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.

Saccharomyces cerevisiae single-copy plasmids for auxotrophy compensation, multiple marker selection, and for designing metabolically cooperating communities

Auxotrophic markers are useful tools in cloning and genome editing, enable a large spectrum of genetic techniques, as well as facilitate the study of metabolite exchange interactions in microbial communities. If unused background auxotrophies are left uncomplemented however, yeast cells need to be grown in nutrient supplemented or rich growth media compositions, which precludes the analysis of biosynthetic metabolism, and which leads to a profound impact on physiology and gene expression. Here we present a series of 23 centromeric plasmids designed to restore prototrophy in typicalSaccharomyces cerevisiaelaboratory strains. The 23 single-copy plasmids complement for deficiencies inHIS3, LEU2, URA3, MET17 or LYS2genes and in their combinations, to match the auxotrophic background of the popular functional-genomic yeast libraries that are based on the S288c strain. The plasmids are further suitable for designing self-establishing metabolically cooperating (SeMeCo) communities, and possess a uniform multiple cloning site to exploit multiple parallel selection markers in protein expression experiments.

Stronger purifying selection against gene conversions in a pathogenicSaccharomyces cerevisiaestrain

Gene conversions most often have no selective impact, but some are selectively disadvantageous whereas others are selectively advantageous. Although gene conversions have been extensively studied in yeasts, very little is known about their selective impact in pathological yeasts. Here, we used the GENECONV software to compare the characteristics of candidate gene conversions found in a pathogenic strain (YJM789) and a nonpathogenic strain (S288c) of Saccharomyces cerevisiae. Interestingly, the pathogenic strain has fewer gene conversions when compared with the nonpathogenic strain. Of the 123 conversions we identified, 27 were identical or similar between the two strains, 62 were specific to the S288c strain, and 34 were specific to the YJM789 strain. Identical and similar conversions likely represent conversions that are under similar levels of purifying selection in both strains. The lower number of gene conversions in most gene families of the pathogenic strain is likely the result of higher purifying selection in this strain. In contrast, the higher number of conversions found in the YRF1 helicase gene family of the pathogenic strain could represent an example of adaptive gene conversions involved in maintaining its telomeres.