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A new way of high-yielding ethyl lactate produced by Saccharomyces cerevisiae and its application

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: 2019-08-13
TIANJIN UNIV OF SCI & TECH
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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 production on the basis of lactic acid production in Saccharomyces cerevisiae

Method used

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  • A new way of high-yielding ethyl lactate produced by Saccharomyces cerevisiae and its application
  • A new way of high-yielding ethyl lactate produced by Saccharomyces cerevisiae and its application
  • A new way of high-yielding ethyl lactate produced by Saccharomyces cerevisiae and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Example 1: Construction of Saccharomyces cerevisiae strain with high yield of ethyl lactate

[0098] The starting strain used in this example is Saccharomyces cerevisiae CICC32315. The E. coli DH5a was purchased from Takara Company. The YPD medium is a universal complete medium, and the solid medium contains 2% imported agar powder.

[0099] In this example, taking the propionyl CoA transferase PctMt gene of Acemulia and the alcohol acyl transferase VAAT gene of strawberry as examples, the overexpression of the lactate dehydrogenase gene ldhL1 of Lactobacillus plantarum was constructed, and the propionyl CoA was expressed heterologously. Genetically engineered bacteria with transferase gene and alcohol acyltransferase gene.

[0100] The main construction process of the strain is as follows:

[0101] (1) Construction of Yep352-PK plasmid

[0102] The Yep352-based plasmid is used to construct the plasmid Yep352-PK. The construction process is as follows figure 1 Shown. Using th...

Embodiment 2

[0154] Example 2: Construction of strains with different combinations of Pct and AAT genes

[0155] The present invention constructs a recombinant strain containing a combination of propionyl-CoA transferase and alcohol acyltransferase from various sources, which are respectively the propionyl-CoA transferase gene PctCp of Clostridium propionate and Alcaligenes eutropha H16 Propionyl-CoA transferase gene PctRe, Propionyl-CoA transferase gene PctMe of Megacoccus escherichia, Propionyl-CoA transferase gene PctMt of M. thermoacetica; Saccharomyces cerevisiae ATF1, EHT1 and EEB1 genes, strawberry SAAT Gene, VAAT gene, Mel2 gene of melon and AeAT9 gene of kiwi fruit to obtain modified strains with different ethyl lactate production capacity;

[0156] 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;

[0157] The Gene...

Embodiment 3

[0165] Example 3: Corn hydrolysate fermentation experiment of high-yield ethyl lactate strain

[0166] Recombinant strains and starting strains were simultaneously subjected to corn hydrolysate fermentation experiment

[0167] 1) Fermentation process route map: corn flour → soaking → liquefaction → saccharification → cooling → filtration → inoculation → fermentation → steaming wine → determination of indicators;

[0168] 2) Process conditions

[0169] Soaking conditions: 60~70℃, immersion for 20min; liquefaction conditions: 85~90℃, adding high temperature resistant α-amylase, liquefying for 90min; saccharification conditions: 55~60℃, adding glucoamylase, saccharification for 20h;

[0170] 3) Ingredients: 1500g corn flour, 4500mL water, stand for 20min, high temperature resistant α-amylase 2×10 4 U / mL, 0.9ml, glucoamylase 1×10 5 U / mL, 3mL.

[0171] 4) Medium configuration

[0172] Primary seed culture medium: 8°Brix corn hydrolysate, add 0.5% yeast extract powder, dispense 5mL in test tube...

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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

[0001] Technical field: [0002] The invention belongs to the technical field of bioengineering, and relates to the breeding of industrial microorganisms, in particular to a new approach and application of a strain of Saccharomyces cerevisiae with high yield of ethyl lactate. [0003] Background technique: [0004] Ester aroma substances are the main flavor substances in liquor. The higher ester content not only imparts important ester aroma, but also effectively expands and relaxes the nerves and reduces the side effects caused by drinking. Ethyl lactate is an important aroma substance in all flavor Chinese liquors, and the appropriate ethyl lactate content is very important to the flavor quality of Chinese liquors. Ethyl lactate is second only to ethyl acetate in the ester compounds of fragrance-type liquor. The content and ratio of ethyl lactate and ethyl acetate have a great influence on the flavor of liquor. For Laobai dry-flavor liquor and rice-flavor liquor, the ethyl lactate...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/81C12P7/62C12R1/865
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 UNIV OF SCI & TECH
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