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

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.