Barcoded reciprocal hemizygosity analysis via sequencing illuminates the complex genetic basis of yeast thermotolerance

Decades of successes in statistical genetics have revealed the molecular underpinnings of traits as they vary across individuals of a given species. But standard methods in the field can’t be applied to divergences between reproductively isolated taxa. Genome-wide reciprocal hemizygosity mapping (RH-seq), a mutagenesis screen in an inter-species hybrid background, holds promise as a method to accelerate the progress of interspecies genetics research. Here we describe an improvement to RH-seq in which mutants harbor barcodes for cheap and straightforward sequencing after selection in a condition of interest. As a proof of concept for the new tool, we carried out genetic dissection of the difference in thermotolerance between two reproductively isolated budding yeast species. Experimental screening identified dozens of candidate loci at which variation between the species contributed to the thermotolerance trait. Hits were enriched for mitosis genes and other housekeeping factors, and among them were multiple loci with robust sequence signatures of positive selection. Together, these results shed new light on the mechanisms by which evolution solved the problems of cell survival and division at high temperature in the yeast clade, and they illustrate the power of the barcoded RH-seq approach.

Reciprocal hemizygosity analysis reveals that the Saccharomyces cerevisiae CGI121 gene affects lag time duration in synthetic grape must

It is standard practice to ferment white wines at low temperatures (10-18 °C). However, low temperatures increase fermentation duration and risk of problem ferments, leading to significant costs. The lag duration at fermentation initiation is heavily impacted by temperature; therefore, identification of Saccharomyces cerevisiae genes influencing fermentation kinetics is of interest for winemaking. We selected 28 S. cerevisiae BY4743 single deletants, from a prior list of open reading frames (ORFs) mapped to quantitative trait loci (QTLs) on chromosomes VII and XIII, influencing the duration of fermentative lag time. Five BY4743 deletants, Δapt1, Δcgi121, Δclb6, Δrps17a, and Δvma21, differed significantly in their fermentative lag duration compared to BY4743 in synthetic grape must (SGM) at 15 °C, over 72 h. Fermentation at 12.5 °C for 528 h confirmed the longer lag times of BY4743 Δcgi121, Δrps17a, and Δvma21. These three candidate ORFs were deleted in S. cerevisiae RM11-1a and S288C to perform single reciprocal hemizygosity analysis (RHA). RHA hybrids and single deletants of RM11-1a and S288C were fermented at 12.5 °C in SGM and lag time measurements confirmed that the S288C allele of CGI121 on chromosome XIII, encoding a component of the EKC/KEOPS complex, increased fermentative lag phase duration. Nucleotide sequences of RM11-1a and S288C CGI121 alleles differed by only one synonymous nucleotide, suggesting that intron splicing, codon bias, or positional effects might be responsible for the impact on lag phase duration. This research demonstrates a new role of CGI121 and highlights the applicability of QTL analysis for investigating complex phenotypic traits in yeast.

Reciprocal hemizygosity analysis reveals that the Saccharomyces cerevisiae CGI121 gene affects lag time duration under enological conditions

It is standard practice to ferment white wines at low temperatures (10-18ºC). However, low temperatures increase fermentation duration and risk of problem ferments, leading to significant costs. The lag duration at fermentation initiation is heavily impacted by temperature; therefore, identification of Saccharomyces cerevisiae genes impacting on fermentation kinetics is of interest for winemaking.We selected 28 S. cerevisiae BY4743 single deletants, from a prior list of open reading frames (ORFs) mapped to quantitative trait loci (QTLs) on chromosomes VII and XIII, influencing the duration of fermentative lag time. Five BY4743 deletants, Δapt1, Δcgi121, Δclb6, Δrps17a, and Δvma21, differed significantly in their fermentative lag duration compared to BY4743 in synthetic grape medium (SGM) at 15ºC, over 72 h. Fermentation at 12.5ºC for 528 h confirmed the longer lag times of BY4743 Δcgi121, Δrps17a, and Δvma21. These three candidate ORFs were deleted in S. cerevisiae RM11-1a and S288C to perform single reciprocal hemizygosity analysis (RHA). RHA hybrids and single deletants of RM11-1a and S288C were fermented at 12.5ºC in SGM and lag time measurements confirmed that the S288C allele of CGI121 on chromosome XIII, encoding a component of the EKC/KEOPS complex, increased fermentative lag phase duration. Nucleotide sequences of RM11-1a and S288C CGI121 alleles differed by only one synonymous nucleotide, suggesting that codon bias or positional effects might be responsible for the impact on lag phase duration. This research demonstrates a new role of CGI121 and highlights the applicability of QTL analysis for investigating complex phenotypic traits in yeast.

QTL-guided metabolic engineering of a complex trait

Engineering complex phenotypes for industrial and synthetic biology applications is difficult and often confounds rational design. Bioethanol production from lignocellulosic feedstocks is a complex trait that requires multiple host systems to utilize, detoxify, and metabolize a mixture of sugars and inhibitors present in plant hydrolysates. Here, we demonstrate an integrated approach to discovering and optimizing host factors that impact fitness ofSaccharomyces cerevisiaeduring fermentation of aMiscanthus x giganteusplant hydrolysate. We first used high-resolution Quantitative Trait Loci (QTL) mapping and systematic Bulk Reciprocal Hemizygosity analysis (bRHA) to discover 17 loci that differentiate hydrolysate tolerance between an industrially related (JAY291) and a laboratory (S288C) strain. We then used this data to identify a subset of favorable allelic loci that were most amenable for strain engineering. Guided by this “genetic blueprint”, and using a dual-guide Cas9-based method to efficiently perform multi-kilobase locus replacements, we engineered an S288C strain with superior hydrolysate tolerance than JAY291. Our methods should be generalizable to engineering any complex trait inS. cerevisiae, as well as other organisms.