Abstract
BackgroundIdentifying the genetic basis of complex microbial phenotypes is currently a major barrier to our understanding of multigenic traits and our ability to rationally design biocatalysts with highly specific attributes for the biotechnology industry. Here, we demonstrate that strain evolution by meiotic recombination-based genome shuffling coupled with deep sequencing can be used to deconstruct complex phenotypes and explore the nature of multigenic traits, while providing concrete targets for strain development.ResultsWe determined genomic variations found within Saccharomyces cerevisiae previously evolved in our laboratory by genome shuffling for tolerance to spent sulphite liquor. The representation of these variations was backtracked through parental mutant pools and cross-referenced with RNA-seq gene expression analysis to elucidate the importance of single mutations and key biological processes that play a role in our trait of interest. Our findings pinpoint novel genes and biological determinants of lignocellulosic hydrolysate inhibitor tolerance in yeast. These include the following: protein homeostasis constituents, including Ubp7p and Art5p, related to ubiquitin-mediated proteolysis; stress response transcriptional repressor, Nrg1p; and NADPH-dependent glutamate dehydrogenase, Gdh1p. Reverse engineering a prominent mutation in ubiquitin-specific protease gene UBP7 in a laboratory S. cerevisiae strain effectively increased spent sulphite liquor tolerance.ConclusionsThis study advances understanding of yeast tolerance mechanisms to inhibitory substrates and biocatalyst design for a biomass-to-biofuel/biochemical industry, while providing insights into the process of mutation accumulation that occurs during genome shuffling.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0241-z) contains supplementary material, which is available to authorized users.
Highlights
Identifying the genetic basis of complex microbial phenotypes is currently a major barrier to our understanding of multigenic traits and our ability to rationally design biocatalysts with highly specific attributes for the biotechnology industry
Large pools of UV mutants and genome-shuffled populations were screened on hardwood spent sulphite liquor (HWSSL) gradient agar plates prior to each round of shuffling. (B) Portions of each population that showed more tolerance than the reference (WT) were scraped from gradient plates and used for genome shuffling (different rounds of genome shuffling are depicted - round 1 (R1), round 3 (R3) and round 5 (R5))
Individual colonies showing the highest tolerance to HWSSL were isolated from the frontier of growth
Summary
Identifying the genetic basis of complex microbial phenotypes is currently a major barrier to our understanding of multigenic traits and our ability to rationally design biocatalysts with highly specific attributes for the biotechnology industry. Current technologies do not allow for the rapid creation of industrially relevant microorganisms or the ability to access and understand multigenic phenotypic traits. DNA sequencing technology allows for rapid sequencing of the genomes of these industrial strains to identify the mutations that confer improved phenotypes. Our ability to deconstruct complex, multigenic traits is still limited Possible solutions to these problems include sequencing pools of independent mutants [5], backcrossing non-productive mutations prior to genome resequencing, or combining intercrossing with pool sequencing to assign quantitative trait loci [6] in order to hone
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