Understanding the P450 enzymes essential for monolignol biosynthesis

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Viable processes for sustainable energy production must be environmentally benign, reduce green-house gas production, and utilize renewable resources. Under the Energy Independence Security Act of 2007, the U.S. has established a national goal of replacing 30% of petroleum-based fuels with plant-based lignocellulosic fuels by the year 2030. However, the efficiency of currently applied methods for the manufacturing of lignocellulosic biofuels is significantly hindered by the presence of lignin in the cell wall. Lignin, a polymer formed from the oxidative polymerization of monolignol and related compounds, shields cellulose from processing with hydrolytic enzymes, posing a major challenge to economically viable biofuel production. The proposed research will provide the fundamental knowledge necessary to tune the chemical composition of the cell walls of plants for optimal use in biofuel production. The broader impacts, beyond application to biofuel production, include the training of undergraduate and graduate students in multidisciplinary and quantitative methods. The accumulated knowledge resulting from this research will guide the formulation of efficient enzymes for the biosynthesis of lignin with properties that make it more amenable to biofuel production. The PIs target sorghum specifically, but the results will be generally applicable to most monolignol pathways in a majority of commercially important crop/forestry species. The study uses state-of-the-art single molecule methods, X-ray crystallography, spectroscopy, and thermochemical techniques to develop a detailed framework that describes critical oxidative steps within the monolignol biosynthetic pathway. The work proposed will focus on: (1) protein-protein interaction between the P450s in the pathway (C4H, C3’H, and F5H) and their primary redox partner cytochrome P450 reductase (CPR), (2) the substrate specificity of the targeted P450s, (3) the effect of redox state and substrate-binding on the complex formation of P450-CPR and C4H-PAL. This study will provide the knowledge necessary to be able to tune the chemical composition of cell walls for use in biofuel and feedstock production.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.