Microbial natural products have served as a major inspiration for the development of novel pharmaceuticals. Cyanobacteria are a largely untapped phylum that produces a multitude of natural products, including toxins, anaesthetics, antibiotics and sunscreens. A limitation for the exploitation of these molecules is the lack of accessibility in the natural host due to slow growth rates, relatively low production levels, and limited resources to genetically manipulate the cyanobacteria. This project will involve the isolation of cyanobacterial natural product biosynthesis genes, engineering them for heterologous expression in Escherichia coli, and gene knockouts to characterise the enzymology of biosynthesis.
Cyanobacteria are renowned for the high proportion of genes dedicated to natural product biosynthesis. However, an advantage of using E. coli is that it does not produce any of these natural products, providing a “clean” background for heterologous expression. In contrast, biomass collected from cyanobacterial blooms in the environment can have an extremely complex intracellular matrix that may interfere with purification and affect bioassay and analytical results. E. coli also has an extremely fast growth rate (20 min doubling time) compared to the very slow growth of cyanobacterial native producers, with a doubling time of days in the laboratory. The heterologous expression of cyanobacterial specialised metabolite biosynthetic gene clusters within E. coli will enable the production of specific compounds and will also afford the directed biosynthesis of novel analogues in a ‘made-to-order’ system. Currently, the drawbacks of existing methods include high production costs, low yields, and limitations regarding the type of analogue being produced. This project aims to increase the yields of specialised metabolites using a more economical system and will also provide specific analogues on-demand without the need for extensive purification steps.
- Genome mining techniques will be used to identify target gene clusters followed by direct Pathway Cloning (DiPac) and refactoring in a fosmid/E. coli system. Target gene clusters will include non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes associated with potential bioactivity.
- Heterologously express putative specialised metabolite biosynthesis pathways in the E. coli host system.
- Purify expression products and perform chemical analyses using a range of bioassays for potential therapeutic value, including anticancer, immunomodulatory, bio-preservative, UV filtering and other cell cycle-regulating activities.
Another significant outcome from this project will be the confirmation of biosynthetic routes of the proposed gene clusters responsible for cyanobacterial specialised metabolite biosynthesis.