Construction method for producing 1,3-trimethylene glycol regrouping saccharomyces cerevisiae with glucose as substrate

A technology for recombining Saccharomyces cerevisiae and propylene glycol, applied in the field of genetic engineering, can solve the problems of increasing fermentation cost and high price

Inactive Publication Date: 2008-02-27
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Increased fermentation costs due to the high price of glycerol compared to other cheap carbon sources such as glucose

Method used

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  • Construction method for producing 1,3-trimethylene glycol regrouping saccharomyces cerevisiae with glucose as substrate
  • Construction method for producing 1,3-trimethylene glycol regrouping saccharomyces cerevisiae with glucose as substrate
  • Construction method for producing 1,3-trimethylene glycol regrouping saccharomyces cerevisiae with glucose as substrate

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1: Construction of plasmid PGAPZB-yqhD:

[0058] EcoR I cuts the vector pHsh-dhaB-yqhD, and releases the 1,3-propanediol oxidoreductase isoenzyme coding gene yqhD with a size of about 1.2kb, which is purified with a PCR fragment gel recovery kit; extracts the plasmid PGAPZB, and uses EcoR I digest the vector PGAPZB, purify by agarose gel electrophoresis, reclaim the 2.9kb target fragment with the PCR fragment recovery kit, and use T 4 Ligase ligated yqhD / EcoR I and PGAPZB / EcoR I, and the ligation product was transformed into Escherichia coli JM109 competent cells, and the transformed product was spread on LB agar plate containing 25 μg / mL Zeocin resistance, and cultured overnight at 37°C; 5 colonies were randomly selected, Inoculate in 20 mL of LB medium containing 25 μg / mL Zeocin resistance and culture overnight at 37°C, extract plasmids, digest with EcoR I and Bgl I respectively, identify positive clones by agarose gel electrophoresis, and store positive re...

Embodiment 2

[0059] Embodiment 2: Construction of plasmid PGAPZB-dhaB:

[0060] The vector pHsh-dhaB-yqhD was digested with Xba I to release the glycerol dehydratase coding gene dhaB with a size of about 2.7kb, which was purified with a PCR fragment gel recovery kit; the plasmid PGAPZB was extracted, the vector PGAPZB was digested with Xba I, and gelatinized in agarose Purified by gel electrophoresis, recovered a 2.9kb target fragment with a PCR fragment recovery kit, and used T 4 Ligase ligated dhaB / Xba I and PGAPZB / Xba I, and the ligation product was transformed into Escherichia coli JM109 competent cells, and the transformed product was spread on LB agar plates containing 25 μg / mL Zeocin resistance, and cultured overnight at 37°C; 5 colonies were randomly selected , inoculated in 20mL LB medium containing 25μg / mL Zeocin resistance and cultured overnight at 37°C, extracted plasmids, digested with Xba I and BamH I respectively, and identified positive clones by agarose gel electrophoresis...

Embodiment 3

[0061] Embodiment 3: Construction of plasmid pYX212-zeocin:

[0062] Using the vector PGAPZB as a template, the resistance gene zeocin with a size of 1.2kb was amplified by PCR, and purified with a PCR fragment gel recovery kit; the plasmid pYX212 was extracted, and the vector pYX212 was digested with EcoR I, purified by agarose gel electrophoresis, and purified with PCR fragments The recovery kit recovers the target fragment of 8.3kb, using T 4 Ligase zeocin / EcoR I and pYX212 / EcoR I, and the ligation product was transformed into Escherichia coli JM109 competent cells, and the transformed product was spread on LB agar plate containing 25 μg / mL Zeocin resistance, cultured overnight at 37°C; 5 colonies were randomly selected , inoculated in 20mL LB medium containing 25μg / mL Zeocin resistance and cultured overnight at 37°C, extracted plasmids, digested with EcoR I respectively, identified positive clones by agarose gel electrophoresis, and stored positive recombinants in a medium...

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Abstract

The invention discloses a constructing method for producing 1, 3-methyl ethylene glycol recombinant Saccharomyces cerevisiae with glucose as substrate in the gene engineering technique field. The invention constructs recombinant plasmid pGAPZB-yqhD, pGAPZB-dhaB and the vector pYX212-zeocin firstly, which constructs corpus expression vector pYX212-zeocin-pGAP-yqhD-pGAP-dhaB of Saccharomyces cerevisiae and expresses in Saccharomyces cerevisiae W303-1A. The recombinant Saccharomyces cerevisiae applies glucose as the substrate, and the content of 1, 3-methyl ethylene is about 1. 5g / l after fermenting 70 hours. The invention achieves the expression of yqhD and dhaB in series successfully in Saccharomyces cerevisiae, which creates the basis for constructing gene engineering bacterial with high-producing 1, 3-methyl ethylene with glucose as the substrate.

Description

technical field [0001] The invention relates to a construction method of a recombinant brewer's yeast producing 1,3-propanediol with glucose as a substrate, which belongs to the technical field of genetic engineering. Background technique [0002] 1,3-Propanediol (1,3-Propanediol, 1,3-PD) is an important chemical raw material, used as an intermediate in medicine and organic synthesis for industries such as food, cosmetics and pharmaceuticals, 1,3-Propanediol is the most An important use is as a monomer for the synthesis of new polyesters such as poly-1,3-trimethylene terephthalate (PTT). The production methods of 1,3-propanediol mainly include chemical synthesis and microbial transformation. At present, only the chemical synthesis method has achieved industrialization, but the chemical synthesis of 1,3-propanediol requires a large investment in equipment, high technical difficulty, and difficulty in product separation and purification; the production of 1,3-propanediol by m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19C12N15/53C12N15/60C12P7/18C12R1/865
Inventor 饶志明马正沈微诸葛斌方慧英诸葛健
Owner JIANGNAN UNIV
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