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Method for constructing yeast strain by protoplast fusion using heat-inactivated parents

A protoplast fusion and protoplast technology, applied in the direction of hybrid cell preparation, etc., can solve the problems of interfering with the normal metabolism of bacterial strains, tedious work, and affecting important traits of bacterial strains, so as to facilitate process control and management, improve economic benefits, and reduce equipment input effect

Inactive Publication Date: 2010-11-17
SICHUAN UNIV
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Problems solved by technology

[0005] Protoplast fusion breeding has been widely carried out in yeast strains. Traditional methods use drug resistance markers or auxotrophic markers to mark parent strains in order to detect fusion sub-strains. The work is quite cumbersome, and these genetic markers often interfere with normal strains Metabolism affects some important traits of strains [Liu Ling, Ye Bo, Liu Changjiang. Screening of drug resistance markers of protoplast fusion parent strains [J]. Jiangsu Agricultural Science, 2007(3): 225-227.]; [Tan Jiujiu, Tan Hong et al. Protoplast fusion between Chaetomium species using antibiotic resistance as a selection marker [J]. Journal of East China University of Science and Technology (Natural Science Edition), 2010, 36(1): 36-41.]; and [B.S. Gunashree, G.Venkateswaran.Enhanced phytaseproduction through interspecific protoplast fusion of Aspergillus niger CFR 335 andAspergillus ficuum SGA 01 auxotrophic mutants[J].Enzyme and Microbial Technology 46(2010)562-567.]

Method used

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  • Method for constructing yeast strain by protoplast fusion using heat-inactivated parents
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  • Method for constructing yeast strain by protoplast fusion using heat-inactivated parents

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Experimental program
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Effect test

Embodiment 1

[0039] (1) Preparation of two parental cells

[0040] Saccharomyces cerevisiae K211 strain and chromogenic yeast PY-1 strain were respectively inoculated on the slant of yeast extract peptone glucose agar medium, that is, YPD solid medium, and after 24 hours of growth, the yeast cells grown on the slant were scraped to obtain Cell concentration was 5 x 10 8 Bacterial suspension per mL, draw 200 μL of bacterial suspension, inoculate in a conical flask filled with 30 mL of YPD liquid medium sterilized at 123°C for 15 minutes, place in a constant temperature shaker at 30°C and 150r / min Cultivate for 18 hours to obtain two parental thallines;

[0041] (2) Preparation of two parental protoplasts

[0042] The two parental thalline culture solutions of step (1) are centrifuged and washed three times in a 4° C. refrigerated centrifuge with a concentration of 0.6 mol / L NaCl solution, and then the washed thalline cells are diluted to a concentration of 1 × 10 7 cells / mL, take 10mL of...

Embodiment 2

[0052] (1) Preparation of parental cells

[0053] Saccharomyces cerevisiae LII-E12 strain and chromogenic yeast PY-1 strain were respectively inoculated on the slant of YPD solid medium. After 18 hours of growth, the yeast cells grown on the slant were scraped to obtain a cell concentration of 5×10 7 Bacterial suspension per mL, draw 200 μL of bacterial suspension, inoculate in a conical flask filled with 30 mL of YPD liquid medium sterilized at 121 °C for 15 minutes, and cultivate in a constant temperature shaker at 30 °C and 150 r / min for 13 hours, the two parental thallines were obtained;

[0054] (2) Preparation of parental protoplasts

[0055] The two parental thalline culture fluids of step (1) are centrifuged and washed three times in a refrigerated centrifuge at 4°C with a concentration of 0.6mol / L NaCl, and then the washed thalline cells are diluted to a concentration of 1 × 10 6 cells / mL, take 10mL of the diluted bacterial suspension, centrifuge at 2200r / min in a r...

Embodiment 3

[0065] (1) Preparation of parental cells

[0066] Saccharomyces cerevisiae K211 strain and chromogenic yeast PY-1 strain were inoculated on the slant of YPD solid medium, and after 20 hours of growth, the yeast cells grown on the slant were scraped to obtain a cell concentration of 1×10 8 Bacterial suspension per mL, draw 200 μL of bacterial suspension, inoculate in a conical flask filled with 30 mL of YPD liquid medium sterilized at 118°C for 20 minutes, and cultivate in a constant temperature shaker at 32°C and 150 r / min for 15 hours, the two parental thallines were obtained;

[0067] (2) Preparation of parental protoplasts

[0068] The two parental thalline culture fluids of step (1) are centrifuged and washed three times in a refrigerated centrifuge at 4°C with a concentration of 0.6mol / L NaCl, and then the washed thalline cells are diluted to a concentration of 5 × 10 6 cells / mL, take 10mL of the diluted bacterial suspension, centrifuge at 2200r / min in a refrigerated ce...

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Abstract

The invention discloses a method for constructing a yeast strain by protoplast fusion using heat-inactivated parents, belonging to the strain preparation biotechnical field. In the method, by utilizing the species difference between chromogenic yeast and liquor-making yeast, a parents-protoplast fuses and regenerates to obtain chromogenic and liquor-making fusion sub-strain from different parents by protoplast fusion using the heat-inactivated parents. The method comprises the following process steps: preparing two parent thalli and two parent protoplasts, fusing the two parent protoplasts; and detecting and selecting a fusant, and testing the fermenting property of the fusant and re-screening. The method of the invention has simple operation, easy realization of process and high breeding success rate; and the obtained yeast fusion strain has positive significance for theoretically studying metabolic control during the alcoholic fermentation and pigment accumulation processes and improving the management control efficiency of alcoholic fermentation production and provides a new way for strain breeding in alcohol industry and pigment production.

Description

technical field [0001] The invention relates to a protoplast fusion technology, in particular to a method for constructing a yeast strain capable of both alcohol fermentation and pigment accumulation through double heat inactivation of parents and the protoplast fusion technology, and belongs to the field of biotechnology bacteria production. Background technique [0002] Alcohol is an important organic solvent and fuel. Today, with the rapid development of the world economy and the deepening of the energy crisis, alcohol as a renewable energy has received more and more attention. At present, the production of fuel alcohol, especially the production of fuel alcohol using cellulose hydrolyzate as raw material, has become a worldwide research hotspot. [0003] At the same time, some yeast cells have a certain ability to accumulate pigments, such as the accumulation of carotenoids and flavonoid pigments. However, in most cases, the pigment content of these yeast cells is low,...

Claims

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Application Information

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IPC IPC(8): C12N15/04
Inventor 张文学王燕吴正云王蓉郭泓辰
Owner SICHUAN UNIV
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