Construction Of Saccharomyces Cerevisiae Engineering Strain With High-producing Medium Chain Fatty Acid Ethyl Esters

Esters are important flavor compounds in alcoholic beverages.Although the content of these compounds are trace level,the presence of these compounds plays a key role in the formation of flavor in beverages especially the fruity aroma.Low esters contents would result in boring beer,and it's bad for the flavor coordination of beer.Fatty acid ethyl esters(FAEEs)are formed as secondary metabolites of Saccharomyces cerevisiae during fermentation,and the C6-C12 medium chain fatty acid(MCFA)ethyl esters have a great contribution to the flavor of beer and other beverages.Further research on the mechanism of medium chain fatty acidsis is conducive to control the content of medium chain fatty acid ethyl esters in beverages,then control the flavor of beer,which will have great economic potential.This study was performed from the perspective of the transformation of yeast strains,increasing the copy numbers of relative genes during esters formation to improve the production of medium chain fatty acid ethyl esters.The results showed that the minimal inhibitory concentration of G418 antibiotic to industrial Saccharomyces cerevisiae TT21 is 25 ?g/mL through antibiotics sensitivity experiment.In order to improve the screening efficiency of yeast transformants,YEPD plates were supplemented with 80?g/ml filter-sterilized G418 antibiotic.Recombinant plasmid pYPGE-ECK was constructed to study the influence of EEB1 gene overexpression onthe formation of MCFA ethyl esters,such as ethyl hexanoate and ethyl caprylate.Then EEB1 overexpression engineered strain TT21/pYPGE-ECK was verified by PCR analysis and 80 ?g/mL G418 resistance assay after the plasmid pYPGE-ECK was transformed into TT21 according to the electroporation method.After fermentation,the results showed that the production of ethyl hexanoate and ethyl hexanoate by the recombinat strain TT21/pYPGE-ECK were increased by 100.0 and 33.3%compared with the parental strain TT21.The content of Acyl-coenzyme A which as a precursor of fatty acid ester synthesis will greatly influence on the formation of the final fatty acid esters.In order to increase the content of Acyl-coenzyme A,ETR1 gene was selected for the research.Recombinat plasmid pYPGE-RCK was constructed for overexpression ETR1 gene and pYPGE-ECK-PR was constructed for overexpression both ETR1 and EEB1 gene.Recombinat strains TT21/pYPGE-RCK and TT21/pYPGE-ECK-PR were verified by PCR analysis and 80 ?g/mL G418 resistance assay after the plasmid pYPGE-RCK and pYPGE-ECK-PR were transformed into TT21 according to the electroporation method.After beer fermentation,the samples were analyzed by headspace gas chromatography(HS-GC).The results indicated that the production of ethyl hexanoate by the recombinant strain TT21/pYPGE-ECK and TT21/pYPGE-ECK-PR were all increased by 120%compared with the parental strain TT21 and theethyl hexanoate production increased by 80.0 and 60.0%,respectively.But there are no differences between recombinant strain TT21/pYPGE-RCK and parental strain TT21 for the production of ethyl hexanoate and ethyl hexanoate.Unexpectedly the overexpression of ETR1 gene did not increase the production of ethyl hexanoate and ethyl caprylate.