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Novel rumen bacteria variants and process for preparing succinic acid employing the same

A technology of mutant and succinic acid, applied in the field of producing succinic acid, can solve the problems of not reaching industrial application, low yield of organic acid or succinic acid, etc.

Active Publication Date: 2007-02-07
KOREA ADVANCED INST OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in this attempt, a large amount of other organic acids was produced or the yield of succinic acid was so low that the results did not reach the level of industrial application

Method used

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  • Novel rumen bacteria variants and process for preparing succinic acid employing the same
  • Novel rumen bacteria variants and process for preparing succinic acid employing the same
  • Novel rumen bacteria variants and process for preparing succinic acid employing the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Construction of pMLKO-sacB

[0044] In order to disrupt the lactate dehydrogenase gene (ldhA) by homologous recombination, a gene exchange vector can be constructed as follows. First, use the genomic DNA of Mannheimia succiniciproducens 55E (KCTC 0769BP) as a template, and perform PCR with the primers listed in the following SEQ ID NO: 1 and SEQ ID NO: 2, and then digest the resulting PCR fragment with SacI and PstI , and introduced into pUC18 (New England Biolabs, Inc., Beverly, Mass.), thereby constructing pUC18-L1.

[0045] SEQ ID NO: 1: 5'-CAGTGAAGGAGCTCCGTAACGCATCCGCCG (LS1)

[0046] SEQ ID NO: 2: 5'-CTTTATCGAATCTGCAGGCGGTTTCCAAAAA (LP1)

[0047] Afterwards, using the genomic DNA of Mannheimia succiniciproducens 55E as a template, PCR was performed with the primers listed in the following SEQ ID NO: 3 and SEQ ID NO: 4, and then, the obtained PCR fragment was digested with PstI and HindIII, and introduced into pUC18 -L1, thereby constructing pUC18-L1-L...

Embodiment 2

[0055] Example 2: Construction of pMPKO-sacB

[0056] To fragment the pyruvate formate lyase gene (pfl) by homologous recombination, a gene exchange vector can be constructed as follows. The pKmobsacB template containing the sacB gene (Genbank 02730) was subjected to PCR with the primers listed in SEQ ID NO:8 and SEQ ID NO:9 below. The resulting sacB product was digested with PstI and BamHI and introduced into pUC19 (Stratagene Cloning System. La Jolla, Calif.) to construct pUC19-sacB.

[0057] SEQ ID NO: 8: 5'-AGCGGATCCCCTTCTATCGCCTTCTTGACG(SBG)

[0058] SEQ ID NO: 9: 5'-GTCCTGCAGGGCTACAAAATCACGGGCGTC (SPR)

[0059] Using the genomic DNA of Mannheimia succiniciproducens 55E as a template, PCR was performed with the primers listed in SEQ ID NO: 10 and SEQ ID NO: 11 below. The resulting PCR fragment was digested with BamHI and fused with pUC19-sacB digested with BamHI to construct pUC19-sacB-pfl.

[0060] SEQ ID NO: 10: 5'-CATGGCGGATCCAGGTACGCTGATTTCGAT (PB1)

[0061] SEQ ...

Embodiment 3

[0065] Embodiment 3: Construction of Mannheimia sp.LPK bacterial strain

[0066]Figure 3 shows the process of constructing a mutant strain (LPK) by breaking the ldhA and pfl genes from Mannheimia succiniciproducens 55E. Mannheimia succiniciproducens 55E was poured onto LB-glucose medium containing 10 g / l glucose and incubated at 37°C for 36 hours. The formed colonies were inoculated in 10 ml of LB-glucose liquid medium and cultured for 12 hours. Inoculate 1% of the fully grown culture solution into 100 ml LB-glucose liquid medium, and culture in a shaking incubator at 200 rpm and 37°C.

[0067] After 4-5 hours, when the OD value of the culture solution reached about 0.2-0.3, the cells were collected by centrifugation at 4000 rpm for 10 minutes at 4°C. Afterwards, the cells were resuspended in 200 ml of 10% glycerol solution at 4°C. The suspension was centrifuged at 4000 rpm for 10 minutes at 4°C to collect the cells, and resuspended in 200 ml of 10% glycerol solution at 4°C...

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Abstract

The present invention relates to novel rumen bacterial mutants resulted from the disruption of a lactate dehydrogenase gene (ldhA) and a pyruvate formate-lyase gene (pfl) (which are involved in the production of lactic acid, formic acid and acetic acid) from rumen bacteria; a novel bacterial mutant (Mannheimia sp. LPK7) having disruptions of a lactate dehydrogenase gene (ldhA), a pyruvate formate-lyase gene (pfl), a phosphotransacetylase gene (pta), and a acetate kinase gene (ackA); a novel bacterial mutant (Mannheimia sp. LPK4) having disruptions of a lactate dehydrogenase gene (ldhA), a pyruvate formate-lyase gene (pfl) and a phosphoenolpyruvate carboxylase gene (ppc) involved in the immobilization of CO2 in a metabolic pathway of producing succinic acid; and a method for producing succinic acid, which is characterized by the culture of the above mutants in anaerobic conditions. The inventive bacterial mutants have the property of producing succinic acid at high concentration while producing little or no organic acids, as compared to the prior wild-type strains of producing various organic acids. Thus, the inventive bacterial mutants are useful as strains for the industrial production of succinic acid.

Description

technical field [0001] The present invention relates to a rumen bacterial mutant capable of producing high concentrations of succinic acid with little or no other organic acids, and a method for producing succinic acid, characterized in that the mutant is grown under anaerobic conditions. Background technique [0002] Various anaerobic microorganisms, including Succinivibrio dextrinosolvens, Fibrobacter succinogenes, Ruminococcus flavefaciens, etc., produce succinic acid as a final product through glucose metabolism (Zeikus, Annu.Rev.Microbiol ., 34:423, 1980). In addition to the known Anaerobiospirillum succiniciproducens in excess CO 2 Except that succinate can be produced from glucose in high concentrations and in high yields in the presence of succinate (David et al., Int. J. Syst. Bacteriol., 26:498, 1976), there are no studies on succinic acid suitable for industrial production. Acid strains were reported. However, since Anaerospirillum succinogenes is an obligate a...

Claims

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Application Information

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IPC IPC(8): C12N1/21
CPCC12N15/74C12P7/40C12N1/205C12R2001/01
Inventor 李相烨李尚俊
Owner KOREA ADVANCED INST OF SCI & TECH
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