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Pyruvic acid producing Saccharomyces cerevisiae gene engineering bacteria and construction method and use thereof

A technology of genetically engineered bacteria and Saccharomyces cerevisiae, applied in microorganism-based methods, biochemical equipment and methods, fermentation and other directions, can solve problems such as loss of cofactors, inability to accumulate pyruvic acid, etc., achieving a simple construction method, good application prospects, Ease of use

Inactive Publication Date: 2011-10-19
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the production of ethanol leads to the loss of a large amount of carbon metabolic flux and cofactors such as NAD+, resulting in the inability of S. cerevisiae to accumulate a large amount of pyruvate in the cell

Method used

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  • Pyruvic acid producing Saccharomyces cerevisiae gene engineering bacteria and construction method and use thereof
  • Pyruvic acid producing Saccharomyces cerevisiae gene engineering bacteria and construction method and use thereof
  • Pyruvic acid producing Saccharomyces cerevisiae gene engineering bacteria and construction method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Producing Saccharomyces cerevisiae Engineering Bacteria of Pyruvate

[0023] A pyruvate-producing Saccharomyces cerevisiae engineering strain, the regulatory gene THI2 in the thiamine synthesis pathway was knocked out.

Embodiment 2

[0024] Example 2 The construction method of the Saccharomyces cerevisiae engineering bacteria producing pyruvate

[0025] 1. Strains and plasmids

[0026] Saccharomyces cerevisiae (S.cerevisiae CEN.PK2-1C) was purchased from EUROSCARF in Europe, and its genotype was MATa ura3-52leu2-3, 112trp1-289 his3ΔMAL2-8c SUC2; plasmid pUG27 was constructed by our laboratory; plasmid pSH47 was donated by Germany.

[0027] 2. Construction of the knockout box

[0028] Plasmid pUG27 was extracted, and the plasmid was diluted 25 times as a template. The primers used to construct the knockout frame were F1:ACCACGTATATATAGCCTATATATATCCGCACTAGAACCAA-CAGCTGAAGCTTCGTACGC.R1:TGGCTTTTTTTTTCTTGAAATGAGTGAAGGGAAGGCTCAATAAGC-GCATAGGCCACTAGTGGATCTG. The obtained PCR product was purified and used to transform Saccharomyces cerevisiae.

[0029] 3. Conversion and verification

[0030] The constructed knockout frame was transformed into Saccharomyces cerevisiae (S.cerevisiae CEN.PK2-1C), verified by PCR of...

Embodiment 3

[0031] Embodiment 3 fermentation produces fumaric acid

[0032] The seeds of genetically engineered bacteria cultivated at 30°C and 200rpm for 12h were transferred to fermentation culture at an inoculum of 5%. Based on the culture at 30°C and 200rpm for 60h, different concentrations of Leu, Trp, Ura, and His were added to the medium according to the experimental requirements. . Seed culture medium: yeast extract-peptone-dextrose (YPD) medium medium, the filling volume is 20ml / 250ml. Fermentation medium (g liter-1): glucose 60, NH4Cl 7, KH2PO45, MgSO4 7H2O 0.8, trace element 10ml, pH=5.0, sterilized at 115oC for 20min, liquid volume 50ml / 250ml, CaCO340, VB1 0.04μM alone Sterilized, additionally added.

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Abstract

The invention discloses pyruvic acid producing Saccharomyces cerevisiae gene engineering bacteria and a construction method and use thereof and belongs to the field of genetic engineering. In the invention, by knocking out a key regulation gene THI2 in the thiamine synthesis process of Saccharomyces cerevisiae CEN.PK2-1C, the anabolism of the thiamine is interfered to lower the enzymatic activity of a key enzyme for the metabolism of pyruvic acid, so that a carbon metabolic flow to ethanol is reduced and the accumulation of pyruvic acid is promoted. Under a condition of limited addition of thiamine, the Saccharomyces cerevisiae gene engineering bacteria can accumulate pyruvic acid, the method is simple, the yield can reach 6.77+ / -0.14g / L, and thus, a foundation is laid for researching the production of C4 dicarboxylic acid by metabolic engineering modified Saccharomyces cerevisiae.

Description

technical field [0001] The invention relates to a pyruvate-producing Saccharomyces cerevisiae genetically engineered bacterium, a construction method and application thereof, and belongs to the field of genetic engineering. Background technique [0002] The pyruvate dehydrogenase bypass metabolism in S. cerevisiae has important physiological functions, including: 1) promoting the regeneration of NAD+ and maintaining intracellular redox balance; 2) synthesizing cytosolic acetyl-CoA, which is used for the synthesis of cell components and amino acids Metabolism provides precursors; 3) produces ethanol. However, the production of ethanol leads to the loss of a large amount of carbon metabolic flux and cofactors such as NAD+, so that S.cerevisiae cannot accumulate a large amount of pyruvate in the cell. The key enzymes in the metabolic bypass of pyruvate dehydrogenase system are pyruvate decarboxylase (pyruvate decarboxylase) and alcohol dehydrogenase (alcohol dehydrogenase). A...

Claims

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Application Information

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IPC IPC(8): C12N1/18C12N15/09C12P7/40C12R1/865
Inventor 刘立明徐国强陈坚
Owner JIANGNAN UNIV
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