Saccharomyces cerevisiae engineering bacterium for highly yielding medium-chain fatty acid ethyl ester as well as construction method thereof

A fatty acid ethyl ester and yeast engineering technology, applied in the field of bioengineering, can solve the problem of low ester production capacity of Saccharomyces cerevisiae

Active Publication Date: 2014-02-12
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the problem that Saccharomyces cerevisiae has lower self-ester-producing ability, a...

Method used

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  • Saccharomyces cerevisiae engineering bacterium for highly yielding medium-chain fatty acid ethyl ester as well as construction method thereof
  • Saccharomyces cerevisiae engineering bacterium for highly yielding medium-chain fatty acid ethyl ester as well as construction method thereof
  • Saccharomyces cerevisiae engineering bacterium for highly yielding medium-chain fatty acid ethyl ester as well as construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Construction of a genetically engineered strain of Saccharomyces cerevisiae with high yield of ethyl caproate

[0034] (1) Construction of genetically engineered strains

[0035] 1) Construction of pUC-APEKB plasmid

[0036] The homologous recombination plasmid pUC-APEKB is constructed with pUC-19 as the basic plasmid, and the construction process is as follows figure 1 As shown, using the haploid a8 or α5 of AY15 as a template, PCR amplification yields a 411 bp upstream homology arm FA and a 409 bp downstream homology arm FB, respectively, through EcoRI / KpnI and PstI / HindIII Double enzyme digestion and ligation into pUC-19 to obtain plasmid pUC-FAB. Using the haploid a8 or α5 of AY15 as the template, the 1356bp alcohol hexanoyltransferase gene EHT1 was obtained by PCR amplification, which was inserted into the promoter PGK1p and terminator PGK1 on the pPGK1 plasmid by XhoI single enzyme digestion T In between, the plasmid pPGK1-E was obtained; using the pPGK1-E ...

Embodiment 2

[0057] Example 2: Simulated corn raw material liquid liquor fermentation experiment

[0058] 1) Fermentation process route:

[0059] Corn flour → soaking → liquefaction → saccharification → cooling → inoculation → fermentation → steaming wine → measurement index

[0060] 2) Process conditions: soaking conditions: 60~70℃, immersion for 20min; liquefaction conditions: 85~90℃, adding high temperature resistant α-amylase, liquefaction for 90min; saccharification conditions: 55~60℃, adding glucoamylase, saccharification for 20min fermentation Conditions: 30°C, 15 days. When steaming wine, take 100mL mash, add 100mL water, and steam 100mL wine sample.

[0061] 3) Ingredients: corn flour: 60g; add water 180mL; high temperature resistant α-amylase: 30μL; glucoamylase: 90μL; acid protease: 1.2mL; nutrient salt: 1mL; inoculation amount: 7.5%;

[0062] According to the above-mentioned simulation process, the fermentation experiment of corn raw material liquid liquor was carried out on the engine...

Embodiment 3

[0070] Example 3: Solid-state Daqu liquor fermentation experiment

[0071] 1) Fermentation process route:

[0072] Sorghum → soaking → steaming → spreading → mixing koji → culture and saccharification for 24 hours → inoculation → fermentation → distillation

[0073] 2) Process conditions: soaking conditions: 95~98℃, fully absorbing water without hard cores; cooking conditions: steaming at normal pressure for about 30 minutes, uniform particles and no white cores. Fermentation conditions: 30°C, 15 days. Steaming conditions: 100g distillers grains, add 100mL water, steam 100mL wine sample.

[0074] 3) Ingredients: 50g sorghum; 5g Daqu; inoculation amount: 5%;

[0075] Perform solid-state Daqu liquor fermentation experiments on Saccharomyces cerevisiae engineering strains a8-1, α5-1, EY15 and starting strains a8, α5, and AY15 according to the above-mentioned simulation process; shake and weigh every 12 hours during the fermentation, and record the weight loss; after the fermentation is o...

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Abstract

The invention provides a saccharomyces cerevisiae engineering bacterium for highly yielding medium-chain fatty acid ethyl ester. The saccharomyces cerevisiae engineering bacterium is realized by selecting a strong promoter PGK1 (Phosphoglycerate kinase 1) for overexpression coding of an EHT1 (Ethanol Hexanoyl Transferase 1) gene of alcohol acyltransferase and knocking out a gene FFA1 (Free Fatty Acid Receptor 1) of an exogenous fatty acid activating enzyme. The preservation number is CGMCC (China General Microbiological Culture Collection Center) No.7937. Under the condition that other fermenting properties are not affected, compared with a parent bacterial strain, the content of ethyl hexanoate can be improved to 2.23mg/L after simulating fermentation of corn raw material liquid white spirit for 15 days, wherein the content is 2.75 times the original bacteria. The contents of ethyl caprylate and ethyl caprate are respectively improved by 52% and 62%. After fermentation for 30 days, the contents of ethyl hexanoate, ethyl caprylate and ethyl caprate are respectively improved by 120%, 16.2% and 16.7%. After simulating fermentation of corn raw material liquid white spirit for 15 days, the content of ethyl hexanoate can be improved to 2.83mg/L which is 2.8 times the original bacteria, and the contents of ethyl caprylate and ethyl caprate are respectively improved by 43.3% and 40.9%.

Description

Technical field [0001] The invention belongs to the technical field of bioengineering, and relates to the breeding of industrial microorganisms, in particular to an engineered Saccharomyces cerevisiae with high yield of medium-chain fatty acid ethyl esters and a construction method thereof. Background technique [0002] Ester aroma substances are the main flavor substances in beverage wines and the main carriers of wine aroma. Increasing the content of ester aroma substances in wine can enhance the flavor of alcoholic beverages and improve the quality of beverage wines. Fatty acid ethyl esters, mainly ethyl caproate, are the main aroma of luzhou-flavor liquors and give important ester aromas (fruit aromas) to beverage wines. Domestic ordinary liquor and rice wine are mainly fermented by pure-bred Saccharomyces cerevisiae, which is characterized by short fermentation period and high yield of raw materials. However, due to the extremely low ability of Saccharomyces cerevisiae to pr...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12R1/865
Inventor 陈叶福肖冬光李锋郭学武张翠英董健杜丽平
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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