Identification of Novel Alleles Conferring Superior Production of Rose Flavor Phenylethyl Acetate Using Polygenic Analysis in Yeast.

Flavor compound metabolism is one of the last areas in metabolism where multiple genes encoding biosynthetic enzymes are still unknown. A major challenge is the involvement of side activities of enzymes having their main function in other areas of metabolism. We have applied pooled-segregant whole-genome sequence analysis to identify novel Saccharomyces cerevisiae genes affecting production of phenylethyl acetate (2-PEAc). This is a desirable flavor compound of major importance in alcoholic beverages imparting rose- and honey-like aromas, with production of high 2-PEAc levels considered a superior trait. Four quantitative trait loci (QTLs) responsible for high 2-PEAc production were identified, with two loci each showing linkage to the genomes of the BTC.1D and ER18 parents. The first two loci were investigated further. The causative genes were identified by reciprocal allele swapping into both parents using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9. The superior allele of the first major causative gene, FAS2, was dominant and contained two unique single nucleotide polymorphisms (SNPs) responsible for high 2-PEAc production that were not present in other sequenced yeast strains. FAS2 encodes the alpha subunit of the fatty acid synthetase complex. Surprisingly, the second causative gene was a mutant allele of TOR1, a gene involved in nitrogen regulation. Exchange of both superior alleles in the ER18 parent strain increased 2-PEAc production 70%, nearly to the same level as in the best superior segregant. Our results show that polygenic analysis combined with CRISPR/Cas9-mediated allele exchange is a powerful tool for identification of genes encoding missing metabolic enzymes and for development of industrial yeast strains generating novel flavor profiles in alcoholic beverages.IMPORTANCE Multiple reactions in flavor metabolism appear to be catalyzed by side activities of other enzymes that have been difficult to identify. We have applied genetic mapping of quantitative trait loci in the yeast Saccharomyces cerevisiae to identify mutant alleles of genes determining the production of phenylethyl acetate, an important flavor compound imparting rose- and honey-like aromas to alcoholic beverages. We identified a unique, dominant allele of FAS2 that supports high production of phenylethyl acetate. FAS2 encodes a subunit of the fatty acid synthetase complex and apparently exerts an important side activity on one or more alternative substrates in flavor compound synthesis. The second mutant allele contained a nonsense mutation in TOR1, a gene involved in nitrogen regulation of growth. Together the two alleles strongly increased the level of phenylethyl acetate. Our work highlights the potential of genetic mapping of quantitative phenotypic traits to identify novel enzymes and regulatory components in yeast metabolism, including regular metabolic enzymes with unknown side activities responsible for biosynthesis of specific flavor compounds. The superior alleles identified can be used to develop industrial yeast strains generating novel flavor profiles in alcoholic beverages.

Enzymatic synthesis of phenethyl ester from phenethyl alcohol with acyl donors.

Phenethyl ester is an important contributors to food, drinks, cosmetics and perfumery. Phenethyl acetate was synthesized enzymatically from phenethyl alcohol with acyl donors catalyzed by Novozym 435 from Candida antarctica lipase B. Through acyl donor screening, acetic anhydride and vinyl acetate were found to be the optimal acyl donors for the reaction. The effects of the molar ratio, temperature and solvents were investigated. The maximum conversion of phenethyl acetate were 99.12%, and 98.44%, using acetic anhydride and vinyl acetate as the acyl donors, respectively. After 20 reuses, the respective conversion of phenethyl acetate were 97.62% and 96.52%. This study may contribute to environment-friendly synthesis of phenethyl ester based on optimal conditions, and enzyme reusability for high conversion of phenethyl acetate.