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

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

Active Publication Date: 2015-06-10
TIANJIN UNIV OF SCI & TECH
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  • 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, and provide a kind of Saccharomyces cerevisiae engineering strain and its construction method of high-yielding medium-chain fatty acid ethyl ester

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] Embodiment 1: Construction of high-yield ethyl caproate Saccharomyces cerevisiae genetically engineered bacteria

[0034] (1) Construction of genetic engineering strains

[0035] 1) Construction of pUC-APEKB plasmid

[0036] Using pUC-19 as the base plasmid to construct the homologous recombination plasmid pUC-APEKB, the construction process is as follows: figure 1 As shown, the 411bp upstream homology arm FA and the 409bp downstream homology arm FB were amplified by PCR using the haploid a8 or α5 of AY15 as a template, which were passed through EcoRI / KpnI and PstI / Hind III Double digestion and ligation into pUC-19 to obtain plasmid pUC-FAB. The 1356bp alcohol hexanoyltransferase gene EHT1 was amplified by PCR using the haploid a8 or α5 of AY15 as a template, and the promoter PGK1p and terminator PGK1 were inserted into the pPGK1 plasmid by XhoI single enzyme digestion T Between, the plasmid pPGK1-E was obtained; using the pPGK1-E plasmid as a template, PCR amplifi...

Embodiment 2

[0057] Embodiment 2: Fermentation experiment of simulated corn raw material liquid liquor

[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°C, immersion for 20 minutes; liquefaction conditions: 85-90°C, add high-temperature-resistant α-amylase, liquefy for 90 minutes; saccharification conditions: 55-60°C, add glucoamylase, saccharify for 20 minutes and ferment 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;

[0062] According to the above simulation process, the Saccharomyces cerevisiae engineering bacteria a8-1, α5-1, EY15 and the starting strains a8, α5, AY15 were respectively subj...

Embodiment 3

[0070] Embodiment 3: Fermentation experiment of solid-state Daqu liquor

[0071] 1) Fermentation process route:

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

[0073] 2) Process conditions: soaking conditions: 95-98°C, fully absorb water without hard core; steaming conditions: steam at normal pressure for about 30 minutes, uniform particles, no white inside. Fermentation conditions: 30°C, 15 days. Wine steaming conditions: 100g distiller's grains, add 100mL water, distill 100mL wine sample.

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

[0075] According to the above simulation process, the Saccharomyces cerevisiae engineering bacteria a8-1, α5-1, EY15 and the starting strains a8, α5, AY15 were respectively subjected to solid-state Daqu liquor fermentation experiments; during the fermentation period, oscillate and weigh every 12 hours, and record the weight loss; a...

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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 a Saccharomyces cerevisiae engineering bacteria with high production of medium-chain fatty acid ethyl ester and a construction method thereof. Background technique [0002] Ester aroma substances are the main flavor substances in drinking wine and the main carrier of wine aroma. Increasing the content of ester aroma substances in wine can enhance the flavor of wine and improve the quality of drinking wine. Fatty acid ethyl esters, mainly ethyl caproate, are the main aroma of Luzhou-flavor liquor, which endows the drinking wine with an important ester aroma (fruit aroma). Domestic ordinary liquor and rice wine are mainly fermented by purebred Saccharomyces cerevisiae, which is characterized by a short fermentation cycle and a high yield of raw materials. However, due to the extremely low ability of Saccharomyces cerev...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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 UNIV OF SCI & TECH
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