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Method for constructing industrial saccharomyces cerevisiae engineering bacteria by integrating metabolism and rearrangement of gene

A technology of Saccharomyces cerevisiae and metabolic engineering, applied in the direction of microorganism-based methods, biochemical equipment and methods, fungi, etc., can solve the problems of difficult to obtain excellent strains with comprehensive performance, degradation and decay of key properties of production strains, and achieve reduction Energy consumption, high alcohol resistance, and low by-product effects

Inactive Publication Date: 2013-05-22
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 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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  • Method for constructing industrial saccharomyces cerevisiae engineering bacteria by integrating metabolism and rearrangement of gene
  • Method for constructing industrial saccharomyces cerevisiae engineering bacteria by integrating metabolism and rearrangement of gene
  • Method for constructing industrial saccharomyces cerevisiae engineering bacteria by integrating metabolism and rearrangement of gene

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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 ascospores were observed under a microscope, the bacterial cells were collected, washed twice with normal saline, and then added with 700 μL Tris-HCl (pH8.0, 0.01 mol / L), 200 μL 100 mg / mL helicase solution and 100 μL 0.1 mol / L mercaptoethanol, Incubate at 30°C for 16 hours at 120r / min 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 strain to be tested and the...

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. 1% (v / v) EMS mutagen was used to treat genetically engineered haploid strains YFG1 (MAT a, fps1Δ::PGKp-gapN) and YFG2 (MATα, fps1Δ::PGKp-gapN) for 30-120 minutes, Add 5% (w / v) sodium thiosulfate to remove EMS contamination every 30 minutes, collect bacteria by centrifugation at 4000rpm for 5 minutes, wash twice with normal saline, and apply gradient dilution to a YPD plate containing 8% (v / v) ethanol, 30 Cultivate for 3 days at ℃, and pick a vigorously growing single colony;

[0040] 2. Under the ultraviolet lamp with a distance of 40cm and a power of 15w, the genetically engineered haploid strains YFG1 (MAT a, fps1Δ::PGKp-gapN) and YFG2 (MATα, fps1Δ::PGKp-gapN) were subjected to ultraviolet mutagenesis , Sampling every 1min, 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] The starting strain Z87, the control strain Y12, the genetically engineered strain YFG12, and the genetically engineered rearrangement FG1 were cultured in YPD liquid medium containing 0% and 10% (v / v) ethanol at 30°C, and samples were taken at different times to determine the dry weight of the strains. 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 growth period was extended to about 10 hours, and the genetic engineering rearrangement FG1 grew the fastest, and the maximum specific growth rate was 11.4% higher than tha...

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Abstract

The invention provides a method for constructing industrial saccharomyces cerevisiae engineering bacteria by integrating metabolism and rearrangement of gene and further provides an excellent industrial saccharomyces cerevisiae engineering bacterial strain obtained by the method disclosed by the invention and provided with the advantages of low by-product and high alcohol resistance as well as an application of the excellent industrial saccharomyces cerevisiae engineering bacterial strain. Due to the adoption of the method disclosed by the invention, a plurality of production properties such as the sugar-alcohol conversion rate, the resistance and fermentation rate of the saccharomyces cerevisiae bacterial strain can be improved; the improved bacterial strain can be applied to the fermentation and the production of industrial thick mash alcohol, so that the energy consumption is reduced and the production cost is reduced; and the method disclosed by the invention can be used for improving properties of other industrial microorganisms.

Description

[0001] This application is a divisional application for an invention patent with application number 201110293244.X and titled "Industrial Saccharomyces cerevisiae Strain with Low Glycerol Synthesis and High Alcohol Resistance and Its Application". (1) Technical field [0002] The invention relates to a method for constructing industrial Saccharomyces cerevisiae engineering bacteria by integrating gene metabolism engineering and whole genome rearrangement. (2) Background technology [0003] 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; the general distinction is as follows: starchy raw material...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12R1/865C12R1/46
Inventor 吴雪昌王品美郑道琼陶香林刘天喆
Owner ZHEJIANG UNIV
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