Expression, characterisation, and engineering of cyanobacterial natural product biosynthetic pathways

Abstract

Microorganisms are a valuable source of natural products with medically and industrially relevant activities. Cyanobacteria are one of the most chemically diverse microbial phyla, but have been largely underexplored due to their slow growth and intractability to genetic engineering. In recent years, genomic and metagenomic investigations of the aquatic environment have uncovered the untapped diversity of cyanobacterial nonribosomal peptide and polyketide biosynthetic gene clusters. This dissertation describes the application of emerging synthetic biology techniques using Escherichia coli as a heterologous host, focussing on translating the bioactive potential of cyanobacteria into industrial applications, while simultaneously characterising and tailoring this biochemical capacity.

E. coli GB05-MtaA was previously shown to be a suitable host for the relatively simple cyanobacterial nonribosomal peptide synthetase (NRPS) pathway lyngbyatoxin (LTX). A synthetic biology approach for characterising and expanding lyngbyatoxin chemical diversity was explored. This thesis reports an in vitro investigation of wild-type LtxA NRPS activity that unravelled the multispecificity of the first adenylation domain to L-Val related amino acids, which correlates with the formation of novel lyngbyatoxin analogues in vivo. Efficient site-directed mutagenesis of the adenylation domain to modulate substrate specificities was performed through Red/ET-based recombineering, resulting in a library of mutated LTX pathways. Investigation of in vitro and in vivo pathway activity revealed the complexity of LTX biosynthesis, dictated by tailoring enzymes.

The benefits of using this approach to probe LTX biosynthesis motivated the use of a similar application for the directed production of neosaxitoxin (neoSTX). NeoSTX is a paralytic shellfish toxin (PST) which has medically-relevant activities. The polyketide synthase (PKS)-like enzyme SxtA, which initiates PST biosynthesis, was expressed in E. coli to assess the suitability of this host. Efficient production of PST intermediates by heterologously expressed SxtA paved the way for the expression of an engineered neoSTX biosynthetic pathway in E. coli. Several variants of E. coli strains were successfully constructed to produce neoSTX. This synthetic biology process, with an E. coli expression system, is a feasible strategy to facilitate the commercial production of neoSTX.