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Method for realizing whole cell transformation to synthesize L-phenyllactic acid by genetic engineering strain

A genetically engineered bacteria and gene technology, applied in the field of genetically engineered strains to realize whole-cell transformation and synthesis of L-phenyllactic acid, can solve problems such as complicated operation, consumption of coenzymes, product separation, etc., and achieve broad application prospects, reduce production costs, and reduce additives. amount of effect

Active Publication Date: 2019-04-09
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The microbial fermentation method takes a long time to ferment, the product needs to be separated, and the operation is complicated
The enzyme-catalyzed method needs to consume coenzyme I, which is costly and complicated for enzyme purification, and is not suitable for industrial production

Method used

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  • Method for realizing whole cell transformation to synthesize L-phenyllactic acid by genetic engineering strain
  • Method for realizing whole cell transformation to synthesize L-phenyllactic acid by genetic engineering strain
  • Method for realizing whole cell transformation to synthesize L-phenyllactic acid by genetic engineering strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Construction of co-expression plasmid pET28a-pdh-ldh

[0030] The phenylalanine dehydrogenase gene pdh and the L-hydroxyisocaproate reductase gene ldh were artificially synthesized. PCR amplification reaction system (50.0μL): dd h 2 O 35.5 μL, 5×Phusion HF Buffer 10.0 μL, dNTPs 1.0 μL, pdh-up / ldh-up 1.0 μL, pdh-down / ldh-down 1.0 μL, template plasmid 1.0 μL, Phusion enzyme 0.5 μL. Reaction conditions: 1) Pre-denaturation at 98°C for 30s; 2) Denaturation at 98°C for 10s; 3) Primer annealing at 68°C (pdh) / 58.5°C (ldh) for 30s; 4) Primer extension at 72°C for 35s (pdh) / 50s (ldh) (Repeat steps 2-4, cycle 30 times); 5) Continue to extend at 72°C for 7 minutes. After the PCR product was verified by agarose gel electrophoresis, impurities were removed with an agarose gel recovery kit, and the purified target gene was stored at 4°C for future use.

[0031] Table 1 Primers for amplifying gene pdh and ldh

[0032] Primer name

Primer sequence

pdh-...

Embodiment 2

[0039] Example 2 Construction and induced expression of co-expression strain E.coli BL21(DE3) / pET28a-pdh-ldh

[0040] After activating and culturing the recombinant strain E.coli DH5α / pET28a-pdh-ldh, extract the plasmid, transfer the co-expression plasmid pET28a-pdh-ldh into E.coli BL21(DE3) competent cells, and name the obtained co-expression strain is E.coliBL21(DE3) / pET28a-pdh-ldh.

[0041] The co-expression strain E.coli BL21(DE3) / pET28a-pdh-ldh was inoculated into LB (containing 1.0mmol / L kanamycin) liquid medium, cultivated overnight at 37°C and 160rpm, as the seed solution. Inoculate the seed liquid into 100.0mL LB (containing 1.0mmol / L kanamycin) liquid medium with 1.0% inoculum size, shake and cultivate to OD 600 0.6, add 0.8mmol / L isopropyl-β-D-thiogalactoside (IPTG), and cultivate at 22°C for 14h. The cultured bacterial solution was centrifuged at 4° C., 8000 rpm for 10 minutes to remove the supernatant and collect the bacterial cells. The bacterial cells were wa...

Embodiment 3

[0042] Example 3 Co-expression strain E.coli BL21(DE3) / pET28a-pdh-ldh whole cell transformation to synthesize L-phenyllactic acid

[0043] Using L-phenylalanine as a substrate to convert and synthesize L-phenyllactic acid is divided into two steps: deamination reaction and reduction reaction ( image 3 ). Deamination reaction: Phenylalanine dehydrogenase deaminates L-phenylalanine to phenylpyruvate, accompanied by NAD + Conversion to NADH. Reduction reaction: L-hydroxyisocaproate reductase reduces phenylpyruvate to L-phenyllactate, at the same time, accompanied by NADH to NAD + change. Therefore, the tandem reaction of phenylalanine dehydrogenase and L-hydroxyisocaproate reductase is used to convert and synthesize L-phenyllactic acid, which can realize the cofactor NAD + and NADH self-circulation.

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Abstract

The invention discloses a method for realizing whole cell transformation to synthesize L-phenyllactic acid by a genetic engineering strain, in particular to a genetic engineering strain capable of co-expressing phenylalanine dehydrogenase and L-hydroxyisocaproic acid reductase to realize self-circulation of cofactors NAD<+> and NADH. The method for synthesizing L-phenyllactic acid by a whole celltransformation substrate L-phenylalanine belongs to the field of bioengineering technology. By using the whole cell transformation of a recombinant strain E.coli BL21(DE3) / pET28a-pdh-ldh to synthesizeL-phenyllactic acid, the whole cell transformation rate can reach 88.9% to 95.6% under the action of an added surfactant. By adopting the method, the self-circulation of the cofactors NAD<+> and NADHis realized, the addition amount of the cofactors is reduced, the production cost is reduced, and the method has a broad application prospect in the field of industrial synthesis of L-phenyllactic acid.

Description

technical field [0001] The present invention relates to a kind of genetic engineering bacterial strain realizes the method that the whole cell transforms and synthesizes L-phenyllactic acid, specifically relates to a kind of method that can realize the cofactor NAD + A genetically engineered strain that co-expresses phenylalanine dehydrogenase and L-hydroxyisocaproate reductase with NADH self-circulation, and a method for synthesizing L-phenyllactic acid through whole-cell transformation of substrate L-phenylalanine, which belongs to bioengineering technology field. Background technique [0002] L-phenyllactic acid (L-PLA), that is, L-2-hydroxy-3-phenylpropionic acid, is one of the two chiral enantiomers of PLA. Isomer D-PLA naturally coexists in lactic acid bacteria fermentation products and honey, has special biological activity, and can be widely used as a chiral intermediate in the fields of chemical industry, medicine, pesticide and biosynthesis. [0003] L-PLA can no...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P7/42C12R1/19
CPCC12N9/0004C12N9/0018C12P7/42C12Y104/0102
Inventor 夏雨茅菁菁王周平刘祖河刘琦
Owner JIANGNAN UNIV
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