Alkaloids synthesis via enzymatic cascades

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.

The 2016 UK Roadmap Bio-design for the Bio-economy highlighted the substantial impact that synthetic biology can bring to the UK and global economies by developing: frontier science and technology; establishing a healthy innovation pipeline; a highly skilled workforce and an environment in which innovative science and businesses can thrive. Synthetic biology promises to transform the UK Bio-economy landscape, bringing bio-sustainable and affordable manufacturing routes to all industrial sectors and will ensure society can tackle many contemporary global Grand Challenges including: Sustainable Manufacturing, Environmental Sustainability Energy, Global Healthcare, and Urban Development. Whilst synthetic biology is burgeoning in the UK, we now need to build on the investments made and take a further lead in training next generation scientists to ensure sustained growth of a capable workforce to underpin the science base development and growth in an advanced UK bio-economy.
This training provided by this CDT will give students from diverse backgrounds a unique synthesis of computational, biomolecular and cellular engineering skills, a peer-to-peer and industrial network, and unique entrepreneurial insight. In so doing, it will address key EPSRC priority areas and Bioeconomy strategic priorities including: Next-generation therapeutics; Engineered biomaterials; Renewable alternatives for fuels, chemicals and other small molecules; Reliable, predictable, and scalable bioprocesses; Sustainable future; Lifelong health & wellbeing.
Advances created by our BioDesign Engineering approach will address major societal challenges by delivering new routes for chemical/pharma/materials manufacture through to sustainable energy, whilst providing clean growth and reductions in energy use, greenhouse gas emissions and carbon footprints. Increased industry awareness of bio-options with better civic understanding will drive end-user demand to create market pull for products. The CDT benefits from unrivalled existing academic-industry frameworks at the host institutions, which will provide direct links to industrial partners and a direct pathway to early economic and industrial impact.

This CDT will develop 80-100 next-generation scientists and technologists (via the funded cohort and wider integration of aligned students at the three institutions) as adept scientists and engineers, instilled with technical leadership, who as broadly trained individuals will fill key skills gaps and could be expected to impact internationally through leadership roles in the medium term. Importantly the CDT addresses key skill-gaps identified with industry, which are urgently required to create and support high value jobs that will enable the UK to compete in global markets. Commercialisation and entrepreneurship training will equip the next generation of visionaries and leaders needed to accelerate and support the creation of new innovative companies to exploit these new technologies and opportunities.

The UK government identified Synthetic Biology as one of the "Eight Great Technologies" that could be a key enabler to economic and societal development. This CDT will be at the forefront of research that will accelerate the clean growth agenda and the development of a resilient circular bioeconomy, and will align with key EPSRC prosperity outcomes including a productive, healthy and resilient nation. To foster wider societal impact, the CDT will expect all students to contribute to public outreach and engagement activities including: open days, schools visits, and science festival events: students will participate in an outreach programme, with special focus on widening participation.

This CDT will contribute to the development of industrial strategy through the Synthetic Biology Leadership Council (SBLC), Industrial Biotechnology Leadership Forum (IBLF), and wider Networks in Industrial Biotechnology and Bioenergy and Professional Institutes.