Understanding the P450 enzymes essential for monolignol biosynthesis

可持续能源生产的可行过程必须对环境无害，减少温室气体的产生，并利用可再生资源。根据2007年的《能源独立安全法案》（Energy Independence Security Act），美国制定了到2030年用植物木质纤维素燃料取代30%的石油基燃料的国家目标。然而，目前用于制造木质纤维素生物燃料的方法的效率受到细胞壁中木质素存在的严重阻碍。木质素是一种由单木质素和相关化合物氧化聚合形成的聚合物，它可以保护纤维素免受水解酶的加工，这对经济上可行的生物燃料生产提出了重大挑战。拟议的研究将提供必要的基础知识，以调整植物细胞壁的化学成分，以最佳地利用生物燃料生产。除了应用于生物燃料生产之外，更广泛的影响还包括对本科生和研究生进行多学科和定量方法的培训。从这项研究中积累的知识将指导有效的酶的配方，用于木质素的生物合成，使其更适合生物燃料生产。pi专门针对高粱，但结果将普遍适用于大多数重要商业作物/林业物种的大多数单素途径。该研究使用最先进的单分子方法、x射线晶体学、光谱学和热化学技术，开发了一个详细的框架，描述了单分子醇生物合成途径中的关键氧化步骤。建议的工作将集中在：(1)途径中P450 （C4H, C3'H和F5H）及其主要氧化还原伙伴细胞色素P450还原酶（CPR）之间的蛋白-蛋白相互作用，(2)靶向P450的底物特异性，(3)氧化还原状态和底物结合对P450-CPR和C4H- pal复合物形成的影响。这项研究将提供必要的知识，能够调整细胞壁的化学成分，用于生物燃料和原料生产。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.