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Saccharomyces cerevisiae genetic engineering strain for producing fumaric acid as well as construction method and application thereof

A technology of genetically engineered bacteria and Saccharomyces cerevisiae, applied in the direction of genetic engineering, microorganism-based methods, applications, etc., to achieve the effects of simple construction methods, good application prospects, and easy use

Inactive Publication Date: 2011-04-27
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Metabolic engineering of Saccharomyces cerevisiae to produce fumaric acid has not been reported at home and abroad

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Saccharomyces cerevisiae engineering bacteria producing fumaric acid

[0020] An engineered strain of Saccharomyces cerevisiae that produces fumaric acid, carrying the malate dehydrogenase gene (RoMDH) sequence as shown in SEQ ID: 1 and the fumarase gene (RoFUM1), the sequence as shown in SEQ ID: 2

Embodiment 2

[0021] Example 2 Construction method of Saccharomyces cerevisiae engineering bacteria producing fumaric acid

[0022] 1. Strains and plasmids

[0023] Rhizopus oryzae (R. oryzae NRRL 1526) was purchased from the American Type Culture Collection (ATCC); Saccharomyces cerevisiae (S. cerevisiae BMA64) was purchased from EUROSCARF in Europe, and its genotype was MAT a / α ura 3-1 ade 2- 1 leu 2-3, 112 his3-11, 15 trp1D can 1-100; Saccharomyces cerevisiae bidirectional expression plasmid pY26TEF / GPD (catalog number YV8124) was purchased from Deerbao Biotechnology Co., Ltd.

[0024] 2. Gene cloning

[0025] The total RNA of Rhizopus oryzae (R. oryzae NRRL 1526) was extracted, the mature malate dehydrogenase gene RoMDH and fumarase gene RoFUM1 were obtained by RT-PCR, and cloned into the expression plasmid pY26TEF / GPD (catalog number YV8124). The sequencing results were submitted to Genbank and published. The number of RoMDH is HM130702.1 and the number of RoFUM1 is HM130701.1. The primers ...

Embodiment 3

[0028] Example 3 Fermentation to produce fumaric acid

[0029] The genetically engineered bacteria seeds cultured at 30°C and 200 rpm for 24 h were transferred to the fermentation medium at 5% inoculum and cultured at 30°C and 200 rpm for 60 h. Different concentrations of Leu and Trp were added to the medium according to the experimental requirements. , Ade, His.

[0030] SC medium: Difco Yeast Nitrogen Base w / o amino acids 1.7g / L, (NH4)2SO4 5 g / L, Glucose 20g / L. Add Leu 100 mg / L, Trp 20 mg / L, Ade 20 mg / L, His 20 mg / L, Ura 20 mg / L as required.

[0031] Seed medium: SC medium, with a liquid volume of 20ml / bottle.

[0032] Fermentation medium: SC medium, calcium carbonate added, 5g / L, and the liquid volume is 50ml / bottle.

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Abstract

The invention discloses a Saccharomyces cerevisiae genetic engineering strain for producing fumaric acid as well as a construction method and application thereof, belonging to the field of genetic engineering. The construction method provided by the invention comprises the following steps: cloning key genes containing dehydrogenase genes (RoMDH) and fumarase genes (RoFUM1) in the accumulation path of the fumaric acid from the Rhizopus oryzae NRRL1526 capable of accumulating a large amount of fumaric acid, and converting the Saccharomyces cerevisiae BMA64 by connecting to the plasmid pY26TEF / GPD capable of realizing high-copy two-way expression in the Saccharomyces cerevisiae to obtain the Saccharomyces cerevisiae genetic engineering strain for producing fumaric acid. The Saccharomyces cerevisiae genetic engineering strain disclosed by the invention can accumulate the fumaric acid in the cytoplasm, the fermentation method is simple, the yield can reach 26.2mg / L, and the invention establishes a basis for industrialization of Saccharomyces cerevisiae for producing fumaric acid in metabolic engineering construction.

Description

technical field [0001] The invention relates to a Saccharomyces cerevisiae gene engineering bacterium producing fumaric acid, a construction method and application thereof, and belongs to the field of genetic engineering. Background technique [0002] Fumaric acid, also known as fumaric acid (IUPAC name fumaric acid), is the simplest unsaturated dicarboxylic acid. It is widely used in industry, medicine, food and animal husbandry. In 2004, the U.S. Federal Department of Energy established a 1,4-dicarboxylic acid group, mainly researching fumaric acid, malic acid and succinic acid, and these three organic acids are contained in the most eye-catching renewable ten The two major chemical structural elements were also listed as C4 compounds that should be developed in the development report of the Organization for Economic Cooperation and Development (OCED) in the same year. [0003] Petrochemical routes are currently the main production method of fumaric acid, but with the co...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/52C12N15/53C12N15/63C12P7/46C12R1/865
Inventor 刘立明徐国强段宁骏庄洪波陈坚
Owner JIANGNAN UNIV
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