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Enzymatic activity improved pyruvate carboxylase mutant N1078F and application thereof

A pyruvate carboxylase and mutant technology, which is applied in the fields of genetic engineering and fermentation engineering, can solve the problems of carbon flow loss, lack of carboxylic acid synthesis pathway, etc., and achieves the effects of improving yield, good industrial application value and prospect.

Active Publication Date: 2016-08-31
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Saccharomyces cerevisiae produces a large amount of ethanol by batch fermentation under the conditions of high concentration of sugar and ventilation. For the target product of carboxylic acid, the accumulation of ethanol causes a large loss of carbon flow, and Saccharomyces cerevisiae itself does not have carboxylic acid synthetic pathway

Method used

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  • Enzymatic activity improved pyruvate carboxylase mutant N1078F and application thereof
  • Enzymatic activity improved pyruvate carboxylase mutant N1078F and application thereof
  • Enzymatic activity improved pyruvate carboxylase mutant N1078F and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Example 1: Localization of RoPYC in Saccharomyces cerevisiae

[0028] 1. TEF1 promoter and RoPYC gene ORF were cloned into pGFP33 vector

[0029] 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.

[0030] Table 1 Primers for amplifying RoPYC gene

[0031]

[0032] Using pY15TEF1-RoPYC as a template (Xu et al. Fumaric acid production in Saccharomyces cerevisiaeby in silico aided metabolic engineering, 2012), RoPYC-F, RoPYC-R (sequences are shown in SEQ ID NO.3, SEQ ID NO.4 respectively ) for primers to amplify the ORF frame of TEF1p and RoPYC (the obtained PCR product contains the sequence of the amino acid sequence SEQ ID NO.1), and the amplified product is connected to the pGFP33 vector (a low-copy expression with green fluorescent protein GFP carrier, using a fluoresc...

Embodiment 2

[0037] Example 2: RoPYC site-directed mutation, expression and production of fumaric acid

[0038] Site-directed mutagenesis was performed by PCR method, and the pY15TEF1-RoPYC plasmid was used as a template, F and R containing the mutation site were used as primers, and the high-fidelity enzyme PrimeSTAR GXL of Takara Company was used to perform PCR to amplify the entire plasmid. 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.

[0039] Among them, the primers F(Asn) and R(Asn) (sequences shown in SEQ ID NO.5 and SEQ ID NO.6 respectively) used for N1078F mutation are shown in Table 2.

[0040] Table 2 Primers for s...

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Abstract

The invention discloses an enzymatic activity improved pyruvate carboxylase mutant N1078F and an application thereof and belongs to the fields of genetic engineering and fermentative engineering. According to the mutant N1078F and the application thereof, a locus N1078 of pyruvate carboxylase of Rhizopus oryzae is mutated into phenylalanine, so that the enzymatic activity of the obtained mutant is improved by 16.5%. A gene FUM1 is knocked out on the basis of knocking out PDC1 and ADH1, and meanwhile, the pyruvate carboxylase mutant N1078F is excessive, so that the yield of fumaric acid is increased by 20.5%. According to the mutant N1078F and the application thereof, a way of synthesis of carbon metabolic flux into fumaric acid from pyruvic acid is effectively strengthened, and conditions for constructing engineering yeasts and efficiently producing fumaric acid and other dicarboxylic acids are created, so that the mutant N1078F has very good industrial application values and prospects.

Description

technical field [0001] The invention relates to a pyruvate carboxylase mutant N1078F 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); Able to tolerate high concentrations of substrates; certified by the FDA as GRAS (General Regarded As Safe) 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 fermenta...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/52C12N1/19C12P7/46C12R1/865
CPCC12N9/93C12P7/46C12Y604/01001
Inventor 徐国强蒋伶活刘洋李佳雨刘维瑾
Owner JIANGNAN UNIV
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