Huge progress in our understanding of the molecular mechanisms of natural product biosynthesis has been made in the last two and a half decades. This has underpinned the development, of genome mining as a tool for the discovery of novel bioactive natural products, leading to renewed industrial interest in such compounds as leads for the development of novel drugs and agrochemicals. Over the past decade, microbial genome sequencing projects have uncovered hundreds of so-called “cryptic” natural product biosynthetic pathways, for which the metabolic products are unknown. Marine actinobacteria have been shown to be a particularly prolific resource of cryptic biosynthetic pathways and, given the provenance of this group of microorganisms, it seems highly likely that many of the metabolic products of such pathways will be novel antimicrobials with the potential to tackle the emerging health threat posed by AMR. Advances in both DNA sequencing technology has made it possible to obtain complete bacterial genome sequences using Pacific Biosciences SMRT sequencing in a matter of weeks. The long sequence reads generated by Pacific Biosciences SMRT sequencing facilitates the correct assembly of natural product biosynthetic gene clusters (BGCs), especially those encoding modular polyketide synthase (PKS) or nonribosomal peptide synthetases (NRPS) multienzymes, which are frequently involved in antibiotic biosynthesis.
In collaboration with the Ocean University of China, the candidate will carry out initial LC-ESI-TOF-MS survey of the metabolites produced by marine microorganism strain selected for genome sequencing. Secondary metabolite biosynthetic gene clusters will be identified through bioinformatic analysis of sequenced genome. Combing bioinformatic prediction with LC-MS analysis data will facilitate rapid de-replication to avoid rediscovery of known compounds. Molecular formulae and UV-Vis spectra generated by these analyses will be compared with databases to ascertain whether they correspond to known natural products. Subsequently, the candidate will investigate fermentation condition and analyse metabolite profiles of both wild type and non-producing mutant strains generated by collaborators in SLS, purify novel compounds for structure elucidation and investigate activity against the ESKAPE panel of pathogens. The purified material will be used for further anti-microbial property investigation.
During this project, you will learn advanced analytical chemistry techniques, including bacterial fermentation optimisation and natural product extraction, normal phase and reverse phase liquid chromatography, dereplication, modern high resolution and high accuracy mass spectrometry and NMR for structural elucidation.
Project Aims:
- Optimizing fermentation conditions, manipulating regulatory factors to activate silent gene cluster or enhance gene expression and improve titer of bioactive compounds.
- De-replication with high resolution LC-MS/MS to identify potential novel compounds.
- Purify novel antibiotics, structure elucidation using a combination of 1D, 2D NMR and high resolution MS, MS/MS techniques.
- Purified bioactive compounds will be evaluated for their antimicrobial activity against a range of Gram-positive and Gram-negative bacteria, including representative members of the ESKAPE panel of pathogens.
BBSRC Strategic Research Priority: Understanding the rules of life – Systems Biology, Plant Science, Soil Science, and Microbiology, Renewable Resources and Clean Growth - Industrial Biotechnology, Sustainable Agriculture and Food - Microbial Food and Safety, and Plant and Crop Science, and Integrated Understanding of Health - Pharmaceuticals.
Techniques that will be undertaken during the project:
- Bioinformatic analysis of bacterial genome
- Microorganism fermentation
- Natural product purification through preparative HPLC
- LC-MS/MS for metabolite profiling and dereplication
- Structural elucidation with MS and NMR techniques
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