Plant cell wall architecture guided design of CBM3-GH11 chimeras with enhanced xylanase activity using a tandem repeat left-handed beta-3-prism scaffold

Author(s)

Pinheiro, P. Matheus ^[1] ; Reis, A. G. Renata ^[2] ; Dupree, Paul ^[3] ; Ward, J. Richard ^[1]

Affiliation

^[1] Univ Sao Paulo, Dept Quim, Fac Filosofia Ciencias & Letras Ribeirao Preto, Av Bandeirantes 3900, Bloco 18, Sala 8, BR-14040901 Ribeirao Preto, SP - Brazil

^[2] Univ Sao Paulo, Dept Quim & Fis, Fac Ciencias Farmaceut, BR-14040901 Ribeirao Preto, SP - Brazil

^[3] Univ Cambridge, Dept Biochem, Cambridge CB2 1QW - England

• Document type: Journal article
• Source: COMPUTATIONAL AND STRUCTURAL BIOTECHNOLOGY JOURNAL; v. 19, p. 1108-1118, 2021.
• Web of Science Citations: 0

Abstract

Effective use of plant biomass as an abundant and renewable feedstock for biofuel production and biorefinery requires efficient enzymatic mobilization of cell wall polymers. Knowledge of plant cell wall composition and architecture has been exploited to develop novel multifunctional enzymes with improved activity against lignocellulose, where a left-handed beta-3-prism synthetic scaffold (BeSS) was designed for insertion of multiple protein domains at the prism vertices. This allowed construction of a series of chimeras fusing variable numbers of a GH11 beta-endo-1,4-xylanase and the CipA-CBM3 with defined distances and constrained relative orientations between catalytic domains. The cellulose binding and endoxylanase activities of all chimeras were maintained. Activity against lignocellulose substrates revealed a rapid 1.6- to 3-fold increase in total reducing saccharide release and increased levels of all major oligosaccharides as measured by polysaccharide analysis using carbohydrate gel electrophoresis (PACE). A construct with CBM3 and GH11 domains inserted in the same prism vertex showed highest activity, demonstrating interdomain geometry rather than number of catalytic sites is important for optimized chimera design. These results confirm that the BeSS concept is robust and can be successfully applied to the construction of multifunctional chimeras, which expands the possibilities for knowledge-based protein design. (C) 2021 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology. (AU)

• FAPESP's process: 12/01066-2 - Design of multifunctional enzymes that combine the hydrolysis of B-glucans and Xylans to evaluate the conformation of these polysaccharides in plant cell walls
• Grantee: Matheus Pinto Pinheiro
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 16/24139-6 - Design and Characterization of Multifunctional Xylanolytic Enzymes
• Grantee: Richard John Ward
• Support Opportunities: Regular Research Grants
• FAPESP's process: 14/13956-8 - Investigation of cell wall polysaccharide architecture from different lignocellulosic feedstocks
• Grantee: Matheus Pinto Pinheiro
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• FAPESP's process: 14/50884-5 - INCT 2014: National Institute of Science and Technology of Bioethanol
• Grantee: Marcos Silveira Buckeridge
• Support Opportunities: Research Projects - Thematic Grants