Synthetic Biology For Green Chemistry: Building In Vivo Enzymatic Cascades Using Carboxylic Acid Reductases (CARs)

Abstract

Biocatalysis has a proven track record of offering replacements for individual chemical reactions with a lower environmental impact. Cascade reactions are an extension of biocatalysis; coupling a series of reactions to provide replacement for more than one chemical step. Herein, this thesis describes the engineering of a multi-enzyme cascade reaction for the production of phenylacetylcarbinol (PAC). Using a carboxylic acid reductase (CAR) from Mycobacterium phlei and a pyruvate decarboxylase from Acetobacter pasteurianus this thesis demonstrated a biocatalytic cascade reaction in which benzoic acid and pyruvate are converted into PAC. This cascade was combined with several other enzymes to recycle spent cofactors and deplete inhibitor by-products. Furthermore, this thesis has highlighted the discovery of five putative enzymes; three ancestral CARs (AncCARs) and two thiamine diphosphate (ThDP) dependent enzymes. CARs typically have poor stability and thus limited tractability in industrial reactions. Within this study ancestral sequence reconstruction was performed on type I CARs. This is a developing engineering tool that can identify stabilizing and enzymatically neutral mutations throughout a protein. A combined algorithm approach was used to reconstruct functional ancestors of the Mycobacterial and Nocardial Type I CAR orthologues. Carboxylic acid reduction by Ancestral CARs was confirmed. Each showed a preference for aromatic carboxylic acids. AncCARs also showed improved tolerance to solvents, pH and in vivo-like salt-like conditions. Compared to well-studied extant CARs, AncCARs had a Tm up to 35 °C higher. Two ThDP enzymes were discovered using metagenomics. These were assessed in silico through homology modelling and docking simulations. Furthermore, this study has demonstrated the importance of each tool in the discovery of new enzymes from within the ThDP family. Our homology models were used in docking simulations with unique carboligation-like intermediates allowing a rationalization of the reactions and stereoisomerism of the products. Two functional enzymes ThDP enzymes were identified that are capable of producing PAC; one from Thermus thermophilus and another from bacterium HR16.