Chhun, Audam (2020) Antimicrobial potential of the marine actinomycete salinispora tropica cnb-440 in co-culture : a metabolomic, proteomic and genome engineering approach. PhD thesis, University of Warwick.

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Abstract

he alarming rise of antimicrobial resistance in pathogenic strains has fuelled tremendous research efforts towards the discovery of novel bioactive molecules from untried ecological niches. In this regard, the world’s oceans have revealed to be a remarkable resource of new bacterial taxa with promising biosynthetic potential. Study of the secondary metabolism of the marine actinobacterium Salinispora, for instance, has shown the genus to be an exceptional trove of unique and bioactive natural products, rivalling the significance of its terrestrial counterpart.

The isolation of new secondary metabolites from members of the genus Salinispora and other microorganism is, however, currently limited because most of the biosynthetic gene clusters (BGCs) encoded in their genomes are not expressed under standard laboratory conditions. This is well exemplified in Salinispora, as a staggering 80% of its BGCs are still orphan (i.e. not linked to their products). This observation warrants the development of new approaches to unlock the biosynthetic potential of the genus.

This thesis is devoted to investigating novel ways to activate, interrogate, and manipulate the biosynthetic potential of Salinispora tropica CNB-440. First, we demonstrate how co-cultivation of S. tropica with phytoplankton could be used to elicit the production of novel cryptic compounds in the actinobacterium. Our data also reveal that S. tropica exhibits antimicrobial activity against a range of eukaryotic and prokaryotic marine phototrophs via an uncharacterized mechanism.

Second, we report the first proteome dataset available in the genus Salinispora, that we explored in order to identify candidate secondary pathways responsible for the biosynthesis of the detected cryptic molecules and/or the antimicrobial activity observed in co-culture. Using high-throughput proteomics, we provide evidence that the orphan nrps1 BGC is active and upregulated upon exposure to phytoplankton. We also suggest a potential mechanistic understanding of the antimicrobial effect seen in co-culture.

Finally, we implemented for the first time the CRISPR/Cas9 system in a member of the genus Salinispora, as a promising tool to link specific BGCs to the detected molecules and/or observed bioactivity described in our study. As a proof of concept, we successfully engineered S. tropica by deleting an entire BGC and a PPTase from its genome.

Item Type:	Thesis or Dissertation (PhD)
Subjects:	Q Science > QH Natural history > QH426 Genetics Q Science > QR Microbiology R Medicine > RM Therapeutics. Pharmacology
Library of Congress Subject Headings (LCSH):	Bioactive compounds, Actinobacteria, Anti-infective agents -- Development, Drug resistance in microorganisms, CRISPR-associated protein 9
Official Date:	October 2020
Dates:	Date Event October 2020 UNSPECIFIED
Institution:	University of Warwick
Theses Department:	School of Life Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Christie-Oleza, Joseph Alexander ; Corre, Christophe
Format of File:	pdf
Extent:	133 leaves : illustrations (chiefly colour), colour map.
Language:	eng

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