Saccharomyces cerevisiae strain producing high-grade alcohols at low yield and built through regulating and controlling cell wall constitutive proteins

A technology of Saccharomyces cerevisiae and higher alcohols, which is applied in the field of bioengineering and can solve the problems of synthesizing too many higher alcohols

Inactive Publication Date: 2019-12-10
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to solve the problem that Saccharomyces cerevisiae synthesizes too much higher alcohol in beer production, and construct a Saccharomyces cerevisiae strain with low yield of higher alcohol by regulating the synthesis of Saccharomyces cerevisiae cell wall constituent proteins

Method used

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  • Saccharomyces cerevisiae strain producing high-grade alcohols at low yield and built through regulating and controlling cell wall constitutive proteins
  • Saccharomyces cerevisiae strain producing high-grade alcohols at low yield and built through regulating and controlling cell wall constitutive proteins
  • Saccharomyces cerevisiae strain producing high-grade alcohols at low yield and built through regulating and controlling cell wall constitutive proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1: Construction of Saccharomyces cerevisiae strain with single knockout of TIR1 gene

[0064] The main construction process of the strain is as follows:

[0065] 1) Amplification of the fragment required to knock out one allele of TIR1

[0066] Using the S17 genome of Saccharomyces cerevisiae as a template, T1A-F and T1A-R were used as primer pairs to PCR amplify the upstream homologous sequence fragment required for TIR1 gene knockout, with a length of 662bp; using the S17 genome of Saccharomyces cerevisiae as a template, T1B-F and T1B-R as the primer pair PCR to amplify the downstream homologous sequence fragment with a length of 566bp; using the plasmid pUC6 genome as the template, T1K-F and T1K-R as the primer pair PCR amplification to amplify the loxP required for TIR1 gene knockout -KanMX3-loxP fragment, 1663bp in length, such as figure 1 ).

[0067] 2) Construction of a recombinant yeast strain that knocks out one allele of TIR11

[0068] After the am...

Embodiment 2

[0075] Example 2: Construction of Saccharomyces cerevisiae strains with double knockout of TIR3 gene

[0076] The main construction process of the strain is as follows:

[0077] 1) Amplification of the fragment required to knock out one allele of TIR3

[0078] Using the S17 genome of Saccharomyces cerevisiae as a template, T3A-F and T3A-R were used as primer pairs to PCR amplify the upstream homologous sequence fragment required for TIR3 gene knockout, with a length of 496bp; using the S17 genome of Saccharomyces cerevisiae as a template, T3B-F and T3B-R as primers to PCR amplify the downstream homologous sequence fragment with a length of 676bp; using the plasmid pUC6 genome as a template, T3K-F and T3K-R as primers to PCR amplify loxP required for TIR3 gene knockout -KanMX3-loxP fragment, length is 1663bp ( Figure 5 ).

[0079] 2) Construction of a recombinant yeast strain that knocks out one allele of TIR13

[0080] After the three amplified fragments were purified and...

Embodiment 3

[0098] Example 3: Wheat beer fermentation experiments of recombinant strains S17-Δtir1-k-p and S17-DΔtir3-k-p with low production of higher alcohols

[0099] 1) The wheat beer fermentation process route is as follows: Figure 14 Shown:

[0100] 2) Saccharification process: Weigh the crushed wheat malt and add it to warm water at 30°C with a material-to-water ratio of 1:4. After stirring well, place it in a constant temperature water bath, keep it at 30°C for 30 minutes, and raise the temperature at 2.0°C / min. to 65°C, keep for 90min, then rapidly raise the temperature to 78°C, and keep for 10min. Fully stir once every 5 minutes during the saccharification process. The saccharified wheat wort is filtered while hot, and washed with hot water at 75°C for 3 times. Place the filtrate on an induction cooker for steaming, add 3‰ of bitter hops (calculated by malt weight) 40 minutes after boiling, and boil for 70 minutes. Naturally cool to room temperature after boiling, centrifug...

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Abstract

The invention belongs to the technical field of biological engineering, and relates to breeding of industrial microorganisms, in particular to a saccharomyces cerevisiae strain which is synthesized and built through regulating and controlling cell wall constitutive proteins and produces high-grade alcohols at a low yield and application of the saccharomyces cerevisiae strain. In order to solve theproblem that excessive high-grade alcohols are synthesized by saccharomyces cerevisiae during production of beer, through regulating and controlling synthesis of the cell wall constitutive proteins,the saccharomyces cerevisiae strain producing the high-grade alcohols at the low yield is built; and under the premise of keeping good fermentation performance, the effect of producing the high-gradealcohols at the low yield is achieved, and a theoretical basis is laid for brewing wheat beer having a good flavor and a unique taste.

Description

Technical field: [0001] The invention belongs to the technical field of bioengineering and relates to the breeding of industrial microorganisms, in particular to a Saccharomyces cerevisiae strain with low production of higher alcohols constructed by regulating the synthesis of cell wall constituent proteins and its application. Background technique: [0002] Higher alcohol is one of the main flavor metabolites produced by Saccharomyces cerevisiae during beer brewing. Higher alcohol, commonly known as fusel oil, refers to a general term for a class of alcohols with 3 or more carbon chain skeletons, and is one of the important chemical substances that form the flavor and taste of beer. Higher alcohols in beer mainly include n-propanol, n-butanol, 2-methyl-1-propanol (isobutanol), 3-methyl-1-butanol (isoamyl alcohol), 2-methanol Base-1-butanol (active pentanol), 2-phenylethanol, tryptophan, p-hydroxyphenylethanol (tyrosol), etc. An appropriate amount of higher alcohol can mak...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12C12/00C12R1/865
CPCC07K14/395C12C12/006C12N15/81
Inventor 肖冬光孙中贯胡智慧张翠英陈叶福杜丽平郭学武
Owner TIANJIN UNIV OF SCI & TECH
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