Dataset Information

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.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.

SUBMITTER: Trindade de Carvalho B

PROVIDER: S-EPMC5676035 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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

Trindade de Carvalho Bruna B Holt Sylvester S Souffriau Ben B Lopes Brandão Rogelio R Foulquié-Moreno Maria R MR Thevelein Johan M JM

mBio 20171107 6

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 <i>Saccharomyces cerevisiae</i> genes affecting production of phenylethyl acetate (2-PEAc). This is a desirable flavor compound of major importance in alcohol ...[more]

PMID: 29114020

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Project description: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.

Project description: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.

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Dataset Information

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

Publications

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

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