Saccharomyces cerevisiae gene engineering bacteria for producing succinic acid and application thereof

A technology of genetically engineered bacteria and Saccharomyces cerevisiae, applied in the field of Saccharomyces cerevisiae genetically engineered bacteria, can solve the problems of difficulty in accumulation and loss of carbon flow, and achieve the effect of achieving accumulation and reducing the loss of carbon flow

Active Publication Date: 2016-08-10
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of Saccharomyces cerevisiae to ferment and produce succinic acid faces the following problems: (1) Batch fermentation produces a large amount of ethanol under conditions of high concentration of sugar and ventilation. (2) Succinic acid is an intermediate metabolite in the TCA cycle. Saccharomyces cerevisiae itself does not have a metabolic pathway for excessive accumulation of succinic acid. It is necessary to construct a synthetic pathway for succinic acid, but succinic acid has a relatively high reducing p...

Method used

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  • Saccharomyces cerevisiae gene engineering bacteria for producing succinic acid and application thereof
  • Saccharomyces cerevisiae gene engineering bacteria for producing succinic acid and application thereof
  • Saccharomyces cerevisiae gene engineering bacteria for producing succinic acid and application thereof

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Embodiment 1

[0028] Example 1 Construction of low-yield ethanol and accumulation of succinic acid Saccharomyces cerevisiae engineering bacteria

[0029] On the basis of the wild type, the pyruvate decarboxylase gene PDC1 was replaced with the malate dehydrogenase gene RoMDH from Rhizopus oryzae. On this basis, the key enzyme gene SDH2 in the succinate metabolic pathway was knocked out.

[0030] 1. Strains and plasmids

[0031] Saccharomyces cerevisiae (S.cerevisiae) CEN.PK2-1C was purchased from EUROSCARF (http: / / web.uni-frankfurt.de / fb15 / mikro / euroscarf / data / cen.html), and its genotype was MATa; ura3- 52; trp1-289; leu2-3,112; his3Δ1; MAL2-8 C;SUC2. For the construction method of knockout cassette template pUG27 plasmid (containing HIS3 marker gene) and Cre expression plasmid pSH47 (used for marker recovery), please refer to the literature "Guldener U, Heck S, Fielder T, et al. A new efficient gene disruption cassette for repeated use in budding yeast[J].Nucleic Acids Res,1996,24(13):...

Embodiment 2

[0038] Embodiment 2 fermentation method produces succinic acid

[0039] The seeds of genetically engineered bacteria cultured at 30°C and 220rpm for 24 hours were used to start the OD 600 The inoculum size of =0.2 was transferred to the fermentation culture based on the conditions of 30°C and 220rpm for 96h, and samples were taken at regular intervals to measure OD, and 1mL samples were taken at the same time and centrifuged for storage to measure the production of ethanol and succinic acid.

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Abstract

The invention discloses saccharomyces cerevisiae gene engineering bacteria for producing succinic acid and application thereof, and belongs to the fields of genetic engineering and fermentation engineering. Malate dehydrogenase gene RoMDH from rhizopus oryzae is adopted to replace PDC1, the yield of ethanol obtained by fermentation of wild saccharomyces cerevisiae reaches the maximum value 13.55 plus or minus 1.062g/L, and the yield of improved strain MP ethanol is 11.09 plus or minus 0.539g/L, and compared with the yield obtained by adopting the wild type, the yield is reduced by 18.15 percent. On the basis, SDH2 gene is knocked out. After culture medium optimization, MP Delta S deleted strain can accumulate succinic acid by 0.698 plus or minus 0.0285g/L, and by contrast, the wild saccharomyces cerevisiae does not accumulate succinic acid. The saccharomyces cerevisiae gene engineering bacteria can effectively reduce the loss of a carbon flow, creates a condition for the engineering yeast to efficiently produce succinic acid, and has a good industrial application value and prospect.

Description

technical field [0001] The invention relates to a succinic acid-producing Saccharomyces cerevisiae genetically engineered bacterium and an application thereof, belonging to the fields of genetic engineering and fermentation engineering. Background technique [0002] Succinic acid is a four-carbon dibasic acid with the scientific name butanedioic acid. Succinic acid has more than 30 important derivative chemicals: tetrahydrofuran, 1,4-butanediol, γ-butyrolactone, maleic acid and fumaric acid, etc., with huge market space. The main sour agent and flavoring agent in the food industry, can be used in the synthesis of sedatives, pH regulators, contraceptives, anticancer drugs, etc. , Foaming agent and lubricant etc. It is worth mentioning that, as the main raw material of biodegradable plastic polybutylene succinate (completely degradable), succinic acid has broad application prospects. In view of the above-mentioned important application value and many advantages of succinic ...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12P7/56C12P7/50C12P7/46C12R1/865
CPCC12N9/0006C12N9/001C12N9/88C12N15/81C12N2800/102C12P7/46C12P7/50C12P7/56C12Y101/01037C12Y103/05001C12Y401/01001
Inventor 徐国强蒋伶活苏珂李佳雨刘维瑾范欧洋赵祯
Owner JIANGNAN UNIV
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