New method for producing high-yield ethyl lactate by using saccharomyces cerevisiae strain and application of strain

A Saccharomyces cerevisiae strain and technology of Saccharomyces cerevisiae are applied in the field of bioengineering to achieve the effect of increasing yield

Active Publication Date: 2018-10-12
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no research on the transformation of ethyl lactate prod

Method used

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  • New method for producing high-yield ethyl lactate by using saccharomyces cerevisiae strain and application of strain
  • New method for producing high-yield ethyl lactate by using saccharomyces cerevisiae strain and application of strain
  • New method for producing high-yield ethyl lactate by using saccharomyces cerevisiae strain and application of strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Embodiment 1: Construction of high-yield ethyl lactate Saccharomyces cerevisiae strain

[0093] The starting strain used in this example is Saccharomyces cerevisiae CICC32315. The Escherichia coli DH5a was purchased from Takara Company. The YPD medium is a universal complete medium, and the solid medium contains 2% imported agar powder.

[0094] In this example, taking the propionyl-CoA transferase PctMt gene of M. acetate and the alcohol acyltransferase VAAT gene of strawberry as examples, the overexpression of the lactate dehydrogenase gene ldhL1 of Lactobacillus plantarum was constructed, and propionyl-CoA was heterologously expressed at the same time. Genetic engineering bacteria of transferase gene and alcohol acyltransferase gene.

[0095] The main construction process of the strain is as follows:

[0096] (1) Construction of Yep352-PK plasmid

[0097] The plasmid Yep352-PK was constructed using Yep352 as the base plasmid, and the construction process was as f...

Embodiment 2

[0149] Embodiment 2: Construction of different Pct and AAT gene combination strains

[0150] The present invention constructs recombinant strains comprising the combination of propionyl-CoA transferase and alcohol acyltransferase from various sources, which are the propionyl-CoA transferase gene PctCp of Clostridium propionicum and the gene of Alcaligenes eutropha H16 respectively. Propionyl-CoA-transferase gene PctRe, propionyl-CoA-transferase gene PctMe of M. erescherii, propionyl-CoA-transferase gene PctMt of M. thermoacetica; Saccharomyces cerevisiae ATF1, EHT1 and EEB1 genes, Strawberry SAAT gene, VAAT gene, muskmelon Mel2 gene and kiwifruit AeAT9 gene to obtain modified strains with different ethyl lactate production abilities;

[0151] Among them, the Protein_ID of the PctCp gene corresponding to propionyl-CoA transferase is CAB77207.1; the GeneID of PctRe: 4250134; the Protein_ID of the PctMe gene corresponding to propionyl-CoA transferase is BAU59368.1;

[0152] Gene...

Embodiment 3

[0160] Embodiment 3: Fermentation experiment of corn hydrolyzate of high-yield ethyl lactate strain

[0161] Fermentation experiments of corn hydrolyzate were carried out at the same time for the recombinant strain and the original strain respectively

[0162] 1) Fermentation process roadmap: corn flour→soaking→liquefaction→saccharification→cooling→filtration→inoculation→fermentation→steaming wine→measurement indicators;

[0163] 2) Process conditions

[0164] 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 hours;

[0165] 3) Ingredients: corn flour 1500g, water 4500mL, let stand for 20min, high temperature resistant α-amylase 2×10 4 U / mL, 0.9ml, glucoamylase 1×10 5 U / mL, 3mL.

[0166] 4) Culture medium configuration

[0167] Primary seed medium: 8°Brix corn hydrolyzate, add 0.5% yeas...

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Abstract

The invention belongs to the technical field of bioengineering, and particularly discloses a new method for producing high-yield ethyl lactate by using a saccharomyces cerevisiae strain and application of the strain. High-yield ethyl lactate is produced through the simultaneous heterologous expression of lactate dehydrogenase, propionyl-CoA transferase and alcohol acyltransferase in saccharomycescerevisiae. Pyruvate decarboxylase is completely knocked out from an original strain, and lactate dehydrogenase is overexpressed to obtain the yeast strain with a certain capability of generating L-lactic acid; a strong promoter TEF1 is selected and adopted for overexpressing propionyl-CoA transferase, a strong promoter PGK1 is selected and adopted for overexpressing alcohol acyltransferase, witha homothallic gene HO as an integration site, a copy number of propionyl-CoA transferase is increased, the saccharomyces cerevisiae strain capable of significantly increasing the yield of ethyl lactate is obtained, and the yield of ethyl lactate obtained by using the saccharomyces cerevisiae strain reaches 353.1 mg/L; compared with the original strain, the yield of isoamyl alcohol is decreased by49.9%, the yield of phenylethyl alcohol is decreased by 59.2%, and the new method meets the higher requirements of yeast in the related field of liquor and has broad application prospects.

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 new method for high-yielding ethyl lactate of Saccharomyces cerevisiae strains and its application. Background technique: [0002] Ester aroma substances are the main flavor substances in liquor. The higher ester content not only gives it an important ester aroma, but also can effectively expand and relax nerves and reduce the side effects caused by drinking. Ethyl lactate is an important aroma substance of all flavor Chinese liquors, and an appropriate content of ethyl lactate is crucial to the flavor quality of Chinese liquors. Ethyl lactate is second only to ethyl acetate in the ester compounds of Fen-flavor liquor, and the content and ratio of ethyl lactate and ethyl acetate have a great influence on the flavor of liquor. For Laobaigan-flavored liquor and rice-flavored liquor, the content of ethyl lactate is...

Claims

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Application Information

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IPC IPC(8): C12P7/62C12N1/19C12N15/81C12R1/865
CPCC12N9/0006C12N9/1029C12N15/81C12P7/62C12Y101/01027C12Y101/01028
Inventor 陈叶福刘港李洁任津莹肖冬光郭学武
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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