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

碳-碳键的形成是合成有机化学中最重要的反应之一，因为它们允许合成包括药物在内的大多数复杂分子所基于的框架。近年来，形成C-C键的酶一直是重要研究的焦点，因为它们在形成C-C键方面具有高活性和选择性的优点，加上可持续合成方法的好处。人们感兴趣的C-C键形成酶包括醛缩酶、羟基腈裂解酶和最近催化生物Friedel-Craft反应的酶。植物代谢物中碳骨架的多样性令人印象深刻，包括萜类、苯丙类和生物碱，每种都是生物活性分子的宝贵来源。因此，植物中的C-C键形成酶已被研究在制备合成中的可能应用。例如，严格苷合成酶(STRs)、去甲黄嘌呤合成酶(NCSs)和黄连素桥酶(BBES)，它们都在生物碱化合物的形成中起作用，并且它们催化的C-C键形成反应具有显著的立体选择性。对植物生物合成和次生代谢的文献研究发现，其他候选的C-C键形成酶还没有实现通过异源表达和蛋白质工程进行制备合成的潜力。在这个项目中，我们将研究植物生物合成中的C-C键形成酶。将获得编码这些酶的基因，并在大肠杆菌或其他表达系统中进行重组表达。这些酶的结构将通过X射线结晶学确定，并用于指导定点突变实验，以研究酶的机制或改变活性/底物范围。将评估这些生物催化剂在工业生物技术中的潜在应用。

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.