QTL mapping of volatile compound production in Saccharomyces cerevisiae during alcoholic fermentation

Abstract

Background: The volatile metabolites produced by Saccharomyces cerevisiae during alcoholic fermentation, which are mainly esters, higher alcohols and organic acids, play a vital role in the quality and perception of fermented beverages, such as wine. Although the metabolic pathways and genes behind yeast fermentative aroma formation are well described, little is known about the genetic mechanisms underlying variations between strains in the production of these aroma compounds. To increase our knowledge about the links between genetic variation and volatile production, we performed quantitative trait locus (QTL) mapping using 130 F2-meiotic segregants from two S. cerevisiae wine strains. The segregants were individually genotyped by next-generation sequencing and separately phenotyped during wine fermentation.

Results: Using different QTL mapping strategies, we were able to identify 65 QTLs in the genome, including 55 that influence the formation of 30 volatile secondary metabolites, 14 with an effect on sugar consumption and central carbon metabolite production, and 7 influencing fermentation parameters. For ethyl lactate, ethyl octanoate and propanol formation, we discovered 2 interacting QTLs each. Within 9 of the detected regions, we validated the contribution of 13 genes in the observed phenotypic variation by reciprocal hemizygosity analysis. These genes are involved in nitrogen uptake and metabolism (AGP1, ALP1, ILV6, LEU9), central carbon metabolism (HXT3, MAE1), fatty acid synthesis (FAS1) and regulation (AGP2, IXR1, NRG1, RGS2, RGT1, SIR2) and explain variations in the production of characteristic sensorial esters (e.g., 2-phenylethyl acetate, 2-metyhlpropyl acetate and ethyl hexanoate), higher alcohols and fatty acids.

Conclusions: The detection of QTLs and their interactions emphasizes the complexity of yeast fermentative aroma formation. The validation of underlying allelic variants increases knowledge about genetic variation impacting metabolic pathways that lead to the synthesis of sensorial important compounds. As a result, this work lays the foundation for tailoring S. cerevisiae strains with optimized volatile metabolite production for fermented beverages and other biotechnological applications.

Keywords: Aroma compounds; Fermentation; Metabolites; QTL mapping; Yeast.

Fig. 1

Simplified synthesis pathways of determined metabolites. Main and secondary metabolites determined in this study by HPLC (green) and GC/MS (red)

Fig. 2

Principle component analysis. PCA of the formation of extracellular metabolites by S. cerevisiae. Traits that are less than 2% explained by the first two dimensions of the PCA were excluded (2-methylbutanol, acetate yield, alpha-ketoglutarate yield, ethanol yield, ethyl acetate, glycerol yield, propyl acetate, and valeric acid)

Fig. 3

Effect of validated variants on medium chain fatty acid formation. Simplified pathway of fatty acid synthesis by the enzymes Fas1 and Fas2, which is dependent on intracellular acetyl transport (a). Allelic effect of the enzymes Agp2, Fas1 and Sir2 on the formation of fatty acids (b) and fatty acid ethyl esters (c) as determined by RHA. Concentrations are given in relation to the heterozygote of the parental strains MTF2621 and MTF2622. (p-value: ns (not significant) > 0.05, * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, **** ≤ 0.0001)

Fig. 4

Effect of validated variants on of higher alcohol and fusel acid formation. Amino acids are transported into the cell by Agp1 and Alp1. The expression of AGP1 is influenced by Sir2 (a). Simplified synthesis pathway of fermentative aromas connected to valine and leucine metabolism (b). Allelic effect of the involved enzymes Agp1, Alp1, Ilv6, Mae1 and Sir2 on the formation of volatiles deriving from α-ketoisovalerate (c) and α-ketoisocaproate (d) as determined by RHA. Concentrations are given in relation to the heterozygote of the parental strains MTF2621 and MTF2622. (p-value: ns (not significant) > 0.05, * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, **** ≤ 0.0001)

Fig. 5

Effect of validated variants on propanol formation. Simplified synthesis pathway of fermentative aromas connected to threonine metabolism (a). Allelic effect of the involved enzymes Alp1 and Nrg1 on the formation of volatiles derived from α-ketobutyrate as determined by RHA (b). Concentrations are given in relation to the heterozygote of the parental strains MTF2621 and MTF2622. (p-value: ns (not significant) > 0.05, * ≤ 0.05, ** ≤ 0.01)