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Saccharomyces cerevisiae engineering bacteria and application thereof in preparation of protocatechuic acid

A strain of Saccharomyces cerevisiae, the technology of Saccharomyces cerevisiae, applied in the field of genetic engineering, can solve the problems of ineffective use of benzene ring structure and waste of resources, etc.

Active Publication Date: 2021-08-03
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, if the benzene ring structure of lignin is not effectively utilized, it will cause a waste of resources

Method used

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  • Saccharomyces cerevisiae engineering bacteria and application thereof in preparation of protocatechuic acid
  • Saccharomyces cerevisiae engineering bacteria and application thereof in preparation of protocatechuic acid
  • Saccharomyces cerevisiae engineering bacteria and application thereof in preparation of protocatechuic acid

Examples

Experimental program
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Effect test

Embodiment 1

[0074] 1. Construction of strains ZR01-ZR03: Obtain 400 bp sequences of Saccharomyces cerevisiae ADH6 by PCR, name them ADH6L and ADH6R, carry out overlap, and obtain ADH6LR as donor DNA. The gRNA plasmid was constructed according to the ADH6 sequence, the gRNA, cas9, and donor DNA were introduced into Saccharomyces cerevisiae BY4742, and the SC screening plate was spread. The obtained transformants were streaked and purified, and then the yeast genome was extracted for PCR verification and sequencing. According to the above method, ADH7 and BDH2 were sequentially knocked out, named ZR01, and MHT1 was further knocked out, named ZR02. On the basis of ZR02, SAM4 was knocked out and named ZR03.

[0075] 2. Construction of yeast engineering bacteria PCA1, PCA2, PCA3:

[0076] PCA1 (transfer of exogenous gene 4CL, ppech in ZR01): amplification of yeast promoter P by PCR TPI1P terminator T GPM1 , overlap with the gene 4CL gene to obtain the module P TPI1P -4CL-T GPM1 . Amplifi...

Embodiment 2

[0085] The ability of different strains of embodiment 2 to synthesize protocatechuic acid

[0086] The strain PCA1 PCA2 constructed in Example 1 was initially added with SC-LEU of 100mg / L ferulic acid (synthetic yeast nitrogen source YNB 6.7g / L, glucose 20g / L, mixed amino acid powder 2g / L lacking leucine) Cultivate under medium for 24h, analyze the output of its vanillic acid and vanillin, the results are shown in figure 1 .

[0087] The strain PCA3 constructed in Example 1 was initially cultured in SC-LEU (synthetic yeast nitrogen source YNB 6.7g / L, glucose 20g / L, leucine-deficient mixed amino acid powder 2g / L) initially added with 100mg / L ferulic acid Production of protocatechuic acid and vanillic acid ( figure 2 ).

[0088] The bacterial strain PCA4, PCA5, PCA6, PCA7, PCA8, PCA9 constructed in Example 1 were cultivated for 24h under the SC medium of the corresponding deficiency of 100mg / L initially added 100mg / L vanillic acid and the production of protocatechuic acid an...

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Abstract

The invention relates to the technical field of genetic engineering, in particular to saccharomyces cerevisiae engineering bacteria and application thereof in preparation of protocatechuic acid. According to the invention, on the basis of saccharomyces cerevisiae without ADH6, ADH7 and BDH2 genes, at least one of exogenous genes 4CL, ECH, vdh, vanA, vanB, metf1 and ligM is transferred; an endogenous gene MET6 is over-expressed; and / or at least one of endogenous genes MHT1 and SAM4 is knocked out, a new lignin bioconversion path is constructed in the yeast body, the path regulates and controls the demethylation of tetrahydrofolate metabolism intensified strains through the modification of the genes to promote the conversion of vanillic acid to synthesize protocatechuic acid, and the purpose of synthesizing protocatechuic acid by using lignin monomers and lignin is achieved. Not only can the utilization problem of the lignin be solved, but also the protocatechuic acid which is an important chemical raw material can be generated, and high-value utilization of the lignin is achieved.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to engineering bacteria of Saccharomyces cerevisiae and its application in the preparation of protocatechuic acid. Background technique [0002] Lignin, a second natural polymer, is present in most land plants at 15% to 40% dry weight (Ragauskas et al., 2014). The annual output of lignin in the global pulp and paper industry is about 50 million tons. However, 98% of lignin is burned for energy production or disposed of in landfills, posing a serious threat to the environment (Garlapati et al., 2020). In nature, there are some microorganisms that can degrade lignin, and these microorganisms can transform and utilize lignin to produce various products. Recently, chemicals such as muconic acid (MA) and polyhydroxyalkanoate (PHA) and oils were produced in engineered lignin-degrading microorganisms by degrading and converting lignin (Kosa & Ragauskas, 2013; Lin et al., 2016...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12P7/42C12R1/865
CPCC12Y602/01012C12Y402/01017C12Y102/01067C12Y114/13082C12N9/0026C12N9/1007C07K14/395C12N9/12C12N9/93C12N9/88C12N9/0073C12N9/0008C12N15/81C12P7/42Y02E50/10
Inventor 元英进李炳志张仁宽
Owner TIANJIN UNIV
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