to produce succinic acid in the absence of oxygen, where the rumen bacteria are selected from a group consisting of the genera Mannheimia, Actinobacillus and Anaerobiospirillum. ----------------------------------------------------------------------------------------------------Rumen mutants in which the lactate dehydrogenase-encoding gene (ldhA) and pyruvate formate-lyse encoding gene (pfl) are destroyed, have the ability to produce high concentrations of succinic acid while producing little or no other organic acids in the absence of oxygen.Rumen mutants in which the lactate dehydrogenase-encoding gene (ldhA), pyruvate formate-lyse encoding gene (pfl), phosphatetransactylase-encoding gene (pta), and acetate kinase-encoding gene (ackA) are destroyed, have the ability to produce high concentrations of succinic acid. While producing little or no other organic acids in the absence of oxygen, the rumen mutant bacteria in which the lactate dehydrogenase-encoding gene (ldhA), pyruvate formate-lyse encoding gene (pfl), and phosphoenolpyruvate carboxylase-encoding gene (ppc) are deletion, and such bacteria are capable of producing high concentrations of succinic acid, while producing little or no other organic acids in the absence of oxygen. The rumen mutant bacteria claimed in any of the preceding claims 1 to 3, wherein the rumen mutant bacteria were selected from among the genus Manheimia, genus Actinobacillus, and genus Anaerobiospirillum. The rumen mutant bacteria claimed in any of the preceding claims 1 to 3. Whereas the rumen bacteria are homo-fermentative bacteria that produce only succinic acid while producing little or no other organic acids; the rumen mutant bacteria claimed in Claim 1, wherein the rumen mutant bacteria is Mannheimia succincproducens LPK; the rumen mutant bacteria claimed in Claim 6, wherein Mannheimia succincproducens LPK is KCTC 10558BP; the rumen mutant bacteria claimed in Claim 2, wherein the rumen mutant bacteria is Mannheimia succinicproducens LPK7Rumen mutant bacteria claimed in claim 6, wherein Mannheimia succincproducens LPK7 is KCTC 10626BP 1Rumen mutant bacteria claimed in claim 3, wherein the rumen mutant bacteria is Mannheimia succinicproducens LPK4 1Method for producing rumen mutant bacteria, wherein the bacteria are capable of producing high concentrations of succinic acid while producing low or no amounts of other organic acids, in the absence of oxygen, comprising the steps of destroying the genes encoding lactate dehydrogenase (ldhA) and pyruvate formate lyase (pfl) from rumen bacteria selected from among the genera Manheimia, Actinobacillus, and Anaerobiospirillum 1Method for producing rumen mutant bacteria, wherein the bacteria are capable of producing high concentrations of succinic acid while producing low or no amounts of other organic acids, in the absence of oxygen. The method involves the ablation of the genes encoding phosphate transacetylase (pta) and acetate kinase (ackA) from selected ruminal bacteria from the genera Manheimia, Actinobacillus and Anaerobiospirillum, and the lactate dehydrogenase (ldhA) and pyruvate formate lyase (pfl) genes were ablated. 1Method for the production of ruminal mutant bacteria, such that they produce high concentrations of succinic acid while producing little or no other organic acids, in the absence of oxygen. The method involves the additional step of ablation of the phosphoenolpyruvate carboxylase (ppc) genes from selected ruminal bacteria from the genera Manheimia, Actinobacillus and Anaerobiospirillum. and the genes encoding lactate dehydrogenase (ldhA) and pyruvate formate lyase (pfl) are disrupted. 1A method for producing mutant rumen bacteria claimed in claim 12 or 13, wherein the mutant rumen bacteria with the lactate dehydrogenase (ldhA) and pyruvate formate lyase (pfl) genes disrupted are Mannheimia succincproducens LPK (KCTC 10558BP). 1A method for producing mutant rumen bacteria claimed in claim 11, wherein the disruption of the ldhA and pfl genes is performed by homologous recombination. 1A method for producing mutant rumen bacteria claimed in claim 15, wherein the homologous recombination method is performed by using a gene exchange vector containing the disrupted ldhA and the exchange vector. 1. A method for producing rumen mutant bacteria claimed in claim 16, wherein the gene exchange vector containing the ldhA gene is pMLKO-sacB, and the gene exchange vector containing the pfl gene is pMPKO-sacB. 1. A method for producing rumen mutant bacteria claimed in claim 12, wherein the ptA and ackA genes are destroyed by homologous recombination. 1. A method for producing rumen mutant bacteria claimed in claim 18, wherein the method Homologous recombination is performed using a gene exchange vector containing disrupted ptA and ackA. 2. Method for producing rumen mutant bacteria claimed in claim 19, wherein the gene exchange vector containing disrupted ptA and ackA is pPTA-sacB. 2. Method for producing rumen mutant bacteria claimed in claim 13, wherein the disruption of the ppc gene is performed using a homologous recombination method. 2. Method for producing rumen mutant bacteria claimed in claim 21, wherein the method The homologous recombination is performed using a gene exchange vector containing a disrupted ppc. 2. The method for producing a rumen mutant bacterium claimed in claim 22, wherein the gene exchange vector containing a disrupted ppc is pPPC-sacB. 2. The pMLKO-sacB gene exchange vector containing a disrupted ldhA. 2. The pMLKO-sacB gene exchange vector containing a disrupted pfl. 2. The pPTA-sacB gene exchange vector containing a disrupted pta and ackA. 2. The pPPc-sacB gene exchange vector containing a disrupted ppc. 2. The method for producing succinic acid. The method comprises the steps of cultivating any one of the rumen mutant bacteria claimed in claims 1 to 3 in the absence of oxygen, and extracting succinic acid from the culture medium. 2. The method for producing succinic acid claimed in claim 28, wherein the culture step is homofermentation. (homo-fermentation) which produces high concentrations of succinic acid while producing only small or no amounts of other organic acids. Three methods for producing succinic acid claimed in claim 28, wherein the rumen bacterium mutants are Mannheimia succinicproducens LPK, LPK7 or LPK4.