title: Directed evolution of a fungal β-glucosidase in Saccharomyces cerevisiae.
abstract: BACKGROUND
  β-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.
  
  
  RESULTS
  We 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 Gln(140) 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 Pro(21) or Val(22) 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.
  
  
  CONCLUSIONS
  Using 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.
authors.affiliation: Department of Biology, Centre for Structural and Functional Genomics, Concordia University, 7141 Sherbrooke West, Montreal, QC H4B 1R6 Canada.
authors.forename: Kane
authors.initials: K
authors.lastname: Larue
authors.affiliation: Department of Biology, Centre for Structural and Functional Genomics, Concordia University, 7141 Sherbrooke West, Montreal, QC H4B 1R6 Canada.
authors.forename: Mindy
authors.initials: M
authors.lastname: Melgar
authors.affiliation: Department of Biology, Centre for Structural and Functional Genomics, Concordia University, 7141 Sherbrooke West, Montreal, QC H4B 1R6 Canada.
authors.forename: Vincent J J
authors.initials: VJ
authors.lastname: Martin
keywords: Biofuels; Cellulase; Consolidated bioprocessing; Directed evolution; Inhibition; β-glucosidase