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Method for improving yield of fumaric acid

A technology of arginine and proline, which is applied in the fields of genetic engineering and fermentation engineering, can solve problems such as carbon flow loss, and achieve the effects of increasing production, good industrial application value and prospects

Inactive Publication Date: 2016-07-13
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the batch fermentation of Saccharomyces cerevisiae under the conditions of high concentration of sugar and aeration will produce a large amount of ethanol. For the fermentation with carboxylic acid as the target product, the large accumulation of ethanol will cause a large loss of carbon flow

Method used

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  • Method for improving yield of fumaric acid
  • Method for improving yield of fumaric acid
  • Method for improving yield of fumaric acid

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Experimental program
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Effect test

Embodiment 1

[0030] Codon optimization of embodiment 1 RoPYC (pyruvate carboxylase from Rhizopus oryzae) gene and expression in Saccharomyces cerevisiae

[0031] 1. Gene acquisition after RoPYC codon optimization

[0032] Submit the RoPYC nucleotide sequence derived from Rhizopus oryzae to the sequence box of the codon optimization website (http: / / www.jcat.de / ), and then select the expression host as Saccharomyces cerevisiae, and the codon can be obtained after submission For the optimized gene sequence, the gene corresponding to this sequence is named RoPYC*.

[0033] The sequence of RoPYC* was sent to Shanghai Bioengineering Co., Ltd. for gene synthesis, and the synthesized gene was connected to the T vector. On this basis, we carried out PCR amplification using RoPYC*+T carrier as a template, and the primers used are shown in Table 1.

[0034] Table 1 Amplifies the primers of RoPYC* gene

[0035]

[0036]The obtained PCR product was double-digested with SpeI and SalI, and then lig...

Embodiment 2

[0039] Example 2 RoPYC* site-directed mutation and expression

[0040] Site-directed mutation was performed by PCR method, and the N315F, R485P, and N1078F sites of RoPYC* were subjected to site-directed mutation. The pY15TEF1-RoPYC plasmid was used as a template, F and R containing mutation sites were used as primers, and Takara's high-fidelity enzyme PrimeSTARGXL was used for PCR amplification. Entire plasmids are amplified. The enzyme digestion system contains 1 μL of PCR product and 1 μL of DpnI 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.

[0041] The primers F(Pro) and R(Pro) (sequences shown in SEQ ID NO.5 and SEQ ID NO.6 respectively) used for RoPYC*R485P mutation are shown in ...

Embodiment 3

[0046] The feeding of embodiment 3 carbon dioxide is to the influence of fumaric acid fermentation

[0047] On the level of 7L fermenter, the influence of the introduction of carbon dioxide on the fermentation of fumaric acid was investigated, and the results are as follows: image 3 shown.

[0048] Culture conditions: Transfer the seeds of genetically engineered bacteria cultured at 30°C and 220rpm for 24 hours into the fermentation medium to make the initial OD 600 = 0.2, and cultured at 30°C for 96 hours, the aeration rate was 1vvm, the stirring speed was 300rpm, and when the fermentation was carried out for 36 hours, 0.1vvm of carbon dioxide was introduced.

[0049] Depend on image 3 It can be seen that after the fermentation was carried out for 36 hours, after the carbon dioxide of 0.1vvm was introduced, compared with the control (no carbon dioxide was introduced during the whole fermentation process), the output of fumaric acid gradually increased, and when the fermen...

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Abstract

The invention discloses a method for improving the yield of fumaric acid and belongs to the fields of genetic engineering and fermentation engineering. The method comprises the following steps: performing codon optimization on a pyruvate carboxylase gene RoPYC of rhizopus oryzae to obtain a gene RoPYC* and then mutating a R485 site of the gene RoPYC* into proline. In comparison with the yield after the expression of a wild type pyruvate carboxylase before codon optimization, the yield is improved by 24.6 percent; 10 percent of carbon dioxide is additionally introduced when genetically-engineered bacteria of an expression mutant is fermented for 36 hours in a fermentation tank of 7L, so that the yield of the fumaric acid reaches 453.6 mg / Lm and is improved by 20 percent through comparison. According to the method disclosed by the invention, a synthetic route through which a carbon metabolic flow enters the fumaric acid from pyruvic acid is effectively enhanced, so that conditions are created for the construction of engineering yeasts to efficiently produce the fumaric acid and other dicarboxylic acid, therefore, the industrial application value and the prospect are good.

Description

technical field [0001] The invention relates to a method for increasing the yield of fumaric acid, 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 operation of metabolic transformation; simple nutritional requirements, low cost of separation and extraction process; good growth under low pH conditions (even pH<3.0); Tolerant to 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-ketoglutadiene Potentially optimal microorganisms for acid, etc.). However, batch fermentation of Saccharomyces cerevisiae under conditions of high sugar concentration and aeration will produc...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/52C12N1/19C12P7/46C12P7/50C12R1/865
CPCC12N9/93C12P7/46C12P7/50C12Y604/01001
Inventor 徐国强蒋伶活刘洋李佳雨刘维瑾
Owner JIANGNAN UNIV
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