Natural and engineered biocatalysts are being employed in the manufacture of high value pharmaceuticals by catalysing the conversion of key building blocks in high yield. The use of enzymes will be more routine once their limitations, such as narrow substrate range, are overcome (Badenhorst & Bornscheuer, 2018, Trends in Biochem. Sci., 43, 180). The range of chemical transformations displayed by enzymes continues to grow, fuelled by the discovery of new enzymes involved in natural product biosynthesis and accelerated by genome sequencing. Once a new enzyme is characterised, modern enzyme engineering techniques (e.g. directed evolution) can be applied to generate a bespoke biocatalyst with broad synthetic utility (e.g. Campopiano DJ et al, 2017, Nature Chem. Biol., 13, 660). Even more powerful is when two or more enzymes are linked together in to generate an efficient cascade optimised for the conversion of simple cheap starting materials into high value products. Such pathway engineering can build complex high value molecules in simple bacterial hosts. In this project we will couple together members of enzymes superfamilies to build an efficient route for the preparation of high value amines. Specifically, we will convert long chain fatty acids into amines by combining a carboxylic acid reductase (CAR) with an aminotransferase (AT) in an efficient process. The CAR enzymes are a growing family of three-domain enzymes that catalyse the reduction of long chain fatty acids (C6-C20) into fatty aldehydes. Recent structural studies have revealed how each CAR domain performs a separate catalytic function (Gahloth et al., 2017, Nature Chem. Biol., 13, 975). The ATs are a family of pyridoxal 5’-phosphate (PLP)-dependent enzymes that reversibly convert aldehydes and ketones into high value amines. Using ketone substrates, the ATs also control the enantioselectivity of the product (either R- or S- enantiomer at >99% e.e.). We have recently discovered a rare Sphingopyxis FumI AT that converts C10-C18 aldehydes and ketones into amines and have determined the crystal structure with the bound PLP cofactor.
The goals of this project are to: (1) screen a range of bacterial CARs from genome libraries and identify lead candidates. (2) Expand the substrate range of the FumI AT by directed evolution/enzyme engineering. (3) Combine the CAR and FumI in vitro to optimise the smooth conversion from acid substrates to amine products. (4) Develop a bacterial host biotransformation for the production of amines by studying metabolic flux via metabolic labelling/pathway engineering. (5) Introduce a branched-chain, substrate-specific AT starter enzyme and optimise production of high value branched-chain amines used in the food and flavour industry.
Training: This project will provide PhD training in modern structural biology techniques, enzyme engineering, metabolic labelling, pathway construction and synthetic biochemistry. We have a long term crystallography collaborator (Dr. Jon Marles-Wright, University of Newcastle), an expert in biocatalysis to provide the CAR enzymes (Prof. Nick Turner, University of Manchester) and Dr. Stephen Wallace has access to the GenomeFoundry labs in the School of Biology, Edinburgh. The EastBio PhD student will spend time in each of these collaborating labs.
To apply for this project, please go to this link.