An engineered GH1 ss-glucosidase displays enhanced glucose tolerance and increased sugar release from lignocellulosic materials

ss-glucosidases play a critical role among the enzymes in enzymatic cocktails designed for plant biomass deconstruction. By catalysing the breakdown of ss-1, 4-glycosidic linkages, ss-glucosidases produce free fermentable glucose and alleviate the inhibition of other cellulases by cellobiose during saccharification. Despite this benefit, most characterised fungal ss-glucosidases show weak activity at high glucose concentrations, limiting enzymatic hydrolysis of plant biomass in industrial settings. In this study, structural analyses combined with site-directed mutagenesis efficiently improved the functional properties of a GH1 ss-glucosidase highly expressed by Trichoderma harzianum (ThBgl) under biomass degradation conditions. The tailored enzyme displayed high glucose tolerance levels, confirming that glucose tolerance can be achieved by the substitution of two amino acids that act as gatekeepers, changing active-site accessibility and preventing product inhibition. Furthermore, the enhanced efficiency of the engineered enzyme in terms of the amount of glucose released and ethanol yield was confirmed by saccharification and simultaneous saccharification and fermentation experiments using a wide range of plant biomass feedstocks. Our results not only experimentally confirm the structural basis of glucose tolerance in GH1 ss-glucosidases but also demonstrate a strategy to improve technologies for bioethanol production based on enzymatic hydrolysis9CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP430350/2018-0sem informação2015/09202-0; 2015/26982-0; 2017/17390-7; 2016/19995-0Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2015/09202-0, 2015/26982-0]; Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq, MCTI/CNPQ/Universal ) [430350/2018-0]; Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)CAPES; Computational Biology Program; Leverhulme Trust Centre for Natural Material Innovation; OpenPlant Synthetic Biology Research Centre; BEPE Postdoctoral fellowship from FAPESP [2017/17390-7]; FAPESPFundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2016/19995-0]; Biotechnology and Biological Sciences Research Council (BBSRC) of the UK as part of the Cambridge BBSRC-DTP Programme [BB/J014540/1]; iCASE studentship from the BBSRC [BB/M015432/1]; CNPqNational Council for Scientific and Technological Development (CNPq