EASTBIO Project: Exploring, evolving and exploiting thioester synthetases for industrial biotechnology

AIM: Thioesters are high value substrates used in the production of various natural products. A goal of biocatalysis is to use “green” or “enzymatic” methods to generate chemical building blocks and catalyse chemical transformations in high yield and specificity. The chemical synthesis of acyl-thioesters requires multiple chemical steps from carboxylic acids. This project will develop a coupling-agent free, one-pot bioconversion of acids to acyl-thioesters in water using an engineered pimeloyl-CoA synthetase (PCAS) biocatalyst.

BACKGROUND: Natural and engineered biocatalysts are already having impact in the manufacture of high value pharmaceuticals by catalysing the conversion of functional groups in high yield. The use of enzymes will be more routine once their limitations, such as narrow substrate range, are overcome. 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 identified, modern enzyme engineering techniques (e.g. directed evolution) can be applied to generate a bespoke biocatalyst with broad synthetic utility. In this project we will exploit members of a large family of enzymes to develop an efficient route for the preparation of high value intermediates.

THIOESTER SYNTHETASE TARGET: Acyl-thioesters are key metabolic building blocks used to drive the production of various natural products (e.g. fatty acids, lipids and antibiotics). Their synthesis involves the conversion of the carboxylic acid to the thioester which is catalysed by an acyl-CoA synthetase enzyme. They use ATP to activate the acid as an adenylate intermediate, then react this with the CoASH thiol to give the thioester. Acyl-CoA synthetase enzymes such as PCAS are part of a large superfamily of ANL enzymes that include firefly luciferase. The PCAS from the bacterium B. subtilis (~260 aa, ~29 kDa, EC:6.2.1.14) is the key first enzyme in the biosynthetic pathway of the vitamin biotin. In collaboration with Prof. Jim Naismith (St. Andrews, co-supervisor) we have made an exciting, recent breakthrough in determining the first x-ray structure and catalytic mechanism of B. subtilis PCAS. The enzyme displays a novel 3D fold and by identifying active site residues, we were able to rationally engineer the enzyme to overcome its narrow substrate specificity and accept novel fatty acid derivatives. Mutant PCAS enzymes were thus capable of generating designer acyl-CoA thioesters. This project now seeks to fully exploit the PCAS enzyme and generate enzymes with broader substrate range and synthetic applications. It will involve cloning/expression/purification of PCAS and other ANL family enzymes and we will work towards understanding the structure of these enzymes with bound substrates, intermediates and products. We will generate novel biocatalysts using directed evolution/site-directed and random mutagenesis methods guided by our structural work. We will also optimise a high-throughput (HTP) screen that will allow us to identify hit PCAS clones with novel activity. We will start with simple substituted fatty acids before moving on to more complex substrates. We will also explore an ATP recycling system to optimise the yield of acid to thioester conversion and explore whether we can interface this with a downstream C-C condensation step to generate novel keto-amines. By introducing –halogens, alkyne and azo functional groups we will also explore chemical couplings to generate a range of thioester derivatives that are outwith the reach of chemical synthesis. This exciting project will train a student in the skills required to work at the forefront of the exciting field of biocatalysis/enzyme engineering. A PhD graduate with these skills will be highly employable in the field of industrial biotechnology in academia or industry. Most drug companies (GSK, Merck, AstraZeneca) have biocatalyst teams playing key roles in the drug discovery process. The EastBio student will join the vibrant EastChem post-graduate school with >150 students and will have opportunities to take additional Masters relevant level courses cherry-picked from other CDTs such as CRITICAT. We expect our work will have high impact and be published in high ranking journals where we publish much of our work.