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 parts 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 QTLs responsible for high 2-PEAc production were identified, with two showing linkage each to the genome of the BTC.1D and ER18 parents. The first two were investigated further. The causative genes were identified by reciprocal allele swapping into both parents using CRISPR/Cas9. The superior allele of the first major causative gene, FAS2, was dominant and contained two unique SNPs responsible for high 2-PEAc production, 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 with 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.