Abstract
ABSTRACTFlavor 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.
Highlights
IMPORTANCE Multiple reactions in flavor metabolism appear to be catalyzed by side activities of other enzymes that have been difficult to identify
We have obtained a pool of haploid strains for flavor profile analysis by sporulating a hybrid diploid Saccharomyces cerevisiae yeast strain, obtained by crossing two descendants from the unrelated industrial yeast strains WLP575, an ale yeast, and Ethanol Red (ER), a bioethanol production yeast
We have identified two new genetic elements, FAS2 and TOR1, that have never previously been connected to production of 2-phenylethyl acetate (2-PEAc), an important flavor compound in alcoholic beverages, imparting a honey- and rose-like flavor
Summary
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. Ethyl esters are formed from ethanol and the acyl-CoA derivative of medium-chain fatty acids (MCFA). Among this group, the most important are ethyl hexanoate (anise seed, apple-like aroma) and ethyl octanoate (apple, pineapple aroma). Quantitative trait locus (QTL) mapping studies have revealed genes involved in production of nerolidol, 2-phenyl ethanol, and ethyl esters [13], ethyl acetate [14], and undesirable sulfur flavor compounds [15]. Given the large variety of flavor compounds and the many parameters affecting their formation, most of the underlying genetic basis of the natural variation in flavor compound production remains unknown
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