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Efficient traceless gene knockout method for saccharomyces cerevisiae and application thereof

A technology of Saccharomyces cerevisiae and gene knockout, which is applied in the field of genetic engineering, can solve the problems of peculiar smell of wine, lower probability of ideal transformants, influence on the flavor and quality of wine, etc., and achieves the effect of reducing isoamyl alcohol content and excellent flavor.

Active Publication Date: 2019-01-04
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2012, Dong et al. applied this method to realize the seamless knockout of yeast genes, but the probability of this method inducing the second-step homologous recombination is random, and more than one kind of transformant is produced, so that the ideal transformant is finally obtained less likely
However, if the content of higher alcohols is too high, the wine will produce strange smells, which will affect the flavor and quality of the wines. Moreover, the oxidation speed of higher alcohols in the human body is slower than that of ethanol, and its poisonous effect on the human body is much higher than that of ethanol.

Method used

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  • Efficient traceless gene knockout method for saccharomyces cerevisiae and application thereof
  • Efficient traceless gene knockout method for saccharomyces cerevisiae and application thereof
  • Efficient traceless gene knockout method for saccharomyces cerevisiae and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Construction of Saccharomyces cerevisiae for the seamless knockout of amino acid transaminase encoding gene BAT2

[0033] The starting strain used in this example is haploid α5 of CICC32315. (For the source of the fragment HERP1.0, see William G. Alexander, Drew T. Doering and Chris Todd Hittinger, High-Efficiency Genome Editing and Allele Replacement in Prototrophic and Wild Strains of Saccharomyces. Genetics, Vol. 198, 859-866 November 2014). The Escherichia coli DH5α was purchased from Takara Company. The YPD medium is a general complete medium; the components of the screening medium YPGly+AF are 5% glycerol, 2% peptone, 1% yeast extract powder, 200mg / mL aminomethylfolate, 5mg / mL sulfonamide, 5 μg / mL thymidine, 50 μg / mL hypoxanthine; the composition of the yeast synthetic medium (SC) is 2% glucose, 0.17% YNB, 0.5% ammonium sulfate, complete amino acid mixture solution, and the solid medium contains 2% imported agar powder.

[0034] According to the yeast genome da...

Embodiment 2

[0056] Determination of Growth Curves of Hα5(500)ΔH and α5 in Saccharomyces cerevisiae Strain Hα5(500) without Scarless BAT2

[0057] Pick a single colony of Hα5(500)ΔH and α5 and inoculate it in 50mL YEPD liquid medium, culture at 30°C with shaking at 180rpm for 24 hours, take the expanded culture and inoculate it into three bottles of 50mL YEPD medium according to the inoculum size of 1:100 , shake culture under the same conditions, and take the bacterial cell culture suspension 0.5 mL, centrifuge at 10000rpm for 1min, then resuspend the sludge with deionized water. Calibrate the zero point of the 7200 visible spectrophotometer with deionized water, and measure the OD of the bacterial solution colorimetrically at a wavelength of 600nm 600 value. The absorbance value (OD) of bacterial solution at each time point 600 ) is the ordinate, and taking the culture time as the abscissa, the growth curve of each bacterial strain is drawn, and the growth curve is as follows: Figur...

Embodiment 3

[0059] Corn mash fermentation experiments of Saccharomyces cerevisiae strains Hα5(500)ΔH and α5 without trace knockout of BAT2

[0060] The recombinant strain Hα5(500)ΔH and the starting strain α5 were respectively subjected to corn thick mash fermentation experiments, and the fermentation process roadmap is as follows:

[0061] Corn flour soaking→liquefaction→saccharification→fermentation with bacteria→weighing for weight loss→distilling alcohol→determination of fermentation indicators

[0062] Process conditions:

[0063] Soaking conditions: 60-70°C, soaking for 20 minutes; liquefaction conditions: 85-90°C, adding high-temperature resistant α-amylase, liquefying for 90 minutes; saccharification conditions, 55-60°C, adding glucoamylase, saccharifying for 20 minutes.

[0064] Ingredients: corn flour 60g, water 130mL, high temperature resistant α-amylase 2×10 4 U / mL, 30μL, glucoamylase 1×105 U / mL, 90μL, 7.5×10 2 U / mL acid protease 1.2mL: nutrient salt 1mL (MgSO 4 150g / L, KH...

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Abstract

The invention discloses an efficient traceless gene knockout method for saccharomyces cerevisiae. By optimizing the forward homologous sequence, galactose concentration and galactose induction time ina traceless gene knockout system, the probability of homologous recombination of the second step reaches 6.86*10<-4>. Haploids alpha 5 of saccharomyces cerevisiae AY15 serves as starting strains, BAT2 genes serves as the target gene, efficient and traceless knockout of wild saccharomyces cerevisiae genes BAT2 is achieved, through a liquor fermentation experiment, the contents of n-propanol, isobutanol and isoamyl alcohol of the modified strains are reduced by 20.32%, 47.85% and 23.14% respectively compared with these of parent strains, and the aim of low yield of higher alcohols is achieved.The method can be widely used in genetic modification of yeast and other microorganisms, because obtained mutants have no exogenous genes left and thus can be safely used in industrial production, anda useful reference is provided for gene knockout directly conducted on industrial strains.

Description

Technical field: [0001] The invention relates to the field of genetic engineering, in particular to constructing an efficient traceless gene knockout method in Saccharomyces cerevisiae and using the method to construct a Saccharomyces cerevisiae strain with low production of higher alcohols. Background technique: [0002] Saccharomyces cerevisiae is a typical eukaryotic cell model and an important model organism. Its characteristics include: fast growth and reproduction, short metabolic cycle, easy isolation and culture, etc. These characteristics make Saccharomyces cerevisiae more convenient for genetic engineering and genetics Research, known as the "E. coli" in eukaryotes. With the continuous development and renewal of molecular biology and genetic engineering technology, the breeding method of Saccharomyces cerevisiae has developed from the initial natural breeding to modern directional genetic engineering breeding. Gene knockout is a genetic engineering technology deve...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N1/19C12R1/865
CPCC07K14/395C12N15/81
Inventor 张翠英肖冬光李凭王建辉郭学武陈叶福董健杜丽平马立娟于爱群
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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