Method for increasing yield of alpha-oxoglutarate produced through whole-cell transformation

A technology of whole cell conversion and ketoglutarate, applied in the direction of microorganism-based methods, biochemical equipment and methods, botany equipment and methods, etc., can solve the problems of pollution, low yield, high cost, etc., and achieve increased production Effect

Active Publication Date: 2014-07-23
JIANGNAN UNIV
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AI Technical Summary

Problems solved by technology

It is to knock out the sucA gene to block the decomposition pathway of L-glutamic acid in the cell, thereby transforming the strain to efficiently convert L-glutamic acid to produce α-ketoglutarate, thereby solving the problem of industrial α-ketoglutarate Problems of high production cost, low yield and serious pollution

Method used

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  • Method for increasing yield of alpha-oxoglutarate produced through whole-cell transformation
  • Method for increasing yield of alpha-oxoglutarate produced through whole-cell transformation
  • Method for increasing yield of alpha-oxoglutarate produced through whole-cell transformation

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Embodiment 1

[0018] The knockout of embodiment 1 sucA gene

[0019] In a previous study, expression of an L-amino acid deaminase gene (SEQ ID NO.1) engineered by error-prone PCR or site-directed saturation mutagenesis in Bacillus subtilis improved whole-cell conversion of L-glutamate to α-ketopentamate Diacid conversion rate.

[0020]

[0021]

[0022] Using this recombinant Bacillus subtilis genomic DNA as a template, using sucA-L-F and sucA-L-R as primers, PCR amplifies about 1000 bp upstream of the sucA gene, and uses sucA-R-F and sucA-R-R as primers, PCR amplifies about 1000 bp downstream of the sucA gene . Using p7Z6 as a template and sucA-Z-F and sucA-Z-R as primers, the resistance marker fragment Lox71-zeo-lox66 was amplified about 1000bp. Fusion PCR was performed on these three fragments. Fusion PCR fragments were transformed into Bacillus subtilis, homologous recombination, and zeor transformants were screened. Transformation of zeo with thermosensitive plasmid pTSC r T...

Embodiment 2

[0026] Preparation of embodiment 2 whole cell catalyst and whole cell transformation process

[0027] The original Bacillus subtilis in Example 1 and the recombinant Bacillus subtilis after knocking out sucA were inoculated into the seed medium (chloramphenicol 10 mg / L), and cultivated overnight at 37°C and 200 rpm. Fermentation was carried out in a 3L NBS fermenter, 1% inoculum was added to 1.8L fermentation medium, the stirring speed, ventilation and temperature were 400rpm, 1.0vvm and 28°C, respectively, when OD 600 When 0.6 was reached, 0.4 mM IPTG was added to induce the expression of L-amino acid deaminase. After 5 hours of induction, centrifuge at 8,000 rpm for 10 minutes at low temperature, collect the bacterial cells, and wash the bacterial cells twice with 20 mM Tris-HCl (pH 8.0) buffer solution. The whole cell transformation system is: L-glutamic acid 15g / L, whole cell catalyst 20.0g / L, the reaction is carried out in 20mM Tris-HCl (pH8.0), 37°C, 200rpm transformati...

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Abstract

The invention discloses a method for increasing yield of alpha-oxoglutarate produced through whole-cell transformation, and belongs to the technical field of biotechnology. The method is characterized in that recombinant bacillus subtilis is used as a producing bacterial strain, and an sucA gene in a genome of the recombinant bacillus subtilis is knocked out. The sucA gene is knocked out so as to block the decomposition approach of intracellular L-glutamic acid, so that the bacterial strain is transformed so as to efficiently transform L-glutamic acid for producing alpha-oxoglutarate, thus the problems of high cost, low yield and serious pollution of industrialized alpha-oxoglutarate are solved.

Description

technical field [0001] The invention relates to a method for improving the production of α-ketoglutarate by transforming whole cells of recombinant Bacillus subtilis by knocking out the sucA gene, and belongs to the field of biotechnology. Background technique [0002] α-ketoglutarate is an important intermediate in the tricarboxylic acid cycle, which plays an important role in coordinating intracellular carbon metabolism and nitrogen metabolism. α-Phenylpyruvate has many applications. It can be used to synthesize heterocyclic compounds of antineoplastic drugs, as an antioxidant and to promote wound healing. In biomedical diagnosis, α-ketoglutarate can be used as the substrate of ketoglutarate dehydrogenase, aspartate aminotransferase and alanine aminotransferase. α-ketoglutaric acid can be used as a raw material for the synthesis of polyethylene, a polymer with biodegradability. The traditional production of α-ketoglutaric acid uses chemical methods. The main disadvantage...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/50C12N15/53C12N15/70C12R1/125
Inventor 陈坚刘龙堵国成李江华嘎子侯赛因侯颖
Owner JIANGNAN UNIV
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