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

Author(s)

Santos, Clelton A. ^{[1, 2]} ; Morais, Mariana A. B. ^[3] ; Terrett, Oliver M. ^[2] ; Lyczakowski, Jan J. ^{[2, 4, 5]} ; Zanphorlin, Leticia M. ^[3] ; Ferreira, Jaire A. ^[1] ; Tonoli, Celisa C. C. ^[6] ; Murakami, Mario T. ^[3] ; Dupree, Paul ^{[2, 4, 5]} ; Souza, Anete P. ^{[7, 1]}

Affiliation

^[1] Univ Estadual Campinas, Ctr Biol Mol & Engn Genet, Campinas, SP - Brazil

^[2] Univ Cambridge, Dept Biochem, Cambridge - England

^[3] Ctr Nacl Pesquisa Energia & Mat, Lab Nacl Ciencia & Tecnol Bioetanol, Campinas, SP - Brazil

^[4] Univ Cambridge, Nat Mat Innovat Ctr, Cambridge - England

^[5] Univ Cambridge, OpenPlant Synthet Biol Res Ctr, Dept Plant Sci, Cambridge - England

^[6] Ctr Nacl Pesquisa Energia & Mat, Lab Nacl Biociencias, Campinas, SP - Brazil

^[7] Univ Estadual Campinas, Inst Biol, Dept Biol Vegetal, Campinas, SP - Brazil

• Document type: Journal article
• Source: SCIENTIFIC REPORTS; v. 9, MAR 20 2019.
• Web of Science Citations: 2

Abstract

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 hydrolysis. (AU)

• FAPESP's process: 16/19995-0 - Analysis of structural and functional diversity of GH43 enzymes from Xanthomonas axonopodis pv. citri: biological implications and potential biotechnological applications
• Grantee: Mariana Abrahão Bueno de Morais
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 15/26982-0 - Exploring novel strategies for depolymerization of plant cell-wall polysaccharides: from structure, function and rational design of glycosyl hydrolases to biological implications and potential biotechnological applications
• Grantee: Mário Tyago Murakami
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 17/17390-7 - Substrate Specificity, Unusual Enzymatic Activity, and Synergistic Relationships of beta-glucosidases from Trichoderma harzianum
• Grantee: Clelton Aparecido dos Santos
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• FAPESP's process: 15/09202-0 - Study of Trichoderma harzianum genomic regions associated to the control of the expression of the enzymes involved in the degradation of biomass
• Grantee: Anete Pereira de Souza
• Support Opportunities: Regular Research Grants