The research proposed aims to provide new sustainable processes for the production of chemicals using nature's catalysts, enzymes. In particular we will focus on an enzyme that is able to catalyse the production of chemicals called esters, which are ubiquitous in chemical products such as pharmaceuticals and also important as chemical intermediates for synthetic processes. The formation of an ester from an acid often involves energy demanding processes or the use of toxic chemicals and the reactions must be done in the absence of water. The enzyme under development in this project will allow the formation of esters under aqueous conditions, making it compatible with other enzyme catalysts and allowing the development of biological synthetic pathways for chemical synthesis, or cascades. In this project, we will develop highly efficient mutants of the ester forming enzyme that are able to catalyse the formation of key intermediates for the syntheis of bioplastics and pharmaceuticals. We will use state-of-the-art screening technology that is already in use in our laboratories to identify the mutants. We will then couple the new enzymes with other smaller enzyme cascades that we have already developed for the synthesis of biodegradable bioplastics from biorenewable materials such as cellulose and lignin. We will also develop new cascades for the synthesis of pharmaceuticals by coupling with other new enzymes we have discovered recently that are surprisingly able to catalyse the formation of amide bonds in water. Amide bonds are important bonds within pharmaceuticals. The combination of the ester formation followed by the amide formation will enable replacement of expensive coupling reagents used in conventional amide bond synthesis. The ester forming enzyme needs a specific molecule found in cells called SAM to act as a catalyst. We will develop optimised cell hosts (so-called microbial cell factories) to produce all the required enzymes and the SAM, such that cell fermentations can be used to carry out the synthesis of the target chemicals. We will demonstrate a fermentation for the conversion of waste plastic, PET, into a pharmaceutical drug. We also will develop cells able to metabolise methanol that will drive the synthesis and a novel strain that can recycle the methanol produced in the amide bond forming reaction which is used by the ester forming enzyme making the whole process able to function with very small amounts of methanol.

Technical Abstract:
The proposed research aims to develop carboxyl methyltransferases (CMTs) for use in sustainable synthesis of bioplastics precursors and pharmaceuticals. Methylation of carboxylic acids is a ubiquitous reaction in synthesis and is used for activation of carboxylic acids or as the final synthetic step. Most methods involve energy intensive processes or use moisture sensitive reagents under dry conditions. CMTs are an unexploited group of S-adenosyl methionine (SAM)-dependent enzymes that provide the opportunity to activate carboxylic acids under aqueous conditions and are compatible with other enzymes to generate artificial biosynthetic pathways. We have recently explored the activity and substrate selectivity of the enzyme FtpM and found that it can methylate and dimethylate mono and dicarboxylic acids. In this project we will improve the activity of FtpM for dimethylation of biomass-derived FDCA and 2,5-PDCA to give the dimethyl esters, known bioplastics precursors, using directed evolution. We have developed a new efficient assay based on release of methanol that will be used to screen mutants and we will adapt this assay to the ultra high throughput FACs based screen currently being used in our laboratory. We will couple FtpM biomethylation with new acyl transferase enzymes for making amides from methyl esters and amines under aqueous conditions, without competing ester hydrolysis. Isolated enzymes will be tested before developing optimised recombinant whole cell strains in which SAM cofactor biosynthesis is upreguated. A fermentation for the conversion of the waste PET plastic into a pharmaceutical drug will show application of the methodology. We also will develop strains where methanol will provide the methyl source for FtpM catalysed biomethylation and a new metabolic circuit will allow the whole cell synthesis of amides from acids that is catalytic in methanol.