Genetic engineering bacterium for synthesizing pyruvic acid and D-alanine as well as construction method and application thereof
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A technology of genetically engineered bacteria and pyruvate, applied in the field of genetic engineering, can solve the problems of consumption product pyruvic acid and low yield of pyruvate, and achieve the effects of simple construction method, good application prospect and easy use.
Active Publication Date: 2020-05-05
JIANGNAN UNIV
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Problems solved by technology
However, in the process of whole-cell catalysis, the cell itself consumes a large amount of the product pyruvate, resulting in a low yield of pyruvate
Method used
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Embodiment 1
[0034] Construction of gene knockout homology arm fragments
[0035] Table 1
[0036]
[0037]
[0038] According to the sequence information of E.coli BL21(DE3), design the primers shown in the table above, and use the above primers to use the E.coli BL21(DE3) genome as a template to amplify btsT, cstA, aceEF, pflB, poxB, pps, The upstream and downstream homology arm fragments of each gene of ldhA were respectively fused by Overlapping PCR to obtain fragments btsT12, cstA12, aceEF12, pflB12, poxB12, pps12, and ldhA12.
Embodiment 2
[0040] Construction of pTarget plasmid
[0041]Select the N20 region (20bp base sequence used to target the gene to be knocked out in the CRISPR / Cas9 system) on each gene to be knocked out, design primers, use pTarget as a template, and replace the N20 region of the template by PCR. The PCR product was transferred into E.coli JM109, and the plasmids were extracted to obtain 7 plasmids, pTarget-btsT, pTarget-cstA, pTarget-aceEF, pTarget-pflB, pTarget-poxB, pTarget-pps, pTarget-ldhA.
Embodiment 3
[0043] gene knockout
[0044] The pCas9 plasmid was transformed into E.coli BL21 (DE3) to obtain the host BL21 (pCas9) expressing Cas9 protein, and then the pTarget-aceEF plasmid and the fragment aceEF12 were electrotransformed into BL21 (pCas9) to obtain BLK01 (pCas9) with the aceEF gene knocked out , eliminate the pTarget-aceEF plasmid, electrotransfer the pTarget-pflB plasmid and the fragment pflB12 into BLK01 (Cas9), knock out the pflB gene, and knock out each gene to be knocked out in sequence according to this method, and finally eliminate the pTarget and pCas9 plasmids to obtain aceEF, pflB, poxB, pps, ldhA gene knockout BLK05 and btsT, cstA, aceEF, pflB, poxB, pps, ldhA gene knockout BLK07. BLK07 gene knockout colony PCR verification results are as follows figure 1 shown.
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Abstract
The invention discloses a genetic engineering bacterium for synthesizing pyruvic acid and D-alanine as well as a construction method and application thereof. Recombinant escherichia coli of the invention is obtained by knocking out apyruvate dehydrogenase complex achEF gene, a pyruvate lyase pflB gene, a pyruvate oxidase poxB gene, a phosphoenolpyruvate synthase pps gene and a lactic dehydrogenaseldhA gene in escherichia coli BL21 (DE3), blocking a key pathway of further metabolism of pyruvic acid and knocking out genes btsT and cstA for encoding a pyruvic acid transport protein. For the genetic engineering bacterium disclosed by the invention, L-amino acid deaminase pm1 is utilized to catalyze D, L-alanine, so that L-alanine can be converted to the pyruvic acid and can be separated to obtain D-alanine at the same time, and a new idea and method is provided to chiral separation of the D, L-alanine; and the recombinant escherichia coli capable of efficiently synthesizing the pyruvic acid and the D-alanine is obtained through modification, the yield of the pyruvic acid reaches 32.0g / L, the conversion rate of the L-alanine reaches up to 80 percent, and the separation rate of the D / L-alanine reaches up to 80 percent.
Description
technical field [0001] The invention relates to a genetic engineering bacterium for synthesizing pyruvate and D-alanine, a construction method and application thereof, and belongs to the technical field of genetic engineering. Background technique [0002] Pyruvic acid (Pyruvic acid) is an important intermediate in the field of drug synthesis, widely used in food, pesticide, biochemical and other industries. At present, the industrial production methods of pyruvate mainly include chemical synthesis, enzymatic conversion and microbial fermentation. Among them, the cost of the chemical synthesis method is relatively high, and its pollution to the environment is relatively large; while in the microbial direct fermentation method, the components of the product are complex and the separation is difficult. Biocatalytic synthesis of pyruvate can overcome the shortcomings and shortcomings of the above two methods, so it has broad prospects in industrial applications. [0003] D-al...
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