Engineering new enzymes for synthesis and biosynthesis

Abstract

Halogenated moieties are massively used as pharmaceuticals, agrochemicals, polymers and many other valuable materials. Biocatalysis has been evolving over the years with interest growing due to the use of naturally occurring enzymes to carry out reactions with chemo- / regio- or stereo- selectivities, which would be otherwise impossible using traditional organic synthesis. That could offer a more efficient ecological and economical advantage during the production of molecular complexity. More selective halogenations can be achieved via biocatalysis. Contrary to traditional chemical reactions which bring about a plethora of side reactions, biocatalysis could bring a more alternative option. In the current project, Vmax™ cells were used instead of E. coli, considering that they would be a more suitable host for halogenases whole cells assays. Despite the fact that detectable activity was achieved in these Vmax™ expressed halogenases, the whole cells assays were unsuccessful. The promising detectable activity though is a novel finding as this halophile has not been reported previously with expression of halogenases. In addition, the identification of a novel tryptophan halogenase, S. chromofuscus found by bioinformatic analysis was screened against a number of substrates and under different conditions, showing halogenation regioselective to the 5 position, showing detectable conversion in most of the substrates. Future work on this enzyme could expand its substrate scope and improve the yield and/or conversion. Finally, with the aim of understanding the biosynthesis pathway involved in the production of one of the major polyketide mycotoxins faced in agriculture, the expression and characterisation of the Ochratoxin halogenase, identified in the Ochratoxin A mycotoxin biosynthesis pathway was attempted but not succeeded. Although many optimisations and alterations within the gene were experimented, it was considered that the expression of the halogenase in E. coli is not suitable as the original system is a fungal host.

Date of Award	1 Aug 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Jason Micklefield (Supervisor) & Anthony Green (Supervisor)