Alkaloids synthesis via enzymatic cascades

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Tetrahydroisoquinoline alkaloids (THIAs) are a structurally and functionally diverse group of plant metabolites with significant therapeutic activities, such as the anti-bacterial berberine, analgesic morphine and anti-psychotic aporphine. Some THIAs can be extracted from plants which is low yielding and chemical total synthesis suffers from multistep procedures and low reaction selectivity. New stereoselective routes to THIAs are highly sought after as they are privileged pharmacophores. We have recently pioneered a cell free enzyme cascade approach where we know the exact properties of each enzyme and thus have a versatile synthetic system to make known and novel THIAs. Key enzymes developed for use in the cascades include tyrosinases and decarboxylases that convert L-tyrosine into dopamine, transaminases that can synthesise aldehydes and the Pictet-Spenglerase norcoclaurine synthase (NCS). In addition, we have other enzymes that can be used in the synthesis of THIA precursors or modify products such as methyl transferases. Our aim in this PhD is to develop the NCS enzymes for use with a wider range of phenethylamines and the use of tailoring enzymes after THIA formation for small molecule generation, via scalable processes.Studies will initially focus on developing the NCS reaction with a range of phenethylamines to probe the substrates accepted as well as NCS mutagenesis, based on our current mechanistic understanding. In silico modelling will be essential for guiding substrate design, synthesis and testing with NCS mutants. We will also develop plant P450s for THIA diversification to access alkaloids and un-natural analogues. Selected enzymes will then be used to construct enzyme pathways to establish efficient sustainable cascades to novel/natural single isomer alkaloids. Enzyme systems will be optimised for scalability in vitro, and we will investigate the co-expression of two or three enzymes on the same plasmid and immobilised enzyme supports. Overall, the end goal is to engineer enzymes and pathways for wider use including scalable processes to this very important family of bioactive alkaloids.