Downregulation of EHT1 and EEB1 in Saccharomyces cerevisiae Alters the Ester Profile of Wine during Fermentation

EHT1 and EEB1 are the key Saccharomyces cerevisiae genes involved in the synthesis of ethyl esters during wine fermentation. We constructed single (Δeht1, Δeeb1) and double (Δeht1Δeeb1) heterogenous mutant strains of the industrial diploid wine yeast EC1118 by disrupting one allele of EHT1 and/or EEB1. In addition, the aromatic profile of wine produced during fermentation of simulated grape juice by these mutant strains was also analyzed. The expression levels of EHT1 and/or EEB1 in the relevant mutants were less than 50% of the wild-type strain when grown in YPD medium and simulated grape juice medium. Compared to the wild-type strain, all mutants produced lower amounts of ethyl esters in the fermented grape juice and also resulted in distinct ethyl ester profiles. ATF2, a gene involved in acetate ester synthesis, was expressed at higher levels in the EEB1 downregulation mutants compared to the wild-type and Δeht1 strains during fermentation, which was consistent with the content of acetate esters. In addition, the production of higher alcohols was also markedly affected by the decrease in EEB1 levels. Compared to EHT1, EEB1 downregulation had a greater impact on the production of acetate esters and higher alcohols, suggesting that controlling EEB1 expression could be an effective means to regulate the content of these aromatic metabolites in wine. Taken together, the synthesis of ethyl esters can be decreased by deleting one allele of EHT1 and EEB1 in the diploid EC1118 strain, which may modify the ester profile of wine more subtly compared to the complete deletion of target genes.

[1]  G. Yan,et al.  Comparing the Effects of Different Unsaturated Fatty Acids on Fermentation Performance of Saccharomyces cerevisiae and Aroma Compounds during Red Wine Fermentation , 2019, Molecules.

[2]  J. Thevelein,et al.  Polygenic Analysis in Absence of Major Effector ATF1 Unveils Novel Components in Yeast Flavor Ester Biosynthesis , 2018, mBio.

[3]  J. Legras,et al.  QTL mapping of volatile compound production in Saccharomyces cerevisiae during alcoholic fermentation , 2018, BMC Genomics.

[4]  Y. Tao,et al.  Increase of medium-chain fatty acid ethyl ester content in mixed H. uvarum/S. cerevisiae fermentation leads to wine fruity aroma enhancement. , 2018, Food chemistry.

[5]  R. Callejón,et al.  Monitoring volatile compounds production throughout fermentation by Saccharomyces and non-Saccharomyces strains using headspace sorptive extraction , 2017, Journal of Food Science and Technology.

[6]  C. Powell,et al.  Production of medium-chain volatile flavour esters in Pichia pastoris whole-cell biocatalysts with extracellular expression of Saccharomyces cerevisiae acyl-CoA:ethanol O-acyltransferase Eht1 or Eeb1 , 2015, SpringerPlus.

[7]  G. Yan,et al.  Effects of Adding Unsaturated Fatty Acids on Fatty Acid Composition of Saccharomyces cerevisiae and Major Volatile Compounds in Wine , 2015 .

[8]  D. Xiao,et al.  Enhanced ethyl caproate production of Chinese liquor yeast by overexpressing EHT1 with deleted FAA1 , 2014, Journal of Industrial Microbiology & Biotechnology.

[9]  C. Varela,et al.  Effect of oxygen and lipid supplementation on the volatile composition of chemically defined medium and Chardonnay wine fermented with Saccharomyces cerevisiae. , 2012, Food chemistry.

[10]  T. Becker,et al.  Function and regulation of yeast genes involved in higher alcohol and ester metabolism during beverage fermentation , 2011 .

[11]  Florian F. Bauer,et al.  Wine flavor and aroma , 2011, Journal of Industrial Microbiology & Biotechnology.

