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Industrial Saccharomyces cerevisiae strain with low glycerol synthesis and high alcohol tolerance and application thereof

A strain of Saccharomyces cerevisiae, a technology for Saccharomyces cerevisiae, applied in the directions of fermentation, fungi, microorganism-based methods, etc., can solve the problems of decreased tolerance, increased synthesis of by-products, decreased ethanol production, etc., and achieves reduced production costs and high alcohol content. Durability, the effect of reducing energy consumption

Inactive Publication Date: 2012-01-25
ZHEJIANG UNIV
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AI Technical Summary

Problems solved by technology

At present, the glycerol metabolism pathway of yeast cells has been thoroughly studied. The application of genetic metabolic engineering technology to modify and transform the genes and pathways related to glycerol synthesis can effectively reduce the synthesis of glycerol, but the modified strains are resistant to high sugar, high The tolerance to stress factors such as concentrated ethanol is reduced, and the growth is slow, which will cause adverse phenomena such as reduced fermentation rate, prolonged fermentation cycle, and reduced ethanol production.
For traits with one or a small number of gene regulation and known mechanisms, genetic metabolic engineering is an easy and direct feasible rational strategy, but for complex traits involving multiple genes and their regulatory networks (such as fermentation rate, tolerance, etc. Fermentation traits), genetic metabolic engineering technology is difficult to achieve the desired effect, and may even cause the degradation and decay of the key performance of the strain
At present, research has applied the blind breeding technology based on the whole genome level—whole genome rearrangement, which has effectively improved the tolerance of strains such as acetic acid resistance and high-concentration ethanol resistance, but other production properties of the modified strains, such as sugar alcohol conversion rate, have been improved. Not significant, and with the increase of mash fermentable carbon source, by-product synthesis also increases, which greatly limits the improvement of ethanol production
[0005] To sum up, although the application of a single breeding method can improve the traits of a certain aspect of the strain, it is difficult to obtain an excellent strain with comprehensive performance, and it is easy to cause the degradation and decay of the key performance of the production strain

Method used

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  • Industrial Saccharomyces cerevisiae strain with low glycerol synthesis and high alcohol tolerance and application thereof
  • Industrial Saccharomyces cerevisiae strain with low glycerol synthesis and high alcohol tolerance and application thereof
  • Industrial Saccharomyces cerevisiae strain with low glycerol synthesis and high alcohol tolerance and application thereof

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Effect test

Embodiment 1

[0026] Example 1: Acquisition of Industrial Saccharomyces cerevisiae Glycerol Metabolism Engineering Strains

[0027] 1. Obtaining the haploid starting strain

[0028] After the industrial Saccharomyces cerevisiae Z87 was activated by YPD at 30°C, it was transferred into the sporulation medium and cultured at 26°C for 3-7 days. When the ascospore formation was observed under a microscope, the bacterial cells were collected, washed twice with normal saline, and then added with 700 μL Tris-HCl (pH8.0, 0.01mol / L), 200 μL 100 mg / mL helicase solution and 100 μL 0.1mol / L mercaptoethanol , 120r / min at 30°C for 16h to rupture the ascus wall and release spores. Treat the vegetative cells at 58°C for 15 minutes to kill the vegetative cells, collect the spores by centrifugation, spread them on a YPD plate, incubate at 30°C for 2-3 days, pick a single colony, and verify the sporulation after activation on the YPD slant. Somatic strains.

[0029]The strains to be tested and the standard...

Embodiment 2

[0037] Embodiment 2: Implement whole genome rearrangement on genetically engineered strains

[0038] Such as image 3 The shown process implements whole genome rearrangement, and the specific steps are as follows:

[0039] 1. Treat genetically engineered haploid strains YFG1 (MAT a, fps1Δ::PGKp-gapN) and YFG2 (MAT α, fps1Δ::PGKp-gapN) with 1% (v / v) EMS mutagen for 30-120 minutes respectively Add 5% (w / v) sodium thiosulfate to remove EMS contamination every 30min, collect the bacterial cells by centrifuging at 4000rpm for 5min, wash twice with normal saline, apply gradient dilution to a YPD plate containing 8% (v / v) ethanol, Cultivate at 30°C for 3 days, and pick a vigorously growing single colony;

[0040] 2. Under the ultraviolet lamp with a distance of 40cm and a power of 15w, carry out ultraviolet induction on genetically engineered haploid strains YFG1 (MAT a, fps1Δ::PGKp-gapN) and YFG2 (MAT α, fps1Δ::PGKp-gapN) Change, every 1min samples were diluted and coated with 8%...

Embodiment 3

[0044] Embodiment 3: Determination of production performance of engineering strains

[0045] 1. Growth determination under high-concentration ethanol stress conditions

[0046] Use YPD liquid medium containing 0% and 10% (v / v) ethanol to cultivate the starting strain Z87, the control strain Y12, the genetically engineered strain YFG12, and the genetically engineered rearrangement FG1 at 30°C, and take samples at different times to measure the dry weight of the strains, and calculate The maximum specific growth rate of the strain. Such as Figure 4 As shown, under the YPD culture condition containing 0% ethanol, there was no significant difference in the growth of the four strains, and the lag period was about 0.4h; under the YPD culture condition containing 10% (v / v) ethanol, the four strains lagged The average period was extended to about 10 hours, and the genetically engineered rearrangement FG1 grew the fastest, and the maximum specific growth rate was 11.4% higher than t...

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Abstract

The invention provides an industrial Saccharomyces cerevisiae strain with low glycerol synthesis and high alcohol tolerance, namely Saccharomyces cerevisiae FG1. The strain was collected in China Center for Type Culture Collection in Wuhan University, Wuhan 430072, China, on August 1, 2011, and the collection number is CCTCC No: M2011274. The invention provides an excellent industrial Saccharomyces cerevisiae engineering strain with few byproducts and high alcohol tolerance and application thereof, and a method for improving various production properties of the industrial strain, namely the combination of genetic and metabolic engineering and complete genome rearrangement. By the method, various production properties of the Saccharomyces cerevisiae strain such as conversion rate of sugar alcohol, tolerance, fermentation rate and the like can be improved; the improved strain can be used for fermentation production of industrial high-gravity alcohol, the energy consumption is reduced, and the production cost is reduced; and the method can also be used for improving the properties of other industrial microorganisms.

Description

(1) Technical field [0001] The present invention relates to an industrial Saccharomyces cerevisiae strain with low glycerol synthesis and high alcohol tolerance—Saccharomyces cerevisiae FG1 and its application; and a method for constructing industrial Saccharomyces cerevisiae engineering bacteria by integrating gene metabolism engineering and whole genome rearrangement . (2) Background technology [0002] Alcoholic mash fermentation, in simple terms, is high-concentration fermentation in the fermentation process, which is specifically manifested in the following characteristics of production: 1. High alcohol content; 2. High osmotic pressure; 3. High yeast count. As far as alcohol production is concerned, there are obvious differences in the boundaries of thick mash fermentation between different raw materials and different periods; roughly, the distinction is as follows: starchy raw materials: alcohol concentration is 14-16% (V / V), molasses raw materials: alcohol concentrat...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12P7/06C12N15/81C12N15/01C12R1/865C12R1/465
CPCY02E50/17Y02E50/10
Inventor 吴雪昌王品美郑道琼陶香林刘天喆
Owner ZHEJIANG UNIV
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