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A strain of Saccharomyces cerevisiae tolerant to high concentration of furfural and its application

A technology of Saccharomyces cerevisiae and high concentration is applied in the field of Saccharomyces cerevisiae to achieve the effects of shortening the fermentation period, shortening the growth delay period and reducing production costs

Active Publication Date: 2019-07-16
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The industrial Saccharomyces cerevisiae strain AS2.489 was purchased from the General Microbiology Center of the China Committee for the Collection of Microbial Cultures

Method used

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  • A strain of Saccharomyces cerevisiae tolerant to high concentration of furfural and its application
  • A strain of Saccharomyces cerevisiae tolerant to high concentration of furfural and its application
  • A strain of Saccharomyces cerevisiae tolerant to high concentration of furfural and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1: Construction of irrE gene mutation library

[0020] The irrE gene was mutated by error-prone PCR technique, and the wild-type irrE gene was used as the amplification template.

[0021] (1) Design of amplification primer sequence

[0022]

[0023] Mn in error-prone PCR system 2+ , Mg 2+ Optimization of Concentration: Mg 2+ Concentration fixed at 2mM, Mn 2+ Set the concentration to 5 gradients (0.25, 0.35, 0.45, 0.55, 0.65mM) for PCR amplification; fix Mn 2+ The concentration is 0.4mM, the Mg 2+ Set the concentration to 5 gradients (1, 1.5, 2, 2.5, 3mM), check the effect of PCR amplification, and preliminarily determine the optimal Mg 2+ Concentration is 2mM, Mn 2+ The concentration is between 0.35-0.65mM.

[0024] (3) Using error-prone PCR amplification to transform the wild-type irrE gene, Mg 2+ Concentration is 2mM, adjust Mn 2+ The concentration is 0.35, 0.55mM (Mn 2+ The concentration is adjusted according to the mutation screening effect ...

Embodiment 2

[0029] Embodiment 2: Construction of Saccharomyces cerevisiae recombinant vector

[0030] (1) The plasmid pYP was constructed with the shuttle vector pYSE2.0 as the backbone, and the PGK gene of the Saccharomycescerevisiae AS2.489 genome was cloned and integrated into the pYES2.0 vector between the SacI and AgeI restriction sites. The PCR amplification system is as follows:

[0031]

[0032]

[0033] The PCR amplification procedure is as follows:

[0034]

[0035] After the PCR reaction, the product was stored at 4°C, identified by 1% (w / v) agarose gel electrophoresis, and the target fragment of about 720 bp was recovered by cutting the gel.

[0036] The PGK recovery product and the pYES2.0 vector were placed at 37°C, and the restriction endonucleases SacI and AgeI were double-digested for 2.5 hours. The enzyme digestion system is as follows:

[0037] Separately cut glue for recycling The above double digestion products were placed in the T4 DNA ligation system and...

Embodiment 3

[0060] Example 3: Screening and Obtaining of Highly Tolerant Saccharomyces cerevisiae Mutants

[0061] (1) Construct the irrE gene mutation library, transfer the above pYPKI plasmid into DH5α for amplification, pick a single colony for detection and sequencing, and calculate the mutation rate to measure the mutation effect. Scrape the colony from the transformed plate and inoculate it into 50 mL of LB medium containing 100 ug / mL ampicillin for culture, and extract the plasmid with a kit to obtain the irrE gene mutation library.

[0062] (2) Screen Saccharomyces cerevisiae transformants, detect G418 resistance of wild-type Saccharomyces cerevisiae AS2.489, set 5 gradients of G418 concentration (respectively 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL mL) YPD plate, the results showed that the growth of AS2.489 was significantly inhibited on the YPD plate with a G418 concentration of 150 μg / mL, so the G418 concentration of 200 μg / mL was used as the initial screening co...

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Abstract

The invention discloses Saccharomyces cerevisiae tolerant to high-concentration furfural and application of the Saccharomyces cerevisiae. The strain is Saccharomyces cerevisiae FR125 which is preserved in China General Microbiological Culture Collection Center on Sep. 9th, 2015, and the preservation number is CGMCC No.11356. According to the tolerance test of the stain under combined pressure, with different gradients, of glucose and furfural, the growth lag phase is significantly shortened compared with a control group, the ethanol yield remains unchanged, but the yield is significantly increased. According to the mutant strain tolerant to combined pressure of the high-concentration glucose and furfural, cheap raw materials can be effectively utilized for large-scale fermentation production of ethanol, and important roles in the aspects of reducing the production cost, protecting the environment and reducing dependence on food crops are achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a strain of Saccharomyces cerevisiae tolerant to high-concentration furfural and its application. Background technique [0002] Saccharomyces cerevisiae, as the most common model eukaryote and industrial strain, has excellent characteristics such as easy cultivation, fast growth and metabolism, high safety, clear genetic background and good fermentation performance, and is widely used in food , medicine and chemical industry and other fermentation industries. [0003] Traditional ethanol fermentation mainly uses crops such as starch as raw materials. However, due to the decrease of cultivated land year by year and the pressure of population is still high, the world is facing the serious threat of food shortage. At this stage, the strategy of large-scale production of ethanol with food crops is not feasible. Be applicable. Therefore, researchers from various countries ar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/81C12P7/10C12R1/865
CPCY02E50/10
Inventor 王菊芳罗平马毅李杉
Owner SOUTH CHINA UNIV OF TECH
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