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High-yield alpha-ketoglutarate engineered saccharomyces cerevisiae and application thereof

A technology of yeast engineering and ketoglutaric acid, applied in application, genetic engineering, fermentation, etc., can solve problems such as high pollution development space, and achieve the effect of promoting excessive accumulation and reducing production

Active Publication Date: 2014-04-09
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the industrial production of α-ketoglutaric acid mainly adopts the method of chemical synthesis, but the disadvantages of high pollution and use of toxic and harmful chemical reagents limit its development space, while microbial fermentation is becoming more and more popular with high yield, high production intensity, Environmental friendliness and other advantages have attracted more and more attention
Domestic and foreign studies on the production of α-ketoglutarate by microbial fermentation mainly focus on the optimization and control of fermentation conditions, but simple fermentation optimization cannot solve the problem of by-product accumulation. Metabolic engineering is a rational method The strain transformation method can more effectively achieve the high yield of the target product
Although there are few reports on the use of metabolic engineering methods to enhance the accumulation of α-ketoglutarate, given the important role of the pyruvate carboxylation pathway in pyruvate metabolism and the TCA cycle, strengthening the pyruvate carboxylation pathway should be a Effective strategies to promote carbon metabolic flux into the TCA cycle

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Replacing the original marker gene on the plasmid to endow the recombinant plasmid with resistance to hygromycin B

[0031] According to the hph gene sequence (Genbank: K01193.1) on the NCBI website, the hph gene sequence was obtained by chemical total synthesis method and cloned into the vector plasmid p0, and the original URA3 marker gene on the plasmid p0 was replaced by the hph gene to obtain the recombinant plasmid p0 (hph). The recombinant plasmid was transformed into Escherichia coli JM109, and the colonies that could grow on the Amp plate were picked, and colony PCR identification was performed with hph-F and hph-R as primers, and a band with a size of about 1.6 kb was obtained. Colony PCR verified that the correct transformants extracted plasmids and sent them to Shanghai Sangong for sequencing. The sequencing results were consistent with expectations, indicating that the plasmid transformation was successful. The plasmid was digested with AvrII, and...

Embodiment 2

[0037] Example 2 Construction and Identification of Recombinant Bacteria Y.lipolytica-RoPYC2

[0038]According to the RoPYC2 gene sequence (Genbank: HM130700.1) on the NCBI website, the RoPYC2 target fragment (about 3.5 kb in size) was obtained by chemical total synthesis. The RoPYC2 gene was ligated with the plasmid p0(hph) digested with SfiI and NotI by enzyme-cut ligation technology to obtain the RoPYC2 gene expression vector p0(hph)-RoPYC2. The constructed recombinant plasmid was digested and analyzed, and DNA sequencing was carried out. The results of gene sequencing were consistent with expectations, indicating that the recombinant plasmid was constructed correctly. The recombinant plasmid p0(hph)-RoPYC2 was digested and purified by AvrII, and transformed into Y. lipolytica WSH-Z06 competent cells by the lithium acetate method. The colonies that can grow on the YPD+HygB plate were continuously transferred for three generations on the YPD+HygB plate to obtain geneticall...

Embodiment 3

[0044] When embodiment 3 glycerol is the sole carbon source, the comparison of recombinant bacteria and control bacteria fermentation characteristics

[0045] Using glycerol as the only carbon source, after 144 hours of fermentation, the recombinant bacteria and the control bacteria compared: (1) The production of α-ketoglutarate in the control bacteria was 36.3g / L, and the production of α-ketoglutarate in the recombinant bacteria was up to 47.2g / L, which is 1.3 times that of the control bacteria; (2) the pyruvate content in the control bacteria is 21.2g / L, while the pyruvate content in the recombinant bacteria is only 10.4g / L, and the accumulation of pyruvate is reduced by 51%. Expression of the RoPYC2 gene effectively diverts the carbon metabolic flux from pyruvate to the production of α-ketoglutarate, and reduces the accumulation of pyruvate while increasing the production of α-ketoglutarate.

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Abstract

The invention discloses a method for promoting the accumulation of alpha-ketoglutarate by strengthening a carboxylation way of pyruvic acid. A gene RoPYC2 which derives from Rhizopus oryzae and encodes pyruvate carboxylase is over-expressed in a strain Yarrowia lipolytica WSH-Z06 of the alpha-ketoglutarate which is produced by a fermentation method to obtain a recombinant strain in which the activity of the pyruvate carboxylase is improved by 10.2 times (the concentration of protein reaches 0.87U / mg); glycerol is taken as an exclusive carbon source, and the yield of the alpha-ketoglutarate reaches 47.2g / L after the strain is fermented for 144h, and is 1.3 times that of an original strain; and the yield of the pyruvic acid is reduced to 10.4g / L and is 49.0 percent of that of the original strain. The carbon metabolic flux successfully flows from a pyruvic acid node to an alpha-ketoglutarate node by regulating and controlling a replenishment pathway of tricarboxylic acid cycle, so that a byproduct, namely the pyruvic acid is reduced while the yield of the alpha-ketoglutarate is increased. The method has certain reference significance for transforming strains to promote target products to be accumulated excessively by using a metabolic engineering means in the future.

Description

technical field [0001] The present invention relates to a high-yield α-ketoglutarate yeast engineered bacteria, in particular to a method of overexpressing the RoPYC2 gene through metabolic engineering means to increase the activity of intracellular pyruvate carboxylase to strengthen the replenishment pathway of the tricarboxylic acid cycle, thereby regulating Carbon metabolism flow from pyruvate into the tricarboxylic acid cycle to realize yeast engineering bacteria with excessive accumulation of α-ketoglutarate. Background technique [0002] α-ketoglutarate (α-KG for short) is an important organic acid, which is widely used in food, medicine, chemical and cosmetic industries. As one of the important intermediate products of the tricarboxylic acid cycle (TCA), α-ketoglutarate participates in important processes such as amino acid, protein, vitamin synthesis and energy metabolism, and plays an important role in the regulation of carbon and nitrogen metabolism in microbial ce...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/60C12P7/50C12R1/645
Inventor 陈坚周景文殷晓霞堵国成李江华胡志杰蒋小东孙福新
Owner JIANGNAN UNIV
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