Studies on the Natural Products from Pseudoalteromonas and the Efforts to Induce the Expression of Biosynthetic Gene Clusters in Pseudoalteromonas

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Authors

Tomm, Hailey

Date

2024-10-02

Type

thesis

Language

eng

Keyword

Natural Products , Pseudoalteromonas , CRISPR-dCas9 , Structural elucidation

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Abstract

Natural products (NPs) are molecules produced by organisms that often have complex structures with inherent bioactivity. Over half of the clinically approved drugs, such as antibiotics, were derived from or inspired by NPs, underscoring the crucial role of NPs in drug discovery. The redundancy in nature often leads to the rediscovery of prominent bioactive NPs. The repeated rediscovery of known NPs led to the belief that many prolific NP-producing bacteria were exhausted. Fortunately, genome sequencing has revealed that organisms are likely capable of producing more NPs than previously thought. In bacterial genomes, the enzymes that biosynthesize NPs are encoded by a series of genes located close together called biosynthetic gene clusters (BGCs).1 Bioinformatic tools have uncovered that most microorganisms harbour more BGCs than detectable NPs, including Pseudoalteromonas, an underexplored genus of marine bacteria that produce pigmented conjugated NPs with interesting bioactivities. This thesis's work focuses on ways to elicit the expression of silent BGCs in Pseudoalteromonas and structurally elucidate an unknown NP. Two approaches were attempted to activate silent BGCs; the first explored the interactions between Pseudoalteromonas and cotton by culturing eighteen Pseudoalteromonas strains with cotton, culturing Pseudoalteromonas luteoviolacea 2ta16 in the presence of glass wool, and attempting to quantify the mRNA production from the violacein BGC. Of the eighteen strains cultured, ten exhibited a change in the metabolic profile and revealed an unknown NP (984 m/z) produced by several Pseudoalteromonas strains. A chapter was dedicated to elucidating the structure, determining it was a peptide with the residues threonine, leucine, glycine, and proline present, as well as a fatty tail eight carbon atoms long. The glass wool culturing confirmed a physical relationship between cotton and Pseudoalteromonas, and attempts to use mRNA quantification were inconclusive. The second approach focused on genetically eliciting the expression of silent BGCs using CRISPR-dCas9 to knock down the production of a prominent NP, prodigiosin, in Pseudoalteromonas rubra. Work toward establishing an in vivo CRISPR system for Pseudoalteromonas, as well as the cultivation of Pseudoalteromonas and structural elucidation of the unknown NP, is presented here.

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