Mapping genetic variants underlying differences in the central nitrogen metabolism in fermenter yeasts

Abstract

Different populations within a species represent a rich reservoir of allelic variants, corresponding to an evolutionary signature of withstood environmental constraints. Saccharomyces cerevisiae strains are widely utilised in the fermentation of different kinds of alcoholic beverages, such as, wine and sake, each of them derived from must with distinct nutrient composition. Importantly, adequate nitrogen levels in the medium are essential for the fermentation process, however, a comprehensive understanding of the genetic variants determining variation in nitrogen consumption is lacking. Here, we assessed the genetic factors underlying variation in nitrogen consumption in a segregating population derived from a cross between two main fermenter yeasts, a Wine/European and a Sake isolate. By linkage analysis we identified 18 main effect QTLs for ammonium and amino acids sources. Interestingly, majority of QTLs were involved in more than a single trait, grouped based on amino acid structure and indicating high levels of pleiotropy across nitrogen sources, in agreement with the observed patterns of phenotypic co-variation. Accordingly, we performed reciprocal hemizygosity analysis validating an effect for three genes, GLT1, ASI1 and AGP1. Furthermore, we detected a widespread pleiotropic effect on these genes, with AGP1 affecting seven amino acids and nine in the case of GLT1 and ASI1. Based on sequence and comparative analysis, candidate causative mutations within these genes were also predicted. Altogether, the identification of these variants demonstrate how Sake and Wine/European genetic backgrounds differentially consume nitrogen sources, in part explaining independently evolved preferences for nitrogen assimilation and representing a niche of genetic diversity for the implementation of practical approaches towards more efficient strains for nitrogen metabolism.

Figure 1. A Principal Component Analysis of ammonium and amino acid consumption variation across segregants.

Repartition of the 14 amino acids and ammonium are shown on the PC1 and PC2 axis. PC1 explains 40% of the variation, while PC2 17%. Amino acids, Red: Non-polar, Blue: Polar uncharged side chain, Black: Polar positively charged side chain, Purple: Polar negatively charged side chain, Green: Aromatic.

Figure 2. Linkage analysis in WE x SA cross.

A. LOD plot from linkage analysis using a nonparametric model for all traits with at least a single QTL. QTLs for the corresponding phenotype are: QTL1, Serine and Threonine; QTL2, Glutamic Acid; QTL3, Lysine and Tryptophane; QTL4, Phenylalanine and Tyrosine; QTL5, Aspartate and Glutamate; QTL6, Ammonium and Lysine; QTL7, Lysine; QTL8, Histidine; QTL9, Isoleucine, Serine, Tyrosine, Glutamine and Threonine; QTL10, Phenylalanine and Tyrosine. B. Amino Acid consumption levels in segregants carrying either WE or SA alleles for QTL XIII.527 underlying Isoleucine, Serine, Tyrosine, Glutamine and Threonine variation.

Figure 3. Reciprocal hemizygosity analysis on GHD2 and GLT1 underlying consumption variation for Aspartic and Glutamic acid.

A. The hybrid hemizygote strains consumption levels (mg/L) for SA and WE GDH2 reciprocal hemizygotes. B. Similarly, GLT1 reciprocal hemizygosity assay. (*), (**) and (***) represents a significant statistical difference between the hemizygote strains for the same gene using ANOVA tests P<0.05, P<0.01 and P<0.001 respectively. Δ/WE denotes hemizygotes carrying the WE allele, while SA/Δdenotes hemizygotes carrying the SA allele.

Figure 4. Reciprocal hemizygosity analysis on AGP1 and ASI1.

A. The hybrid hemizygote strains serine and threonine consumption levels (mg/L) for SA and WE AGP1 reciprocal hemizygotes. B. Aspartic acid, glutamic acid, serine, threonine and tryptophane consumption levels (mg/L) for ASI1 reciprocal hemizygotes. (*), (**) and (***) represents a significant statistical difference between the hemizygote strains for the same gene using an ANOVA test with P<0.05, P<0.01 and P<0.001 respectively. Δ/WE denotes hemizygotes carrying the WE allele, while SA/Δ denotes hemizygotes carrying the SA allele.

Figure 5. Overview of Nitrogen metabolism QTLs in the WE x SA cross.

Genes highlighted in bold were validated by reciprocal hemizygosity and contribute to genetic variation in nitrogen consumption in fermenter yeasts. GDH2, which has a FDR = 13%, is shown in light grey. The black arrow represent amino acids differentially consumed in AGP1 reciprocal hemizygotes, dashed arrow those amino acids produced from glutamic acid and those amino acids with significantly different consumption levels in ASI1 reciprocal hemizygotes are highlighted in red. Red arrow indicates the suggested differential allelic regulation on those genes encoding for amino acid permeases through the indirect effect of ASI1.