Saccharomyces cerevisiae strain for expressing xylose isomerase and construction method

A technology of Saccharomyces cerevisiae strain and xylose isomerase, which is applied in microorganism-based methods, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of slow growth rate of recombinant bacteria and inability to meet fermentation requirements, etc., Achieve the effect of fast xylose fermentation and high ethanol yield

Active Publication Date: 2016-03-02
SICHUAN UNIV
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Problems solved by technology

[0005] The purpose of the invention is to solve the existing xylA After the 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 the xylose isomerase pathway and capable of solving the above problems is provided. Industrial Saccharomyces cerevisiae Efficiently Fermenting Xylose to Ethanol

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  • Saccharomyces cerevisiae strain for expressing xylose isomerase and construction method
  • Saccharomyces cerevisiae strain for expressing xylose isomerase and construction method

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Embodiment 1

[0026] A strain of Saccharomyces cerevisiae expressing xylose isomerase, the Saccharomyces cerevisiae ( Saccharomycescerevisiae The SEB7) strain is deposited in the China Common Microbial Species Collection and Management Center, and the deposit number is CGMCC11327. The specific construction method of the Saccharomyces cerevisiae strain SEB7 is as follows:

[0027] 1) Construct the starting strain:

[0028] 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. Pick single spores on the four-spore analyzer, and obtain haploid cells after verification. In the present invention, only haploids with sex α are selected, and the haploid strain SEB3α25 is finally screened.

[0029] Using the plasmid pBlu-LTKTL-TDH3 as a template, it was amplified with primers Fg3 / Rg3 loxP-KanMX-loxP The fragments were then introduced into the strain SEB3α...

Embodiment 2

[0057] This example is a comparative example of example 1. The construction steps and construction conditions in this example are the same as those of example 1, except that the primers PXYLA1-F / PXYLA1-R and PXYLA2-F / are used in step 2.4). PXYLA2-R the original gene sequence of xylose isomerase PXYLA1 Amplify it and combine it with plasmid pRS426-P TDH3 -T TDH3 through EcoR After digestion with restriction enzyme I, ligation with T4 ligase, and verification by restriction enzyme digestion and sequencing, the multi-copy expression vector pRS426-PXYLA1 was obtained.

[0058] After step 3) construction, industrial Saccharomyces cerevisiae YCPA1 is obtained. Strain YCPA1E was obtained by performing step 4) aerobic growth acclimation and microaerobic fermentation acclimation on strain YCPA1.

[0059] Through the growth evaluation of YCPA1 and YCPA2, before the 5th generation, the strain needs to be cultured for 36h to reach the mid-log growth phase. In the first five generations ...

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Abstract

The invention discloses a Saccharomyces cerevisiae strain for expressing xylose isomerase and a construction method and solves the problem that after heterogenous expression of xylA gene in Saccharomyces cerevisiae, a recombinant bacterium constructed thereby has a low growth rate in xylose, causing the fermentation requirement to be not met. The invention includes a Saccharomyces cerevisiae strain SEB7, collected in China General Microbiological Culture Collection Center under CGMCC11327. The invention also provides a construction method of the Saccharomyces cerevisiae strain SEB7. The strain SEB7 obtained by the invention can generate ethanol 6.98 g / L within a fermenting time of 48 h by using xylose 16.95 g / L, with ethanol yield up to xylose 0.412 g / g; the strain has the advantages such as high xylose fermentation speed and high ethanol yield.

Description

Technical field [0001] The invention relates to a yeast strain, in particular to a Saccharomyces cerevisiae strain expressing xylose isomerase and a method for constructing the strain. Background technique [0002] In recent years, fuel ethanol, as one of the most promising clean energy sources, has received widespread attention. Lignocellulose, which uses agricultural and forestry wastes as raw materials, has the advantages of wide sources, large reserves, low price, and renewable, and is regarded as an ideal raw material for fuel ethanol production. Saccharomyces cerevisiae( Saccharomycescerevisiae ) Due to its fast glycolysis rate, high ethanol output, and good environmental tolerance, it is the most commonly used microorganism in the industrial ethanol production process. 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/61C12N15/81C12R1/865
Inventor 汤岳琴李云成苟敏孙照勇木田建次夏子渊
Owner SICHUAN UNIV
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