[12]  H. Heymann,et al.  Influence of yeast strain, canopy management, and site on the volatile composition and sensory attributes of cabernet sauvignon wines from Western Australia. , 2011, Journal of agricultural and food chemistry.

[13]  P. Grbin,et al.  Microbial modulation of aromatic esters in wine: Current knowledge and future prospects , 2010 .

[14]  L. Bisson,et al.  Genetics of yeast impacting wine quality. , 2010, Annual review of food science and technology.

[15]  K. Verstrepen,et al.  Production and biological function of volatile esters in Saccharomyces cerevisiae , 2010, Microbial biotechnology.

[16]  E. Matallana,et al.  Btn2p is involved in ethanol tolerance and biofilm formation in flor yeast. , 2008, FEMS yeast research.

[17]  J. Pronk,et al.  The Ehrlich Pathway for Fusel Alcohol Production: a Century of Research on Saccharomyces cerevisiae Metabolism , 2008, Applied and Environmental Microbiology.

[18]  K. Verstrepen,et al.  Parameters Affecting Ethyl Ester Production by Saccharomyces cerevisiae during Fermentation , 2007, Applied and Environmental Microbiology.

[19]  I. S. Pretorius,et al.  The effect of increased yeast alcohol acetyltransferase and esterase activity on the flavour profiles of wine and distillates , 2006, Yeast.

[20]  Arnout R. D. Voet,et al.  The Saccharomyces cerevisiae EHT1 and EEB1 Genes Encode Novel Enzymes with Medium-chain Fatty Acid Ethyl Ester Synthesis and Hydrolysis Capacity* , 2006, Journal of Biological Chemistry.

[21]  Eveline J. Bartowsky,et al.  Yeast and bacterial modulation of wine aroma and flavour , 2005 .

[22]  K. Verstrepen,et al.  Expression Levels of the Yeast Alcohol Acetyltransferase Genes ATF1, Lg-ATF1, and ATF2 Control the Formation of a Broad Range of Volatile Esters , 2003, Applied and Environmental Microbiology.

[23]  H. Mizoguchi,et al.  Increased ethyl caproate production by inositol limitation in Saccharomyces cerevisiae. , 2003, Journal of bioscience and bioengineering.

[24]  A. Debourg,et al.  Involvement of branched-chain amino acid aminotransferases in the production of fusel alcohols during fermentation in yeast , 2001, Applied Microbiology and Biotechnology.

[25]  P. J. Trotter,et al.  The genetics of fatty acid metabolism in Saccharomyces cerevisiae. , 2001, Annual review of nutrition.

[26]  J. Dufour,et al.  Alcohol acetyltransferases and the significance of ester synthesis in yeast , 2000, Yeast.

[27]  A. Iwamatsu,et al.  Cloning and nucleotide sequence of the alcohol acetyltransferase II gene (ATF2) from Saccharomyces cerevisiae Kyokai No. 7. , 1998, Bioscience, biotechnology, and biochemistry.

[28]  H. Yoshimoto,et al.  Effect of aeration and unsaturated fatty acids on expression of the Saccharomyces cerevisiae alcohol acetyltransferase gene , 1997, Applied and environmental microbiology.

[29]  Thomas Fiedler,et al.  A new efficient gene disruption cassette for repeated use in budding yeast , 1996, Nucleic Acids Res..

[30]  H. Yoshimoto,et al.  Acetate ester production by Saccharomyces cerevisiae lacking the ATF1 gene encoding the alcohol acetyltransferase , 1996 .

[31]  P. Henschke.  Yeasts-metabolism of nitrogen compounds , 1993 .

[32]  A. H. Rose,et al.  Plasma-membrane phospholipid unsaturation affects expression of the general amino-acid permease in Saccharomyces cerevisiae Y185. , 1985, Journal of general microbiology.

[33]  S. Wakil,et al.  Fatty acid synthesis and its regulation. , 1983, Annual review of biochemistry.

[34]  Thomas D. Schmittgen,et al.  Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2 2 DD C T Method , 2022 .