Backgroundβ-glucosidases (BGLs) catalyze the hydrolysis of soluble cellodextrins to glucose and are a critical component of cellulase systems. In order to engineer Saccharomyces cerevisiae for the production of ethanol from cellulosic biomass, a BGL tailored to industrial bioconversions is needed.ResultsWe applied a directed evolution strategy to a glycosyl hydrolase family 3 (GH3) BGL from Aspergillus niger (BGL1) by expressing a library of mutated bgl1 genes in S. cerevisiae and used a two-step functional screen to identify improved enzymes. Twelve BGL variants that supported growth of S. cerevisiae on cellobiose and showed increased activity on the synthetic substrate p-nitrophenyl-β-D-glucopyranoside were identified and characterized. By performing kinetic experiments, we found that a Tyr → Cys substitution at position 305 of BGL1 dramatically reduced transglycosidation activity that causes inhibition of the hydrolytic reaction at high substrate concentrations. Targeted mutagenesis demonstrated that the position 305 residue is critical in GH3 BGLs and likely determines the extent to which transglycosidation reactions occur. We also found that a substitution at Gln140 reduced the inhibitory effect of glucose and could be combined with the Y305C substitution to produce a BGL with decreased sensitivity to both the product and substrate. Using the crystal structure of a GH3 BGL from A. aculeatus, we mapped a group of beneficial mutations to the β/α domain of the molecule and postulate that this region modulates activity through subunit interactions. Six BGL variants were identified with substitutions in the MFα pre-sequence that was used to mediate secretion of the protein. Substitutions at Pro21 or Val22 of the MFα pre-sequence could produce up to a twofold increase in supernatant hydrolase activity and provides evidence that expression and/or secretion was an additional factor limiting hydrolytic activity.ConclusionsUsing directed evolution on BGL1, we identified a key residue that controls hydrolytic and transglycosidation reactions in GH3 BGLs. We also found that several beneficial mutations could be combined and increased the hydrolytic activity for both synthetic and natural substrates.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0470-9) contains supplementary material, which is available to authorized users.

BackgroundGenome shuffling (GS) is a widely adopted methodology for the evolutionary engineering of desirable traits in industrially relevant microorganisms. We have previously used genome shuffling to generate a strain of Saccharomyces cerevisiae that is tolerant to the growth inhibitors found in a lignocellulosic hydrolysate. In this study, we expand on previous work by performing a population-wide genomic survey of our genome shuffling experiment and dissecting the molecular determinants of the evolved phenotype.ResultsWhole population whole-genome sequencing was used to survey mutations selected during the experiment and extract allele frequency time series. Using growth curve assays on single point mutants and backcrossed derivatives, we explored the genetic architecture of the selected phenotype and detected examples of epistasis. Our results reveal cohorts of strongly correlated mutations, suggesting prevalent genetic hitchhiking and the presence of pre-existing founder mutations. From the patterns of apparent selection and the results of direct phenotypic assays, our results identify key driver mutations and deleterious hitchhikers.ConclusionsWe use these data to propose a model of inhibitor tolerance in our GS mutants. Our results also suggest a role for compensatory evolution and epistasis in our genome shuffling experiment and illustrate the impact of historical contingency on the outcomes of evolutionary engineering.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1283-9) contains supplementary material, which is available to authorized users.