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A Saccharomyces cerevisiae strain expressing xylose isomerase and its construction method

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

Active Publication Date: 2020-10-16
SINOPEC SHANGHAI ENG +2
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  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • A Saccharomyces cerevisiae strain expressing xylose isomerase and its construction method
  • A Saccharomyces cerevisiae strain expressing xylose isomerase and its construction method
  • A Saccharomyces cerevisiae strain expressing xylose isomerase and its construction method

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Embodiment 1

[0031] A recombinant industrial Saccharomyces cerevisiae strain expressing xylose isomerase, said Saccharomyces cerevisiae SEB8 strain is preserved in China General Microorganism Culture Collection and Management Center, and the preservation 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 sporulation medium (0.5g / L glucose, 20g / L potassium acetate, 2g / L yeast powder, pH5.5) for 2 days, and treated with lysozyme at 30°C for 10-20min, Single spores were picked by a micro operating system, and haploid cells were obtained after verification. The present invention only selects the haploid SEB3α25 with sex α, and screens out the haploid strain SEB3α25.

[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 by the lithiu...

Embodiment 2

[0053] In this embodiment, the fermentation and enzyme activity of the bacterial strain SEB8 were measured, wherein the fermentation method was the same as that in the above step 4), and will not be repeated here.

[0054] Determination of enzyme activity: Take 2 mL of the fermentation broth after 24 hours of fermentation, and collect the bacteria by centrifugation at 4°C. After washing the cells with 1 mL of TEA buffer for 3 times, suspend the cells with 1 mL of TEA buffer, transfer the cell suspension into a broken tube containing 0.5 g of glass beads with a diameter of 0.5 mm, and add the final solution to the broken tube. Concentrations were 1 mM of PMSF and 0.5 mM of DTT. Then, it was crushed on the crusher at 4500rpm for 30s, then placed on ice for 2min, repeated crushing in this way 5 times, and then centrifuged at 12000g for 15min at 4°C, and the supernatant was taken, which was the crude protein solution. The total protein concentration was determined by the Brabende...

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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 construction method of the strain. Background technique [0002] The production of fuel ethanol from lignocellulose 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 has been widely used in the production of first-generation fuel ethanol because of its fast sugar metabolism, high ethanol yield, and good environmental tolerance. However, in the sugar solution obtained from lignocellulose hydrolysis, about 20%–30% of the sugar is xylose, and wild-type Saccharomyces cerevisiae cannot directly use xylose for growth and fermentation, but Saccharomyces cerevisiae itself can express xylulokinase (Xylulose kinase, XK), which can phosphorylate xylulose to ge...

Claims

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Application Information

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Patent Type & Authority Patents(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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