Enzymatic oxidation of sugarcane bagasse 2: exploring the interaction between LPMOs and its redox partner CDHs aiming for the development of more efficient enzymes for insertion into an engineered cell factory

• Grant number: 21/06679-1
• Support Opportunities: Program for Research on Bioenergy (BIOEN) - Young Investigators Grants - Phase 2
• Duration: April 01, 2022 - March 31, 2027
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Fernando Segato
• Grantee: Fernando Segato
• Host Institution: Escola de Engenharia de Lorena (EEL). Universidade de São Paulo (USP). Lorena , SP, Brazil
• Associated researchers: Igor Polikarpov ; Leandro Cristante de Oliveira ; Michael A. Marletta
• Associated research grant: 14/18714-2 - Enzymatic oxidation of sugarcane bagasse: discovery, characterization and new application of oxidative enzymes active in carbohydrates, applied to the enhancement of a fungal cell factory, AP.BIOEN.JP
• Associated grant(s): 22/00539-6 - Computational analysis and synthetic biology approaches to understand the interaction and electron transfer between CDH and LPMO, AP.R SPRINT; 22/04227-9 - Multiuser equipment granted in the proposal 2021/06679-1: Dionex ICS600 Capillary HPIC system, AP.EMU
• Associated scholarship(s): 22/14376-1 - Functional analysis of disordered regions associated to lytic polysaccharide monooxygenases (LPMO): Biochemical characterization and interaction with cellobiose dehydrogenase (CDH), BP.IC; 22/09945-7 - Evaluation of interaction sites in the recombinant cellobiohydrolase Cel7D from Phanerochaete chrysosporium with low molecular weight phenolic compounds, BP.IC; 22/04713-0 - Exploring the interaction between lytic polysaccharide monooxygenases and its redox partner cellobiose dehydrogenases, BP.DD

Abstract

Lignocellulose is an abundant source of polysaccharides that can be used as a renewable raw material for the production of biofuels and other compounds of interest. The conversion of lignocellulose into its subunits is a slow and costly process, therefore, more study is needed to make this process feasible. Until recently, the available enzymatic cocktails were composed of hydrolytic enzymes that act on the cellulose components of lignocellulose. These enzymes cocktails are low and inefficient. In recent years, oxidative proteins have been discovered that act directly on crystalline cellulose have been described, and are named LPMOs. Since the discovery, LPMOs have been added to the commercial cocktails improving their performance, as well as, have been the focus of many studies related to their mode of action, interaction with substrate and electron donors including the enzyme CDH. Through the analysis of amino acid, sequence of LPMOs from different filamentous fungi an extra-region was found to the structure described so far. Previous molecular docking analysis of the interaction between CDHs and LPMOs provided evidenced that these extra-regions serve to anchor location of the CDH for electron transfer pathway. Since we have been developing a filamentous fungus for the enzymes production the current proposal aim to 1) understanding the coupling mechanism between CDH and LPMO, as well as describing the electron transfer pathway to the copper active site; 2) use a multiplex RNAi system to improve the production of proteins in filamentous fungi; 3) insert the synthetically modified proteins from previous computational analysis into our expression system in order to improve the biomass saccharification, as well propose the use of this oxidative enzymes in biosensors. (AU)