Recombinant saccharomyces cerevisiae strain for efficiently co-fermenting glucose and xylose and application of recombinant saccharomyces cerevisiae strain

A Saccharomyces cerevisiae strain and co-fermentation technology, applied in the field of microorganisms, can solve the problems of natural Saccharomyces cerevisiae metabolizing xylose, etc., and achieve the effect of optimizing fermentation ability and improving xylose fermentation ability

Active Publication Date: 2015-12-30
SHANDONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, its real use in the production of second-generation fuel ethanol needs to solve two problems: the first is the problem of xylose metabolism. It can significantly reduce the production cost of second-generation fuel ethanol and improve its economic competitiveness. However, due to the lack of genes related to xylose metabolism, natural Saccharomyces cerevisiae cannot metabolize xylose very well; the second is the problem of inhibitors, lignocellulosic raw materials In the process of pretreatment and enzymatic hydrolysis, a large number of compounds that inhibit microbial growth and fermentation will be produced, so increasing the tolerance of Saccharomyces cerevisiae to these inhibitors can increase the production rate of ethanol in the fermentation process

Method used

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  • Recombinant saccharomyces cerevisiae strain for efficiently co-fermenting glucose and xylose and application of recombinant saccharomyces cerevisiae strain
  • Recombinant saccharomyces cerevisiae strain for efficiently co-fermenting glucose and xylose and application of recombinant saccharomyces cerevisiae strain
  • Recombinant saccharomyces cerevisiae strain for efficiently co-fermenting glucose and xylose and application of recombinant saccharomyces cerevisiae strain

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1 culture medium, enzyme and reagent and relevant microorganism and molecular biology technique

[0053] (1) culture medium

[0054] LB medium used for Escherichia coli culture: 10gL -1 Peptone, 5gL -1 Yeast extract, 10gL -1 NaCl; add 20gL of solid medium -1 Agar powder; sterilization conditions: 115°C, 30min; when in use, add ampicillin (Amp) to 100μgmL -1 Used to screen E.coli transformants.

[0055] YEP medium used for Saccharomyces cerevisiae culture: 20gL -1 Peptone, 10gL -1 Yeast powder; add 20gL of solid medium -1 Agar powder; Sterilization conditions: 115°C, 30min. When using, add different concentrations of glucose or xylose as carbon source to make YEPD or YEPX medium respectively, or add glucose and xylose as carbon source to make mixed sugar medium, which is used for strain growth and fermentation performance detection. Add 400-800 μg mL if necessary -1 G418 or 200 μg mL -1 HygromycinB was used for screening and culturing of correspond...

Embodiment 2

[0066] Construction of embodiment 2 plasmid YEp-CH

[0067] (1) Ordinary PCR amplification of DNA fragments

[0068] The following fragments were amplified by PCR: fragment GAL1p-Cre-CYC1t (Cre enzyme gene under the control of GAL1 promoter and CYC1 terminator), TEF1 promoter, TEF1 terminator, hygromycin B resistance gene (hygB).

[0069]Using the plasmid pSH47 as a template, use primers 1 and 2 to amplify the fragment GAL1p-Cre-CYC1t; using the plasmid pUG6 as a template, use primers 3 and 4 to amplify the TEF1 promoter; using the plasmid pUG6 as a template, use primers 5 and 6 Amplify the TEF1 terminator; use the plasmid pUCATPH (Luetal., 1994) as a template, and use primer 7 and primer 8 to amplify fragment hygB.

[0070] (2) fusion PCR amplified DNA fragment

[0071] Amplify the fragment TEF1p-hygB-TEF1t by fusion PCR: the DNA fragment TEF1 promoter obtained in step (1), the TEF1 terminator and the fragment hygB are simultaneously used as templates for PCR, and PCR ampli...

Embodiment 3

[0077] Example 3 Removal of screening marker genes by Cre-loxP recombination

[0078] The plasmid Yep-CH obtained in Example 2 was transferred into a recombinant yeast strain containing a selection marker gene (with loxP sites at both ends), and transformants were selected on a YEPD plate containing hygromycin B.

[0079] Cultivate the obtained transformant overnight in liquid YEPD medium containing hygromycin B, then collect the cells by centrifugation and wash twice with sterile water, resuspend the cells in galactose medium, culture with shaking at 30°C for two hours to induce Cre recombination Enzyme expression, then streak the bacterial solution on the YEPD plate, culture at 30°C for two days to isolate a single colony, drop the single colony on the YEPD plate without G418 and the YEPD plate containing G418, and select no G418 resistance after culturing at 30°C for two days The colonies obtained by PCR verified that the Cre-loxP recombination occurred correctly.

[0080]...

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Abstract

The invention discloses a recombinant saccharomyces cerevisiae strain for efficiently co-fermenting glucose and xylose. The strain is (Saccharomyces cerevisiae) LF1 which is collected with the serial number of CGMCC (China General Microbiological Culture Collection Center) No.11331 in CGMCC on September 8, 2015. The invention also discloses an application of the recombinant saccharomyces cerevisiae strain to ethanol production through fermentation in a mixed sugar culture medium with glucose and xylose as carbon sources or a lignocellulose hydrolysate. Experiments prove that the recombinant saccharomyces cerevisiae strain LF1 disclosed by the invention has relatively high glucose and xylose co-fermentation capacity, and after the glucose and the xylose are fermented for 12 hours, the glucose is completely consumed, and meanwhile about 77.6% (33.24gL<-1>) of xylose is utilized, so that the strain LF1 basically has the actual industrialized potential for producing fuel ethanol by fermenting lignocellulose raw materials.

Description

technical field [0001] The invention relates to a strain of Saccharomyces cerevisiae and its application, in particular to a recombinant Saccharomyces cerevisiae strain for co-fermenting glucose and xylose with high efficiency and its application, belonging to the technical field of microorganisms. Background technique [0002] At present, the development and utilization of renewable energy has become an important measure for countries around the world to ensure energy security, strengthen environmental protection, and address climate change. Biofuel ethanol, due to its unique properties for vehicles, is recognized as one of the promising renewable bio-energy sources. Since the production of first-generation and 1.5-generation fuel ethanol, which uses corn, wheat, cassava, sweet potato and other grains and non-grain starches as raw materials, inevitably has the problems of "competing with people for food" and "competing with food for land", neither can Therefore, the produc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12P7/10C12R1/865
CPCY02E50/10
Inventor 鲍晓明沈煜李洪兴侯进
Owner SHANDONG UNIV
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