Construction method for producing fumaric acid based on pichia stipitis synthetic strain fermented xylose

A technology of Pichia stipitis, fumaric acid, applied in the fields of synthetic biology and bioenergy

Inactive Publication Date: 2015-06-10
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the use of codon-optimized fumaric acid reductive synthesis pathway to ferment and

Method used

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  • Construction method for producing fumaric acid based on pichia stipitis synthetic strain fermented xylose
  • Construction method for producing fumaric acid based on pichia stipitis synthetic strain fermented xylose
  • Construction method for producing fumaric acid based on pichia stipitis synthetic strain fermented xylose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Construction of uracil and leucine auxotrophs

[0033] (1) Using the S. stipitis genome as a template, using primers URA3-5F / URA3-5R, URA3-3F / URA3-3R, the sequences of which are SEQ ID NO: 28 and SEQ ID NO: 29, PCR amplified uracil respectively The upstream and downstream homologous fragments URAF and URAR of the key gene URA3 gene were synthesized, and a homologous recombination knockout cassette was obtained by fusion PCR. Homologous integration in yeast knocked out URA3, screened uracil-deficient strains on a screening medium containing 5-FOA, and finally obtained uracil-auxotrophic strains were verified by PCR.

[0034] (2) Using the S. stipitis genome as a template, using primers LEU2-5F / LEU2-5R, LEU2-3F / LEU2-3R, the sequences of which are SEQ ID NO: 31 and SEQ ID NO: 32, respectively PCR amplified to obtain LEU2 The upstream and downstream homologous fragments LEU2F and LEU2R of the gene are connected with URA3 with the loxP site sequence by fusion PCR ...

Embodiment 2

[0037] The knockout of embodiment 2 fumarase gene

[0038] (1) On the basis of uracil and leucine auxotrophic strains, using the S.stipitis genome as a template, design primers FUM1-5F / FUM1-5R, FUM1-3F / FUM1-3R, the sequence of which is SEQ ID NO : 35 and SEQ ID NO: 36, the upstream and downstream homologous fragments FUM1F and FUM1R of the PSfum1 gene were amplified by PCR respectively, and connected with URA3 with the loxP site sequence by fusion PCR to form a PSfum1 gene homologous recombination knockout box , PSfum1 was knocked out by homologous integration in yeast, and PSfum1-deficient strains were identified by screening on SD solid medium. The plasmid pSSH02 with Cre recombinase was transformed to remove the marker gene URA3, and a PSfum1-deficient strain was obtained. The obtained strains were verified by PCR detection.

[0039] (2) On the basis of the PSfum1-deficient strain obtained in (1), using the S. stipitis genome as a template, design primers FUM2-5F / FUM2-5R,...

Embodiment 3

[0043] The construction of embodiment 3 vector plasmids pYSS01, pBSS01 and pSSH02

[0044] (1) Extract the S.stipitis CBS6054 genome template

[0045] S.stipitis CBS6054 was cultured in YPD medium (20-25g / L peptone, 10-20g / L yeast powder, 20-25g / L glucose) at 30°C and 150-200rpm for 40-48 hours, then the genome extraction kit ( Tiangen) to extract total DNA.

[0046] (2) Using the S.stipitis CBS6054 genome as a template to design primers URA3-F / URA3-R, its sequence is SEQ ID NO: 17, using PCR to obtain gene URA3, its sequence is SEQ ID NO: 11, using ApaI and NdeI double After digestion, it was connected to the vector pY26TEF-GPD to obtain the plasmid pYSS; primers TDH3-1F / TDH3-1R and TEF1-1F / TEF1-1R were designed using the S. stipitis CBS6054 genome as a template, and its sequence is SEQ ID NO: 15 and SEQ ID NO: 16, the constitutive strong promoters TDH3p and TEF1p were amplified respectively, and the sequences thereof were SEQ ID NO: 7 and SEQ ID NO: 8. The fragment TDH3-T...

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Abstract

The invention provides a construction method for producing fumaric acid based on pichia stipitis synthetic strain fermented xylose. The method comprises the following steps: using pichia stipitis having a natural xylose metabolic capability as a host; deleting genes PSfum1 and PSfum2 that encode fumarase in tricarboxylic acid cycle of pichia stipitis, to acquire fumarase deficient mutants; heterologously expressing a modified fumaric reduction synthesis module on the basis of the acquired mutants, using xylose to ferment yeast synthesis strains of fumaric acid; on the basis of the yeast synthesis strains, expressing translocator YMAE that is modified with codons and originated from Chestnut wine fission yeasts, to acquire pichia stipitis synthesis strains for producing fumaric acid by fermented xylose; efficiently fermenting the acquired strains in a fully synthesized fermentation culture medium to produce fumaric acid by fermentation of xylose; cultivating by fermentation for 3-4 days at a temperature of 30 degrees centigrade with a stirring speed of 150-200rpm. The content of fumaric acid is greater than 4g/L.

Description

technical field [0001] The invention belongs to the technical field of synthetic biology and bioenergy, and specifically relates to a construction method for producing fumaric acid based on pichia stipitis synthetic strains fermenting xylose. Using the Pichia stipitis (Scheffersomyces stipitis CBS6054) as the host, the fumarase gene (PSfum1, PSfum2) was knocked out, and the codon-optimized fumaric acid reduction synthesis pathway from Rhizopus oryzae was expressed heterologously and codon-optimized The fumaric acid transporter YMAE from Schizosaccharomyces pombe, obtained a synthetic strain of Pichia stipitis, and a method for efficiently fermenting xylose to produce fumaric acid in a fermentation medium. Background technique [0002] Fumaric acid is a typical C 4 Dicarboxylic acid platform compounds are listed by the U.S. Department of Energy as one of the 12 priority platform compounds for development in the 21st century. They can be widely used in food, chemical, pharmac...

Claims

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Application Information

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IPC IPC(8): C12P7/46C12N15/81C12R1/84
CPCC07K14/39C12N9/0006C12N9/88C12P7/46C12Y101/01037C12Y401/01031C12Y402/01002
Inventor 闻建平魏亮刘蛟齐海山
Owner TIANJIN UNIV
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