Saccharomyces cerevisiae strain expressing xylose isomerase and construction method of saccharomyces cerevisiae strain

A technology of Saccharomyces cerevisiae strain and xylose isomerase, applied in the direction of isomerase, microorganism-based method, method using microorganism, etc., can solve the problems of unable to meet the fermentation requirements, slow growth rate, etc., and achieve high ethanol yield , fast fermentation of xylose, and the effect of reducing production costs

Active Publication Date: 2017-02-22
SINOPEC SHANGHAI ENG +2
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Problems solved by technology

[0005] The object of the present invention is to solve the problem that after the existing xylA gene is heterologously expressed in Saccharomyces cerevisiae, the growth rate of the recombinant bacteria constructed by it is relatively slow in xylose, which cannot meet the needs of fermentation; a strain based on Flocculating industrial Saccharomyces cerevisiae capable of efficiently fermenting xylose to ethanol through the xylose isomerase pathway

Method used

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  • Saccharomyces cerevisiae strain expressing xylose isomerase and construction method of saccharomyces cerevisiae strain
  • Saccharomyces cerevisiae strain expressing xylose isomerase and construction method of saccharomyces cerevisiae strain
  • Saccharomyces cerevisiae strain expressing xylose isomerase and construction method of saccharomyces cerevisiae strain

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Embodiment 1

[0031] A recombinant industrial Saccharomyces cerevisiae strain expressing xylose isomerase, the Saccharomyces cerevisiae SEB8 strain is deposited in the China Common Microbial Species Collection Management Center, and the deposit number is CGMCC11328. The specific construction method of the Saccharomyces cerevisiae strain SEB8 is as follows:

[0032] 1) Construct the starting strain:

[0033] Saccharomyces cerevisiae SEB3 was cultured on a sporulation medium (0.5g / L glucose, 20g / L potassium acetate, 2g / L yeast powder, pH 5.5) for 2 days, and then treated with lysozyme at 30°C for 10-20 minutes. Single spores were picked using the micromanipulation system, and haploid cells were obtained after verification. In the present invention, only the haploid SEB3α25 with sex α is selected, and the haploid strain SEB3α25 is screened out.

[0034] Using the plasmid pBlu-LTKTL-TDH3 as a template, the loxP-KanMX-loxP fragment was amplified using primers Fg3 / Rg3, and then introduced into SEB3α25...

Embodiment 2

[0053] In this example, the fermentation condition and enzyme activity of the strain SEB8 were determined, and the fermentation method is the same as the method in step 4) above, and will not be repeated here.

[0054] Enzyme activity determination: Take 2 mL of the fermentation broth after 24 hours of fermentation, and collect the bacteria by centrifugation at 4°C. After washing the bacteria with 1 mL of TEA buffer three times, suspend the bacteria with 1 mL of TEA buffer, transfer the bacterial suspension into a crushing tube containing 0.5 g glass beads with a diameter of 0.5 mm, and add the final tube to the crushing tube. The concentration is 1 mM PMSF and 0.5 mM DTT. Then it was crushed on a crusher at 4500 rpm for 30 seconds and placed on ice for 2 minutes. The crushing was repeated 5 times, and then centrifuged at 12000g for 15 minutes at 4°C, and the supernatant was taken, which is the crude protein solution. The Brabender method was used to determine the total protein ...

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Abstract

The invention discloses a saccharomyces cerevisiae strain expressing xylose isomerase and a construction method. The saccharomyces cerevisiae (SEB8) strain expressing the xylose isomerase is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC11328. The invention further provides the construction method of the saccharomyces cerevisiae (SEB8). The obtained strain (SEB8) can generate ethanol with the concentration of 14.73 g/L from xylose with the concentration of 33.71 g/L in 48 hours of fermentation time, and the ethanol yield reaches 0.44 gram per gram of the xylose. The strain has the advantages of being high in xylose fermentation speed and ethanol yield and the like, can produce the fuel ethanol from saccharification liquid prepared by taking lignocellulose as a raw material, greatly reduces the production cost of the fuel ethanol and has a wide application prospect.

Description

Technical field [0001] The invention relates to a recombinant Saccharomyces cerevisiae strain, in particular to a Saccharomyces cerevisiae strain expressing xylose isomerase and a method for constructing the strain. Background technique [0002] The production of fuel ethanol with lignocellulose as a raw material is regarded as a promising way of obtaining bioenergy in the future, mainly because lignocellulose has the advantages of wide sources, large reserves, low price, and renewable. Saccharomyces cerevisiae (Saccharomyces cerevisiae) has been widely used in the production of first-generation fuel ethanol because of its fast sugar metabolism, high ethanol production, and good environmental tolerance. However, in the sugar solution obtained by hydrolysis of lignocellulose, about 20%-30% of the sugar is xylose. Wild-type Saccharomyces cerevisiae cannot directly use xylose for growth and fermentation, but Saccharomyces cerevisiae itself can express xylulose kinase. (Xylulose kin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12N1/36C12P7/10C12P7/06C12R1/865
CPCC12N1/36C12N9/92C12N15/81C12P7/06C12P7/10C12Y503/01005Y02E50/10
Inventor 汤岳琴李云成缪晡丁伟军陈栋
Owner SINOPEC SHANGHAI ENG
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