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L-lactate dehydrogenase gene-deleted engineering bacterium and construction method and application thereof

A technology of lactate dehydrogenase and genetically engineered bacteria, applied in the field of D-lactic acid, L-lactate dehydrogenase gene deletion mutants and their construction, can solve the problems of L-lactate dehydrogenase gene deletion and other problems, and achieve broad application Foreground effect

Inactive Publication Date: 2011-06-22
FUSHUN RES INST OF PETROLEUM & PETROCHEMICALS SINOPEC CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to develop a strain of L-lactate dehydrogenase gene-deficient engineering bacteria for producing D-lactic acid with high optical purity, and to provide a kind of L-lactate Method for constructing hydrogenase gene deletion

Method used

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  • L-lactate dehydrogenase gene-deleted engineering bacterium and construction method and application thereof
  • L-lactate dehydrogenase gene-deleted engineering bacterium and construction method and application thereof
  • L-lactate dehydrogenase gene-deleted engineering bacterium and construction method and application thereof

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Embodiment 1

[0032] Embodiment 1: Construction of gene knockout vector:

[0033] 1. Splicing of upstream and downstream sequences of L-lactate dehydrogenase gene

[0034](1) According to the upstream and downstream DNA sequences of the ldh gene (NCBI-GI: 58036263) in Corynebacterium glutamicum, design primers to amplify its upstream and downstream sequences by PCR. The primer sequences are as follows:

[0035] uldh1: 5′-CCAAGGTGCCGACACTAAT-3′

[0036] dldh1: 5′-CGGTGATTTCGCAACTCCAACATCTCCTG-3′

[0037] uldh2: 5′-TTGGAGTTGCGAAATCACCGACCACGAGA-3′

[0038] dldh2: 5′-GCTTCCAGACGGTTTCATC-3′

[0039] Using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, under the guidance of primers uldh1, dldh1 and uldh2, dldh2, the upstream and downstream homology arm sequences of the ldh gene were cloned by PCR. The PCR amplification conditions and system were as follows:

[0040] Reaction system Amplification conditions

[0041] Composition Volume Temperature Time

[0042] uldh1(...

Embodiment 2

[0054] Example 2: Screening of single exchange strains after homologous recombination

[0055] The constructed L-lactate dehydrogenase gene knockout vector pK18mobsacB-ldh was electrotransformed into competent cells of Corynebacterium glutamicum ATCC13032, and the transformation conditions were: 25μF, 600Ω, 2.5kV electric shock, pulse time 10-12ms, Spread the bacterial solution on a BHIS plate with 25 μg / ml kanamycin and culture it for 24 to 48 hours, select positive colonies, and use 10% sucrose plates containing 25 μg / ml kanamycin to replicate the transformed colonies that were initially screened. To confirm by screening, pick the colonies that can grow on the kanamycin-resistant plate but cannot grow on the kanamycin-resistant plate containing 10% sucrose, extract the genomic DNA of the positive colony after re-screening, and amplify ldh1 - The upstream and downstream primers dldh1 and uldh2 of ldh2 were used for PCR amplification. In addition, primers s1 and s2 for amplif...

Embodiment 3

[0056] Embodiment 3: Screening of ldh gene knockout strain

[0057] Insert the strain C.glutamicum Res167L with single exchange into the liquid LB medium without antibiotics for overnight culture, then collect the bacterial liquid, concentrate it by centrifugation, spread it on the LB medium containing 10% sucrose, and pick out the outgrowth Positive strains were placed on solid LB plates containing kanamycin and kanamycin, respectively, and the colonies that could grow on the plate without antibiotics but could not grow on the plate containing antibiotics were picked and shaken. Colony PCR was further performed with upstream and downstream primers dldh1 and uldh2 for amplifying ldh1-ldh2. Additional PCR amplification was performed with primers for amplifying ldh and internal amplification primers up1 and down1 for the ldh1-ldh-ldh2 fragment. The amplification results of the three pairs of PCR primers were consistent with the expected values, indicating that the screened stra...

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Abstract

The invention relates to an L-lactate dehydrogenase gene-deleted engineering bacterium and a construction method and application thereof. Upstream and downstream sequences of ldh1 and ldh2 of L-lactate dehydrogenase (ldh) gene are subjected to polymerase chain reaction amplification, cloned segments are connected to a gene knock-out vector, the constructed knock-out vector is inoculated into corynebacterium glutamicum, and the gene engineering bacterium C.glutamicum Res167 delta ldh is obtained by silencing and screening L-lactate dehydrogenase (ldh) genes in the strain through a homologous recombination method. By the homologous recombination method, the L-lactate dehydrogenase genes in the corynebacterium glutamicum are silenced, so that the gene engineering bacterium for producing pureD-lactic acid is obtained. When the engineering bacterium is used for lactic acid fermentation production, the yield of the D-lactic acid is more than 20g / L, and the purity is over 99 percent. The invention has important significance for industrial production of the D-lactic acid and has wide application prospect.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an L-lactate dehydrogenase gene deletion mutant strain and a construction method thereof for producing D-lactic acid with high optical purity. Background technique [0002] As one of the three major organic acids, lactic acid can be divided into D-lactic acid, L-lactic acid and D, L lactic acid according to its optical activity. At present, lactic acid is mainly produced by microbial fermentation in the world. Among them, L-lactic acid has been developed and produced earlier because of its wide biocompatibility, and its production technology and products have tended to mature. With the discovery of new functions of D-lactic acid, the research on its production and application has attracted more and more attention. [0003] D-lactic acid is widely used in food, medicine, chemical industry, agriculture, etc., especially the polymer of lactic acid - polylact...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N15/09C12P7/56C12R1/15
Inventor 闻建平李爽贾晓强
Owner FUSHUN RES INST OF PETROLEUM & PETROCHEMICALS SINOPEC CORP
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