Escherichia coli coenzyme metabolic-pathway transformation and biotransformation method

A biotransformation and metabolic pathway technology, applied in the field of biotransformation, can solve the problems such as the inability to repeat the work reported in the literature and the inability of coenzymes to be recycled and reused, and achieve the effects of increasing the concentration, reducing the cost of industrialization and high-efficiency transformation.

Inactive Publication Date: 2015-04-29
郁庆明
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AI Technical Summary

Problems solved by technology

Using whole-cell bacteria for transformation, the source of enzymes is relatively cheap, but usually requires the addition of coenzymes, and coenzymes cannot be recycled and reused
There have been reports in foreign countries that high-effic

Method used

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] We deleted the yjaD and yrfE genes in JM109(DE3) by homologous recombination. For the deletion method, select the two sides of the 500bp coding region of the yjaD gene, the upstream and downstream 1kb sequences, amplify from the genomic DNA by PCR, obtain fragments, splice, and subclone on the pCVD442 vector (in Escherichia coli DH5α pir strain The constructed plasmid was transformed into Escherichia coli JM109(DE3), positively selected with the ampicillin resistance gene, and the obtained clone was negatively selected with sucrose to obtain JM109(DE3)ΔyjaD knocked out of the yjaD gene , the gene knockout strains were verified by PCR method. Using the same method as above, in the JM109(DE3)ΔyjaD strain, the yrfE gene was deleted to obtain JM109(DE3)ΔyjaD yrfE. The above wild-type and deficient Escherichia coli were cultured with LB medium, and the content of endogenous coenzymes was determined. The coenzyme content in the deficient JM109(DE3)ΔyjaD yrfE cells was 6.5...

Embodiment 2

[0013] Example 2: Using expression leucine dehydrogenase and formate enzyme JM109 (DE3) ΔyjaD yrfE for the preparation of L-neopentylglycine

[0014] The leucine dehydrogenase and formate enzyme genes were transferred into JM109(DE3)ΔyjaD yrfE through two expression vectors with different copy numbers and resistances for co-expression, and neopentylketoacid was used as a substrate for biotransformation. Under the condition of not adding coenzyme, the conversion efficiency is above 90%. As a control experiment, using wild-type bacteria to express the above two enzymes for biotransformation, the efficiency is only 15%, but after adding coenzymes, 100% high-efficiency transformation can still be achieved. The L-neopentylglycine obtained through conversion is purified by cation exchange resin, the yield is 85%, and the EE value reaches 99.6%.

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Abstract

The invention relates to an escherichia coli coenzyme metabolic-pathway transformation and biotransformation method. According to the method, a coenzyme metabolic pathway is transformed by a homologous recombination method, a coenzyme degradation related gene yjaDyrfE in protein expression host bacterium escherichia coli JM109 (DE3) is removed and the concentration of endogenous coenzyme during reproduction of the host bacterium is increased. When a host bacterium expression enzyme system transformed by the method is used for preparing chiral molecules by biotransformation by a biological reduction method, the addition of coenzyme in a transformation system can be remarkably reduced or no coenzyme is added in the transformation system.

Description

technical field [0001] The invention relates to a method for transforming the coenzyme metabolic pathway in the main genus of Escherichia coli for protein expression to increase its endogenous coenzyme and using the modified whole-cell bacterium of the main genus for biotransformation. Background technique [0002] Asymmetric bioreduction for the preparation of chiral natural and unnatural amino acids, chiral ammonia and chiral alcohols is a very effective method for preparing chiral molecules with high optical activity. Early approaches used bio-extracted enzymes, coenzymes and coenzyme regeneration systems to carry out this reaction. Because the coenzyme is expensive, the membrane technology is used for the reaction, and the coenzyme is coupled with the PEG of the macromolecule (Separations Using Aqueous Phase Systen, 1989, p345-347), so that the coenzyme, like the biotransformation enzyme, can be retained by a translucent membrane , the small molecule of the substrate en...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12P13/04C12R1/19
Inventor 郁庆明
Owner 郁庆明
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