Exploring the structure, function, and engineering of bacterial UbiD (de)carboxylases

Abstract

The UbiD family of prenylated-flavin (prFMN) dependent reversible (de)carboxylases substrates specificity ranges from phenylacrylic acids to (polycyclic) heteroaromatic compounds. Recent work with fungal members of the UbiD family, the ferulic acid decarboxylases (FDCs), has applied these enzymes to the production of value-added aldehydes and primary amines via multi-enzyme biocatalytic pathways, while campaigns of directed evolution have expanded the substrate scope of these enzymes, and enabled FDC-mediated in vivo production of industrially relevant products such as isobutene. Here we apply these approaches to bacterial members of the UbiD family, demonstrating the production of pyrrole-2-carbaldehyde from pyrrole by linking Pseudomonas aeruginosa PA0254 to a carboxylic acid reductase from Segniliparus rotundus. Utilising a mixedformat biocatalyst system in the presence of elevated bicarbonate, we demonstrate a 21% yield of aldehyde product via the UbiD-reaction mediated CO2 fixation. We then describe the biochemical and structural characterisation of a UbiD family member from Kitsatospora viridis (KvCAD) homologous to PA0254. We demonstrate KvCAD can catalyse (de)carboxylation of both phenylacrylic- and heteroaromatic acids and determined the structure of both the apo- and holoenzyme, which revealed how intriguing structural conformation of the key catalytic glutamic acidcontaining loop region implicates our existing understanding of domain motion in UbiD-mediated decarboxylation. We use the newly characterised KvCAD as a chassis for the exploration and development of tools for the directed evolution of the UbiD family, producing analytical techniques and screening vectors for this purpose. We explore the use of a phenotypic screen based on the pHdependent bacteriostatic properties of typical UbiD substrates such as cinnamic acid, and describe how bacterial carbon utilisation interferes with the applicability of such systems. We describe how modification of key residues Y278, W322 and D318 affects KvCAD activity, and isolate D318N with a 2-fold increase the decarboxylation of cinnamic acid in vivo.

Date of Award	1 Aug 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Nicholas Turner (Supervisor), David Leys (Supervisor) & Neil Dixon (Supervisor)