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A pyruvate carboxylase mutant p474n with improved enzymatic activity and its application

A technology of pyruvate carboxylase and pyruvate decarboxylase is applied in the fields of genetic engineering and fermentation engineering to achieve the effects of improving yield, good industrial application value and prospects

Active Publication Date: 2018-01-30
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Any Saccharomyces cerevisiae fermentation process with dicarboxylic acid as the target product will face the same problem: how to strengthen the pyruvate carboxylation reaction and promote the carbon flow from pyruvate to the synthesis pathway of the target carboxylic acid? Carry out directional transformation to pyruvate carboxylase to improve the output of dibasic carboxylic acids such as fumaric acid and have not been reported at present, therefore, the scheme provided by the present invention has general significance for the research utilizing Saccharomyces cerevisiae to produce carboxylic acid

Method used

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  • A pyruvate carboxylase mutant p474n with improved enzymatic activity and its application
  • A pyruvate carboxylase mutant p474n with improved enzymatic activity and its application
  • A pyruvate carboxylase mutant p474n with improved enzymatic activity and its application

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Embodiment 1

[0031] Example 1: Selection of mutation sites

[0032] How to improve the activity of pyruvate carboxylase by site-directed mutagenesis technology, the difficulty lies in the determination of the mutation site.

[0033] The present invention compares the amino acid sequences of pyruvate carboxylases from different species such as Pseudomonas, Bacillus, Rhizobium, Mycobacterium, Staphylococcus, Archaea, and eukaryotic cells, and finally selects P474 for RoPYC 19 saturation mutations were carried out, and the mutant strains were subjected to fermentation experiments to investigate the effects of mutations on the accumulation of fumaric acid.

Embodiment 2

[0034] Example 2: Research on the localization of RoPYC in Saccharomyces cerevisiae

[0035] 1. TEF1promoter and RoPYC gene ORFs were cloned into pGFP33 vector

[0036]According to the principle of the homologous recombination kit, design primers with more than 15 bases homologous to the vector at both ends, as shown in Table 1, the lowercase letters are homologous arms, and the uppercase letters are PCR primers.

[0037] Table 1 Primers for amplifying the RoPYC gene

[0038]

[0039] Using pY15TEF1-RoPYC as a template (Xu et al. Fumaric acid production in Saccharomyces cerevisiae by in silico aided metabolic engineering, 2012), RoPYC-F, RoPYC-R (sequences are shown in SEQ ID NO.3, SEQ ID NO.4, respectively ) as primers to amplify the ORF frame of TEF1p and RoPYC (the obtained PCR product contains the sequence of amino acid sequence SEQ ID NO.1), the amplified product is connected to the pGFP33 vector, transformed into large intestine competent cells, coated with ampicilli...

Embodiment 3

[0044] Example 3: RoPYC site-directed mutation and expression

[0045] Site-directed mutagenesis was performed by PCR method, saturation mutation was performed on the P474 site of RoPYC, and the proline at the 474th site was mutated into other 19 amino acids. The entire plasmid was amplified by PCR using the pY15TEF1-RoPYC plasmid as a template, F and R containing mutation sites as primers, and Takara's high-fidelity enzyme PrimeSTAR GXL. The enzyme digestion system contains 1 μL of PCR product and 1 μL of Dpn I enzyme, with a total volume of 20 μL, digested overnight at 37°C. Fragment purification of digested products. Take 5 μL of the purified product and transform it into 30 μL of competent cells Trans1-T1, smear the LA plate, inoculate the grown transformant in the LA medium, extract the plasmid and send it to Shanghai Sangon for sequencing.

[0046] Among them, the primers F(Asn) and R(Asn) (sequences shown in SEQ ID NO.5 and SEQ ID NO.6 respectively) used for P474N mut...

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Abstract

The invention discloses a pyruvic carboxylase mutant P474N with improved enzymatic activity and an application thereof, belonging to the fields of genetic engineering and fermentation engineering. According to the invention, the P474 locus of the pyruvic carboxylase of rhizopus oryzae is mutated to obtain asparaginase and the enzymatic activity of the obtained mutant is improved by 12.8%; on the basis of knocking out PDC1 and ADH1, FUM1 is knocked out; the yield of fumaric acid of excessive pyruvic carboxylase mutant P474N is improved by 13.3%; and moreover, by adding 64mu g / L biotin, the yield of fumaric acid is up to 357mg / L, improved by 37% in comparison with the yield of fumaric acid in case of no addition of biotin (256.7+ / -3.0mg / L). According to the invention, the synthesis route from carbon metabolism flow into fumaric acid via pyruvic acid is effectively reinforced and a condition under which engineering yeast is constructed to efficiently produce fumaric acid and other dicarboxylic acids is created; and therefore, good industrial application value and prospect are achieved.

Description

technical field [0001] The invention relates to a pyruvate carboxylase mutant P474N with improved enzyme activity and application thereof, belonging to the fields of genetic engineering and fermentation engineering. Background technique [0002] As a eukaryotic model microorganism, Saccharomyces cerevisiae has the following advantages: rich genetic information, convenient metabolic transformation operation; simple nutritional requirements, low cost of separation and extraction process; good growth under low pH conditions (even pH<3.0); Can tolerate high concentrations of substrates; certified by the FDA as GRAS (General Regarded AsSafe) microorganisms, fermented products have the advantages of safety and become fermented to produce carboxylic acids (lactic acid, pyruvic acid, malic acid, fumaric acid, succinic acid, α- Potentially optimal microorganisms for ketoglutarate, etc.). However, Saccharomyces cerevisiae produces a large amount of ethanol by batch fermentation un...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/00C12N1/19C12P7/46C12P7/50C12R1/865
CPCC12N9/93C12P7/46C12P7/50C12Y604/01001
Inventor 徐国强蒋伶活吴满珍李鹏越
Owner JIANGNAN UNIV
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