Engineering Carbon-carbon Bond Forming Enzymes from Plants for Applications in Industrial Biotechnology

工程化植物碳-碳键形成酶在工业生物技术中的应用

• 批准号: 2267474
• 金额: --
• 依托单位: University of York
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2267474
• 关键词: Engineering; Carbon; carbon; Bond; Forming

Carbon-carbon bond formations are some of the most significant reactions in synthetic organic chemistry as they permit the synthesis of the frameworks upon which most complex molecules, including pharmaceuticals, are based. Enzymes that form C-C bonds have been the focus of significant research in recent years as they offer advantages of high activity and selectivity in C-C bond formation, coupled with the benefits of sustainable synthetic methodology. C-C bond forming enzymes of interests have included aldolases, hydroxynitrile lyases and more recently enzymes that catalyze biological Friedel-Crafts reactions.The variety of carbon skeletons in plant metabolites is impressively diverse, and include terpenes, phenylpropanoids and alkaloids, each of which are valuable sources of bioactive molecules. Accordingly, C-C bond forming enzymes from plants have been studied for possible applications in preparative synthesis. Examples include strictosidine synthases (STRs), norcoclaurine synthases (NCSs) and berberine bridge enzymes (BBEs), each of which have roles in the formation of alkaloid compounds and are remarkable for the stereoselectivity of the C-C bond forming reaction that they catalyze.An examination of the literature in plant biosynthesis and secondary metabolism reveals other candidate C-C bond forming enzymes, the potential of which for preparative synthesis through heterologous expression and protein engineering has not yet been realised.In this project, C-C bond forming enzymes from plant biosynthesis will be investigated. Genes encoding the enzymes will be obtained and expressed recombinantly in E. coli or other expression systems. Structures of these enzymes will be determined through X-ray crystallography and used to inform site-directed mutagenesis experiments to investigate the enzyme mechanism or alter the activity/substrate scope. The potential applications of these biocatalysts for industrial biotechnology will be assessed.

碳-碳键的形成是合成有机化学中一些最重要的反应，因为它们可以合成包括药物在内的大多数复杂分子所依赖的框架。形成C-C键的酶近年来一直是重要的研究热点，因为它们在C-C键形成中具有高活性和选择性的优点，再加上可持续合成方法的好处。C-C键形成酶包括醛缩酶、羟基腈裂解酶和最近催化生物弗里德尔-克拉夫特反应的酶。植物代谢物中的碳骨架种类繁多，包括萜烯、苯丙素和生物碱，它们都是生物活性分子的宝贵来源。因此，从植物中提取的C-C键形成酶已被研究用于制备合成的可能应用。例如，strictosidine合成酶（STRs）、norcoclurine合成酶（NCSs）和小檗碱桥酶（BBEs），它们都在生物碱化合物的形成中发挥作用，并且在它们催化的C-C键形成反应中具有显著的立体选择性。通过查阅植物生物合成和次生代谢方面的文献，发现了其他候选的C-C键形成酶，这些酶通过异源表达和蛋白质工程进行制备性合成的潜力尚未实现。本项目将研究植物生物合成中的C-C键形成酶。编码酶的基因将被获得并在大肠杆菌或其他表达系统中重组表达。这些酶的结构将通过x射线晶体学确定，并用于指导定点诱变实验，以研究酶的机制或改变活性/底物范围。将对这些生物催化剂在工业生物技术中的潜在应用进行评估。