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Recombined saccharomyces cerevisiae strain for producing ethyl alcohol, construction method of recombined saccharomyces cerevisiae strain and method for producing ethyl alcohol through recombined saccharomyces cerevisiae strain

A technology of Saccharomyces cerevisiae strains and recombinant Saccharomyces cerevisiae, which is applied in the direction of recombinant DNA technology, fermentation, fungi, etc., can solve the problems that the specific relationship is not clear enough, and achieve the effect of fast and efficient consumption and efficient xylose metabolism

Active Publication Date: 2017-04-05
COFCO NUTRITION & HEALTH RES INST +2
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Problems solved by technology

[0009] However, although the prior art has successfully achieved co-fermentation of C5 sugars and C6 sugars by introducing the xylose isomerase gene into yeast, or by up-regulating the xylose reductase gene and xylitol dehydrogenase gene in yeast produce ethanol, but these recombinant yeasts have a lot of room for improvement in terms of xylose metabolism ability and efficiency
One of the key factors is that the metabolism of carbohydrates is usually linearly related to the copy number of related genes, but too many copies of genes will inevitably bring metabolic burden
In the prior art, the ability of the target strain to metabolize xylose is usually enhanced by increasing the copy number of the xylose isomerase gene, but so far, the specific relationship between the copy number of the xylose isomerase gene and the metabolic effect is still not clear enough Clearly, the balance relationship still needs to be further explored

Method used

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  • Recombined saccharomyces cerevisiae strain for producing ethyl alcohol, construction method of recombined saccharomyces cerevisiae strain and method for producing ethyl alcohol through recombined saccharomyces cerevisiae strain
  • Recombined saccharomyces cerevisiae strain for producing ethyl alcohol, construction method of recombined saccharomyces cerevisiae strain and method for producing ethyl alcohol through recombined saccharomyces cerevisiae strain
  • Recombined saccharomyces cerevisiae strain for producing ethyl alcohol, construction method of recombined saccharomyces cerevisiae strain and method for producing ethyl alcohol through recombined saccharomyces cerevisiae strain

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preparation example Construction

[0077] Preparation of recombinant strains

[0078] The XI-XK-TTRR fragment was integrated into the genome of Saccharomyces cerevisiae by homologous recombination using methods known in the art. Wherein, the method for transforming Saccharomyces cerevisiae can use various transformation methods known to those skilled in the art, such as electrotransformation, lithium acetate chemical transformation, and the like.

[0079] In one embodiment of the present invention, lithium acetate chemical transformation method is used to convert Figure 5 The XI-XK-TTRR fragment shown in was transformed into the starting strain of Saccharomyces cerevisiae (wild-type Saccharomyces cerevisiae strain, Saccharomyces cerevisiae industrial strain without genetic modification) to obtain a recombinant Saccharomyces cerevisiae strain, which was named COFCO-1 engineering strain. In this recombinant S. cerevisiae strain, 2 copies of the XI gene and 2 copies of the XK gene and 1 copy of the TTRR gene wer...

Embodiment 1

[0086] Example 1 Preparation of recombinant Saccharomyces cerevisiae strain COFCO-1 engineering strain

[0087] The starting strain was an industrial strain of Saccharomyces cerevisiae (wine yeast) from Angel Yeast.

[0088] 1.1 Construction of plasmid pBS-XI-XK-1

[0089] The XI-6m gene with the DNA sequence (SEQ ID NO: 9) of the first promoter pPGK1, the 3' end (the end of the stop codon) encoding the farnesyl membrane localization group CAAX, and the first terminator PGK1t were constructed into the XI-6m gene expression cassette pPGK1-XI-6m-CAAX-PGK1t; at the same time, the second promoter pADH1, the 3' end with the DNA sequence (SEQ ID NO: 9) encoding the farnesyl membrane localization group CAAX The XK gene and the second terminator ADH1t are constructed into the XK gene expression cassette pADH1-XK-CAAX-ADH1t, and the above-mentioned XI-6m gene expression cassette and the XK gene expression cassette are sequentially spliced ​​into the cloning vector pbluescript to form ...

Embodiment 2

[0142] Example 2 Preparation of recombinant yeast strain COFCO-2 engineering strain

[0143] 2.1 Construction of plasmid 5-FINAL-2

[0144] According to the method disclosed in Xiong M, Chen G, Barford J., Alteration of xylose reductase coenzymepreference to improve ethanol production by Saccharomyces cerevisiae from highxylose concentrations, Bioresour Technol., 2011, 102(19): 9206-15, the plasmid 5- FINAL-2.

[0145] 2.2 Acquisition of upstream and downstream homology arms for knocking out PHO13 gene

[0146] For knocking out the upstream homology arm of the PHO13 gene: According to the G418 resistance gene sequence, the gene was fully synthesized by Beijing Qingke Xinye Biotechnology Co., Ltd., and then, with primers PHO13up-G418-F (SEQ IDNO: 54) and G418 -pgk1-R (SEQ ID NO: 55) amplifies the G418 resistance gene, wherein the primer PHO13up-G418-F has a sequence complementary to the following PHO13-up fragment, and the primer G418-pgk1-R has a sequence complementary to th...

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Abstract

The invention provides a recombined saccharomyces cerevisiae strain for producing ethyl alcohol, a construction method of the recombined saccharomyces cerevisiae strain and a method for producing ethyl alcohol through the recombined saccharomyces cerevisiae strain. According to the recombined saccharomyces cerevisiae strain for producing ethyl alcohol, a wild type saccharomyces cerevisiae strain is used as an original strain, an XI xylose metabolism pathway is introduced into the original strain, the expression of four key genes in a PPP pathway is enhanced, meanwhile, the aldose reductase gene GRE3 is knocked out, the nitrobenzene phosphatase gene PHO13 is optionally knocked out, and XI protein and XK protein introduced exogenously are positioned to a yeast cell membrane through the membrane positioning effect of a membrane positioning group. The recombined saccharomyces cerevisiae strain for producing ethyl alcohol can efficiently carry out xylose metabolism, so that co-fermentation of C5 sugar and C6 sugar is achieved, and therefore the higher ethyl alcohol conversion rate is achieved.

Description

technical field [0001] The present invention relates to recombinant yeast strains for the production of ethanol from biomass. Specifically, the present invention relates to a co-fermentation recombinant Saccharomyces cerevisiae strain capable of simultaneously utilizing five-carbon sugar and six-carbon sugar to efficiently produce ethanol, a method for constructing the strain, and a method for producing ethanol using the strain. Background technique [0002] In recent years, the depletion of many non-renewable fossil energy such as petroleum has led to more and more attention to renewable energy, especially biofuels, and has brought huge business opportunities and social significance. Ethanol, known as "green oil" and "liquid gold", is a new type of clean and renewable liquid fuel. Many countries have begun to use gasoline-gasoline alcohol added with a certain proportion of ethanol to reduce gasoline consumption. This new type of fuel can not only alleviate the consumption ...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12P7/06
CPCY02E50/10
Inventor 李凡张子剑安泰沈乃东陈博李文钊武国庆王春才袁敬伟李春玲熊强
Owner COFCO NUTRITION & HEALTH RES INST
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