GPD1 and ADH3 Natural Variants Underlie Glycerol Yield Differences in Wine Fermentation

Abstract

Glycerol is one of the most important by-products of alcohol fermentation, and depending on its concentration it can contribute to wine flavor intensity and aroma volatility. Here, we evaluated the potential of utilizing the natural genetic variation of non-coding regions in budding yeast to identify allelic variants that could modulate glycerol phenotype during wine fermentation. For this we utilized four Saccharomyces cerevisiae strains (WE - Wine/European, SA – Sake, NA – North American, and WA – West African), which were previously profiled for genome-wide Allele Specific Expression (ASE) levels. The glycerol yields under Synthetic Wine Must (SWM) fermentations differed significantly between strains; WA produced the highest glycerol yields while SA produced the lowest yields. Subsequently, from our ASE database, we identified two candidate genes involved in alcoholic fermentation pathways, ADH3 and GPD1, exhibiting significant expression differences between strains. A reciprocal hemizygosity assay demonstrated that hemizygotes expressing GPD1WA, GPD1SA, ADH3WA and ADH3SA alleles had significantly greater glycerol yields compared to GPD1WE and ADH3WE. We further analyzed the gene expression profiles for each GPD1 variant under SWM, demonstrating that the expression of GPD1WE occurred earlier and was greater compared to the other alleles. This result indicates that the level, timing, and condition of expression differ between regulatory regions in the various genetic backgrounds. Furthermore, promoter allele swapping demonstrated that these allele expression patterns were transposable across genetic backgrounds; however, glycerol yields did not differ between wild type and modified strains, suggesting a strong trans effect on GPD1 gene expression. In this line, Gpd1 protein levels in parental strains, particularly Gpd1pWE, did not necessarily correlate with gene expression differences, but rather with glycerol yield where low Gpd1pWE levels were detected. This suggests that GPD1WE is influenced by recessive negative post-transcriptional regulation which is absent in the other genetic backgrounds. This dissection of regulatory mechanisms in GPD1 allelic variants demonstrates the potential to exploit natural alleles to improve glycerol production in wine fermentation and highlights the difficulties of trait improvement due to alternative trans-regulation and gene-gene interactions in the different genetic background.

Quantitative Trait Nucleotides Impacting the Technological Performances of Industrial Saccharomyces cerevisiae Strains

“…These genetic variants do not affect the protein sequence but can impact the transcription level, mRNA processing, translation, export, and decay. Mutations altering a functional motif in the promoter region have been identified many times (Shahsavarani et al, 2012; Zimmer et al, 2014; Cubillos, 2016; Salinas et al, 2016; Tapia et al, 2018). They constitute allele-specific expression (ASE) changes that are certainly a source of phenotypic variation (see Cubillos, 2016, for a review).…”

Fungal Diversity Analysis of Grape Musts from Central Valley-Chile and Characterization of Potential New Starter Cultures

“…Fermentations were carried out as previously described [ 37 ]. Briefly, each strain was fermented in quadruplicate in SWM MS300 (Synthetic must contain 300 mgN/L) and prepared according to Rossignol et al [ 38 ].…”

“…20 µL were injected in a Shimadzu Prominence HPLC equipment (Shimadzu, Tokyo, Japan) using a Bio-Rad HPX–87H column to estimate final concentrations of: glucose, fructose, acetic acid, glycerol, and ethanol [ 39 ]. Glycerol and ethanol yields were estimated as previously described [ 37 ].…”

Transcriptional Activity and Protein Levels of Horizontally Acquired Genes in Yeast Reveal Hallmarks of Adaptation to Fermentative Environments

“…Transcriptional activity was assayed, replacing the target gene with the luciferase reporter, and generating a transcriptional fusion of the promoter region. We replaced the entire coding sequence of the target horizontally acquired genes, from the start codon to the stop codon, using the constructs Luc-HphMx or Luc-NatMx, which carry the hygromycin (HphMx) or nourseothricin (NatMx) cassettes as selective markers, respectively (Salinas et al, 2016;Tapia et al, 2018). These genetic constructs encode a destabilized version of the luciferase gene, which contains an ARE sequence for mRNA destabilization and a PEST sequence for proteasome mediated degradation of the protein (Rienzo et al, 2012).…”

“…The protein levels were assayed by replacing the stop codon of the target gene by the gene encoding the mCherry fluorescent protein, generating a translational fusion. The stop codon was replaced using the mCherry-HphMx or mCherry-NatMx constructs, which carry either the hygromycin (HphMx) or the nourseothricin (NatMx) cassettes as selective markers (Tapia et al, 2018). The mCherry-HphMx and mCherry-NatMx constructs were amplified by PCR using Phusion flash high-fidelity master mix (Thermo Fisher Scientific, United States) according to the manufacturer's instructions.…”