Hybridisation between species often leads to inviable or infertile offspring, yet examples of evolutionary successful interspecific hybrids have been reported in all kingdoms of life. However, many questions on the ecological circumstances and evolutionary aftermath of interspecific hybridisation remain unanswered. In this study, we sequenced and phenotyped a large set of interspecific yeast hybrids isolated from the brewing environments to uncover the influence of interspecific hybridisation in yeast adaptation and domestication. Our analyses demonstrate that several hybrids between Saccharomyces species originated and diversified in industrial environments by combining key traits of each parental species. Furthermore, post-hybridisation evolution within each hybrid lineage reflects sub-specialisation and adaptation to specific beer styles, a process that was accompanied by extensive chimerisation between subgenomes. Our results reveal how interspecific hybridisation provides an important evolutionary route that allows swift adaptation to novel environments.

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.

d‐Galacturonic acid is a major component of pectins but cannot be metabolized by Saccharomyces cerevisiae. It is assumed not to be taken up. We show that yeast displays surprisingly rapid low‐affinity uptake of d‐galacturonic acid, strongly increasing with decreasing extracellular pH and without saturation up to 1.5 M. There was no intracellular concentration above the extracellular level and transport was reversible. Among more than 160 single and multiple deletion mutants in channels and transporters, no strain was affected in d‐galacturonic acid uptake. The uptake was not inhibited by any compound tested as candidate competitive inhibitor, including d‐glucuronic acid, which was also transported. The characteristics of d‐galacturonic acid uptake are consistent with involvement of a channel‐type system, probably encoded by multiple genes.

Supplementary Fig. 1. GC-based method is anti-correlated with absorbance-based phenolic off-flavor (POF) measurement represented is a correlation plot, comparing the produced amount 4-vinyl guaiacol (4VG) (GC-based method [10]) and the postfermentation amount of ferulic acid, measured with the absorbance-based technique. The red dashed line represents the obtained linear trend line. The corresponding Pearson correlation coefficient and P value are depicted on the figure. A.U. = arbitrary units.