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

Abstract

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.

Bioprospecting for brewers: Exploiting natural diversity for naturally diverse beers

“…For this, the phenotypic study of new wild Saccharomyces strains represents the first step towards determining whether the industrial potential of wild stocks is promising (Peter et al, ; Warringer et al, ). Among the industrially relevant traits of interest are ethanol and glycerol production, sugar utilization, hop tolerance and low‐temperature resistance, and the relative production of aroma compounds of interest, such as esters and higher alcohols, as well as low levels of acetic acid and hydrogen sulphide (Gallone et al, ; Gibson et al, ; Krogerus, Seppanen‐Laakso, Castillo, & Gibson, ; Senkarcinova, Dias, Nespor, & Branyik, ; Steensels, Meersman, et al, ; Trindade de Carvalho et al, ). The precise wort sugar profile depends on the raw ingredients (mostly barley malt) and method of wort preparation, but the predominant sugar sources in wort in decreasing order are maltose, maltotriose, glucose, and fructose.…”

Disentangling the genetic bases of Saccharomyces cerevisiae nitrogen consumption and adaptation to low nitrogen environments in wine fermentation

“…In this context, several QTLs have been mapped for phenotypes associated with the fermentation process, such as ethanol production, residual sugar and acidity [76][77][78][79][80], as well as QTLs involved in nitrogen-limited fermentations and in nitrogen consumption and utilization [41,42,78,[81][82][83][84][85] (Table 1). Overall, QTL mapping has proved to be an efficient tool for the detection of genes responsible of the phenotypic variation observed in populations generated by crosses, particularly for fermentative phenotypes.…”

Polygenic Analysis in Absence of Major EffectorATF1Unveils Novel Components in Yeast Flavor Ester Biosynthesis

“…Pooled-segregant QTL analysis is an unbiased technique that is used in both applied and fundamental yeast studies to identify causative genetic elements ( 11 , 12 ). It has been used successfully to determine genes underlying several metabolic traits, including production of central metabolites for yeast metabolism ( 13 ), glycerol ( 14 ), acetic acid ( 15 ), sulfite ( 16 ), and the aroma compounds 2-phenylethyl acetate ( 17 ), isoamyl alcohol, isobutanol, 2-phenyl ethanol, ethyl octanoate and decanoate ( 18 , 19 ), and 4-sulfanyl-methylpentan-2-one ( 20 ). In general, little work has been reported on the introduction of the identified alleles in other strain backgrounds in spite of its importance for evaluating the general applicability of the alleles for industrial strain improvement.…”