High temperature-resistant Saccharomyces cerevisiae strain and constructing method thereof

A strain of Saccharomyces cerevisiae, the technology of Saccharomyces cerevisiae, applied in the field of genetic engineering, can solve the problems of no marketed products, etc., and achieve the effects of increased cell survival rate, increased ethanol production, and low cost

Inactive Publication Date: 2018-03-30
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no relatively mature marketable produc

Method used

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  • High temperature-resistant Saccharomyces cerevisiae strain and constructing method thereof
  • High temperature-resistant Saccharomyces cerevisiae strain and constructing method thereof
  • High temperature-resistant Saccharomyces cerevisiae strain and constructing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Simultaneous saccharification and fermentation to determine parental strains

[0025] After activating Angel high temperature resistant active dry yeast with 20g / L sugar water, draw three areas on the YEPD solid plate to obtain a single colony of Saccharomyces cerevisiae. Named AY12-G. AY12-G and the strains AY12 and AY15 preserved in our laboratory were used for synchronous saccharification and fermentation of corn raw materials to select the strain with the best performance as the parent strain for subsequent experiments. Saccharomyces cerevisiae was inserted into the fermentation medium and cultured at 30°C for 12 hours, then transferred to 35°C and fermented at 160r / min. The ethanol-producing ability and reducing sugar utilization ability of each strain were tested at 33°C and 35°C respectively. At 33°C as the main fermentation temperature, the ethanol production capacity of the strain AY12-G was slightly higher than that of AY12 and AY15; at 35°C as th...

Embodiment 2

[0041] Example 2: ARTP plasma mutagenesis

[0042] Pick a ring of bacteria slime and put it into 5mL YEPD liquid medium, culture it on a shaker at 30°C and 180r / min for 12h, then put 500μL of the above bacterial solution into 5mL of fresh YEPD liquid medium, culture it on a shaker at 30°C and 180r / min for 4 -6h. Take an appropriate amount of the above bacterial solution diluted 1000 times with normal saline, take 10 μL of the diluted bacterial solution and spot on the ARTP mutagenesis slide for plasma mutagenesis treatment, the treatment time is 20s, 30s, 40s, 50s, 60s, 70s respectively. After the mutagenesis treatment is completed, place the treated slides in a 2mL centrifuge tube with 1mL sterile saline, shake vigorously with a vortex shaker for 3min, take the shaken bacteria solution and spread it on a YEPD plate, and place it at 30°C cultured in a constant temperature incubator. The first round of lethal curve is drawn, and the time period of mortality ≥ 60% to 100% is t...

Embodiment 3

[0044] Example 3: Genome Rearrangement

[0045] Genome rearrangement was performed on 150 strains screened after ARTP ion mutagenesis to further improve the high temperature resistance of the strains. The obtained 150 positive mutant cell populations were all inoculated into fresh YEPD liquid medium, cultured to the logarithmic phase at 30°C and 180 r / min, and the cells were collected by centrifugation. The collected bacteria were washed with sterile water, and then the washed bacteria sludge was inserted into the YPK pre-sporulation medium, cultured at 28°C and 180r / min for 12 hours, and the bacteria were collected by centrifugation. After washing the collected bacteria three times with sterile water, they were inserted into high-efficiency liquid sporulation medium, and cultured at 28° C. and 180 r / min for 5 days. Bacteria were collected by centrifugation, followed by spore purification.

[0046] First, resuspend the spore-forming bacteria of the mutant population in softe...

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Abstract

The invention discloses a screening and constructing method for high temperature-resistant Saccharomyces cerevisiae. The method comprises the following steps: carrying out ARTP plasma mutagenesis on astrain AY12-G used as an original strain, carrying out primary screening at 37 DEG C to obtain 150 strains, carrying out genome rearrangement on the 150 strains, and primarily screening the 150 strains at 37 DEG C to obtain 137 strains; and carrying out 35 DEG C corn hydrolysate fermentation on the 137 strains to obtain 14 strains, carrying out 35 DEG C simultaneous saccharification and fermentation on the 14 strains and 48 h cell survival rate determination to obtain 7 strains having improved high temperature fermentation performances and 48 h cell survival rate, and carrying out 41 DEG C and 42 DEG C high temperature domestication on the 7 strains to finally obtain a strain X-130 having excellent high temperature fermentation performances and significantly improved 48 h cell survival rate. Compared with the original strain AY12-G, the strain X-130 has the advantages of improvement of the 48 h cell survival rate by 84.68%, no obvious change of the 35 DEG C simultaneous saccharification and fermentation performances, and shortening of the fermentation cycle by 12 h through adding acidic protease.

Description

Technical field: [0001] The invention relates to the field of genetic engineering, in particular to a high-temperature-resistant Saccharomyces cerevisiae strain and a construction method thereof. Background technique: [0002] Fuel ethanol is currently the most mature gasoline alternative fuel recognized globally. As a renewable liquid fuel, fuel ethanol plays a very important role in alleviating air pollution, reducing greenhouse gas emissions, reducing dependence on imported oil, activating the rural economy, and increasing farmers' income. Therefore, it is widely used in many countries around the world. and regions have been promoted. Fuel ethanol has become one of the most concerned renewable energy sources in the world. In 2015, the global fuel ethanol production reached 98.3 billion liters, accounting for 75% of the total bio-liquid fuel (including ethanol, biodiesel and hydrogenated vegetable oil) production (about 130.7 billion liters), becoming the world's most im...

Claims

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Application Information

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IPC IPC(8): C12N1/18C12N13/00C12N15/01C12N15/09C12P7/06C12R1/865
CPCC12N13/00C12N15/01C12N15/09C12P7/06C12N1/185C12R2001/865Y02E50/10
Inventor 董健付肖蒙肖冬光郝爱丽郭学武张翠英
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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