Polygenic Analysis of Tolerance to Carbon Dioxide Inhibition of Isoamyl Acetate "Banana" Flavor Production in Yeast Reveals MDS3 as Major Causative Gene.

The introduction in modern breweries of tall cylindroconical fermentors, replacing the traditional open fermentation vats, unexpectedly revealed strong inhibition of flavor production by the high CO2 pressure in the fermentors. We have screened our collection of Saccharomyces cerevisiae strains for strains displaying elevated tolerance to inhibition of flavor production by +0.65 bar CO2, using a laboratory scale CO2 pressurized fermentation system. We focused on the production of isoamyl acetate, a highly desirable flavor compound conferring fruity banana flavor in beer and other alcoholic beverages, from its precursor isoamyl alcohol (IAAc/Alc ratio). We selected the most tolerant Saccharomyces cerevisiae strain, saké yeast Kyokai no. 1, isolated a stable haploid segregant seg63 with the same high IAAc/Alc ratio under CO2 pressure, crossed seg63 with the unrelated inferior strain ER7A and phenotyped 185 haploid segregants, of which 28 displaying a high IAAc/Alc ratio were pooled. Mapping of Quantitative Trait Loci (QTLs) by whole-genome sequence analysis based on SNP variant frequency revealed two QTLs. In the major QTL, reciprocal hemizygosity analysis identified MDS3 as the causative mutant gene, a putative member of the TOR signaling pathway. The MDS3Seg.63 allele was dominant and contained a single causative point mutation, T2171C, resulting in the F274S substitution. Introduction of MDS3Seg.63 in an industrial tetraploid lager yeast with CRISPR/Cas9 enhanced isoamyl acetate production by 145% under CO2 pressure. This work shows the strong potential of polygenic analysis and targeted genetic modification for creation of cisgenic industrial brewer's yeast strains with specifically improved traits. IMPORTANCE The upscaling of fermentation to very tall cylindroconical tanks is known to negatively impact beer flavor. Most notably, the increased CO2 pressure in such tanks compromises production by the yeast of the desirable fruity "banana" flavor (isoamyl acetate). The cause of the CO2 inhibition of yeast flavor production has always remained enigmatic. Our work has brought the first insight into its molecular-genetic basis and provides a specific gene tool for yeast strain improvement. We first identified a yeast strain with superior tolerance to CO2 inhibition of flavor production, and applied polygenic analysis to identify the responsible gene. We narrowed down the causative element to a single nucleotide difference, MDS3T2171C, and showed that it can be engineered into brewing yeast to obtain strains with superior flavor production in high CO2 pressure conditions, apparently without affecting other traits relevant for beer brewing. Alternatively, such a strain could be obtained through marker-assisted breeding.

Interspecific hybridization facilitates niche adaptation in beer yeast.

Hybridization between species often leads to non-viable or infertile offspring, yet examples of evolutionarily successful interspecific hybrids have been reported in all kingdoms of life. However, many questions on the ecological circumstances and evolutionary aftermath of interspecific hybridization remain unanswered. In this study, we sequenced and phenotyped a large set of interspecific yeast hybrids isolated from brewing environments to uncover the influence of interspecific hybridization 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, posthybridization evolution within each hybrid lineage reflects subspecialization and adaptation to specific beer styles, a process that was accompanied by extensive chimerization between subgenomes. Our results reveal how interspecific hybridization provides an important evolutionary route that allows swift adaptation to novel environments.

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.

Comparison of genome engineering using the CRISPR-Cas9 system in C. glabrata wild-type and lig4 strains.

Candida glabrata is reported as the second most prevalent human opportunistic fungal pathogen in North America and is threatening patients all over the world. Its incidence is rising, while it has developed resistance to the most widely used antifungal drugs, necessitating new approaches based on better insight into the biology of the organism. Despite its close phylogenetic relationship with Saccharomyces cerevisiae, generating precise genomic alterations in this species is problematic. Previously we have shown that deletion of LIG4, which encodes an enzyme involved in Non-Homologous End Joining (NHEJ), strongly enhances the probability of obtaining correctly modified transformants. In this work we used the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated protein 9 (Cas9) system to genetically engineer the C. glabrata genome, targeting the genes ADE2, MET15 and SOK2, located on different chromosomes. We used the CRISPR-Cas9 technology to replace the open reading frame (ORF) by the SAT1 selective marker or introduced a premature stop codon in ADE2 and MET15, as they are easily scored by their adenine or methionine auxotrophy, respectively. The SOK2 gene was modified by insertion of a triple HA-tag sequence and the transformants were verified in a western blot. The CRISPR-Cas9 mediated targeting efficiency varies depending on the gene targeted and the genetic modification performed. We show that CRISPR-Cas9 mediated genome editing is more efficient than the conventional method in the wild-type strain, moreover it has the big advantage being marker-free. In previous work, we showed that the targeting efficiency is highly increased in the lig4Δ strain using the conventional way to delete genes in C. glabrata. Using the CRISPR-Cas9 system in this strain, the percentage of correct transformants is consistently higher compared to the wild-type strain. This indicates that using the lig4 mutant as such is already a strong improvement, while the CRISPR-Cas9 gives the additional advantage of not leaving a scar or marker and that it therefore can be used to generate multiple modifications.

Genomic saturation mutagenesis and polygenic analysis identify novel yeast genes affecting ethyl acetate production, a non-selectable polygenic trait.

Isolation of mutants in populations of microorganisms has been a valuable tool in experimental genetics for decades. The main disadvantage, however, is the inability of isolating mutants in non-selectable polygenic traits. Most traits of organisms, however, are non-selectable and polygenic, including industrially important properties of microorganisms. The advent of powerful technologies for polygenic analysis of complex traits has allowed simultaneous identification of multiple causative mutations among many thousands of irrelevant mutations. We now show that this also applies to haploid strains of which the genome has been loaded with induced mutations so as to affect as many non-selectable, polygenic traits as possible. We have introduced about 900 mutations into single haploid yeast strains using multiple rounds of EMS mutagenesis, while maintaining the mating capacity required for genetic mapping. We screened the strains for defects in flavor production, an important non-selectable, polygenic trait in yeast alcoholic beverage production. A haploid strain with multiple induced mutations showing reduced ethyl acetate production in semi-anaerobic fermentation, was selected and the underlying quantitative trait loci (QTLs) were mapped using pooled-segregant whole-genome sequence analysis after crossing with an unrelated haploid strain. Reciprocal hemizygosity analysis and allele exchange identified PMA1 and CEM1 as causative mutant alleles and TPS1 as a causative genetic background allele. The case of CEM1 revealed that relevant mutations without observable effect in the haploid strain with multiple induced mutations (in this case due to defective mitochondria) can be identified by polygenic analysis as long as the mutations have an effect in part of the segregants (in this case those that regained fully functional mitochondria). Our results show that genomic saturation mutagenesis combined with complex trait polygenic analysis could be used successfully to identify causative alleles underlying many non-selectable, polygenic traits in small collections of haploid strains with multiple induced mutations.

Evidence for rapid uptake of D-galacturonic acid in the yeast Saccharomyces cerevisiae by a channel-type transport system.

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.