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Mutant nucleic acid, expression vector, yeast strain and preparation method and application thereof

A technology of expression vectors and yeast strains, which is applied in the fields of mutated nucleic acids, expression vectors, yeast strains and their preparation and application, can solve the problems of being unable to become dominant strains of lignocellulosic ethanol, inhibiting cell fermentation performance, and inhibiting component sensitivity, etc. Achieve the effects of strong practicability, high sugar utilization rate and stable genetic performance

Active Publication Date: 2015-04-29
CHINA PETROLEUM & CHEM CORP +1
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  • Abstract
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AI Technical Summary

Problems solved by technology

Although Saccharomyces cerevisiae can efficiently use glucose to produce ethanol, it has poor tolerance to inhibitors in fermentation broth and high concentration of ethanol (Almeida J R M et al., 2007), especially sensitive to inhibitory components in cellulose hydrolyzate , preventing Saccharomyces cerevisiae from becoming the dominant strain for lignocellulosic ethanol production
Secondly, in the production of cellulosic ethanol, the degradation products produced during the pretreatment of cellulose raw materials and the residual degradation products will inhibit the subsequent fermentation of strains to varying degrees. Among them, acetic acid, furfural, phenols and SO 4 2- and other inhibitory factors have a strong toxic effect on yeast, which will significantly inhibit the growth of cells and their fermentation performance

Method used

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  • Mutant nucleic acid, expression vector, yeast strain and preparation method and application thereof
  • Mutant nucleic acid, expression vector, yeast strain and preparation method and application thereof
  • Mutant nucleic acid, expression vector, yeast strain and preparation method and application thereof

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Embodiment approach

[0027] According to a preferred embodiment of the present invention, the expression vector is an expression vector obtained by inserting a mutated nucleic acid whose sequence is as described above between the BamHI restriction site and the XhoI restriction site of the plasmid pAUR123. When the expression vector in the preferred embodiment of the present invention is transformed into Saccharomyces cerevisiae cells, higher expression efficiency can be obtained, so that the purpose of the present invention can be better achieved.

[0028] In addition, the present invention also provides a method for preparing highly stress-resistant yeast strains, the method comprising: using the above-mentioned expression vector of the present invention to transform yeast cells to obtain transformed yeast cells.

[0029] In the present invention, various transformation methods commonly used in the art can be used to transform yeast cells with expression vectors, for example, electroporation or ch...

Embodiment 1

[0055] Entrust Shanghai Gemma (GenePharma) company to synthesize base sequence such as the mutated nucleic acid lsm1 shown in SEQ ID NO: 2, with reference to the method in the "Molecular Cloning Experiment Guide (Third Edition)" (Sambrook J, 2001), the lsm1 It was connected between the BamHI restriction site and the XhoI restriction site of Saccharomyces cerevisiae plasmid pAUR123 to constitute the recombinant expression vector pAUR-lsm1.

Embodiment 2

[0057] (1) Pick a single colony of Saccharomyces cerevisiae RIPP-0 into YEPD medium, culture at 30°C and 180r / min until mid-logarithmic late stage (about 16h), centrifuge at 4000r / min for 5min to collect the cells, and use 4°C sterile water and Wash twice with pH 7.5, 10mM Tris-HCl buffer, add 0.6M sorbitol solution to suspend the cells, and dilute to a concentration of about 10 5 -10 6 1 cell / mL, take 0.2ml bacterial liquid, transfer the recombinant expression vector pAUR-lsm1 obtained in Example 1 into S. -0 host bacteria, add 1 mL of YEPD medium to the bacterial solution, and incubate at 30 °C for 1 h.

[0058] (2) Evenly spread the bacterial solution after step (1) incubation for 1 hour on the solution containing 3g / L acetic acid and 30g / L NH 4 Ac, and two composite screening plates of 3g / L acetic acid and 40g / L NaAc were cultured at 30°C for 60h to obtain 64 primary screened strains.

[0059] (3) Introduce the primary screened strains into the acidic seed medium, cultu...

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Abstract

The invention relates to mutant nucleic acid, an expression vector, a yeast strain and a preparation method and application thereof. The invention discloses mutant nucleic acid with a sequence as shown in SEQ ID NO: 1 or mutant nucleic acid with a sequence as shown in SEQ ID NO: 1 connecting a Kozak sequence at 5' end; an expression vector inserted with the nucleic acid, and a method for preparing a yeast strain with high stress resistance by using the expression vector; the yeast strain with high stress resistance; and the method for preparing the yeast strain with high stress resistance and application of the yeast strain with high stress resistance in production of ethanol. Through the technical scheme, after yeast is transformed by using the expression vector constructed by the mutant nucleic acid disclosed by the invention, the yeast strain with high stress resistance and multiple stress tolerance can be obtained, the heredity of the yeast strain can be stable, the utilization rate of sugar in production of ethanol and the yield of ethanol are relatively high, the yeast strain can be applied to an existing fuel ethanol production process and a lignocellulose ethanol new process, and the practicality is relatively strong.

Description

technical field [0001] The present invention relates to a mutated nucleic acid, an expression vector, a method for preparing highly stress-resistant yeast strains, a yeast strain, and a preparation method and use thereof, in particular, to a mutated nucleic acid, an expression vector inserted with the nucleic acid, and the use of The expression vector prepares a method for highly stress-resistant yeast strains, a highly stress-resistant yeast strain, a method for preparing highly stress-resistant yeast strains, and an application of the highly stress-resistant yeast strains in producing ethanol. Background technique [0002] Due to the rapid increase in the use of traditional fossil energy, energy shortage has become a worldwide problem. The development of energy diversification and the acceleration of renewable energy development have become the research and development priorities of countries all over the world. Among them, the production and application of fuel ethanol are...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/81C12N1/19C12P7/06C12R1/865
CPCY02E50/10
Inventor 高岚郭勇刘金胜刘颖李宝石蔺建民
Owner CHINA PETROLEUM & CHEM CORP
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