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Directed evolution of a fungal β-glucosidase in Saccharomyces cerevisiae

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....

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Autores principales: Larue, Kane, Melgar, Mindy, Martin, Vincent J. J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4778352/
https://www.ncbi.nlm.nih.gov/pubmed/26949413
http://dx.doi.org/10.1186/s13068-016-0470-9
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author Larue, Kane
Melgar, Mindy
Martin, Vincent J. J.
author_facet Larue, Kane
Melgar, Mindy
Martin, Vincent J. J.
author_sort Larue, Kane
collection PubMed
description 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13068-016-0470-9) contains supplementary material, which is available to authorized users.
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spelling pubmed-47783522016-03-05 Directed evolution of a fungal β-glucosidase in Saccharomyces cerevisiae Larue, Kane Melgar, Mindy Martin, Vincent J. J. Biotechnol Biofuels Research 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13068-016-0470-9) contains supplementary material, which is available to authorized users. BioMed Central 2016-03-03 /pmc/articles/PMC4778352/ /pubmed/26949413 http://dx.doi.org/10.1186/s13068-016-0470-9 Text en © Larue et al. 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Larue, Kane
Melgar, Mindy
Martin, Vincent J. J.
Directed evolution of a fungal β-glucosidase in Saccharomyces cerevisiae
title Directed evolution of a fungal β-glucosidase in Saccharomyces cerevisiae
title_full Directed evolution of a fungal β-glucosidase in Saccharomyces cerevisiae
title_fullStr Directed evolution of a fungal β-glucosidase in Saccharomyces cerevisiae
title_full_unstemmed Directed evolution of a fungal β-glucosidase in Saccharomyces cerevisiae
title_short Directed evolution of a fungal β-glucosidase in Saccharomyces cerevisiae
title_sort directed evolution of a fungal β-glucosidase in saccharomyces cerevisiae
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4778352/
https://www.ncbi.nlm.nih.gov/pubmed/26949413
http://dx.doi.org/10.1186/s13068-016-0470-9
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