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Development of a pipeline for investigating fungal natural products, and investigation of the biosynthesis of pleurotin production in heterologous hosts
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Weaver, Jack (2023) Development of a pipeline for investigating fungal natural products, and investigation of the biosynthesis of pleurotin production in heterologous hosts. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b3985223
Abstract
During this PhD, a modular, robust, pipeline has been developed for sequencing, assembling, and annotating fungal genomes. The pipeline can use short and long-read sequencing as inputs, alongside RNA sequencing. This has been used to create the foundations from which work on identifying the biosynthetic gene clusters for a number of natural products has been conducted. Within this thesis 8 organisms have been sequenced, assembled, and annotated using this pipeline. Four in collaboration with the John Innes Centre, 3 in collaboration with both the Universiti Kebangsaan Malaysia and Malaysian Agricultural Research and Development Institute, and one genome for a strain of Hohenbuehelia atrocaerulea - a known producer of the anticancer antibiotic pleurotin.
Pleurotin was first discovered in 1947. During the ‘Golden age’ of antibiotic discovery, it has not been utilised industrially to date as was not from an organism that could be fermented easily at scale as well as being toxic to human cells and unstable. Several intermediates are proven, and a predicted metabolic pathway developed. Attempts to create the chemical synthetically had failed to produce it stereo-selectively until recently, and continue to fail to efficiently produce it.
The annotated genome for Hohenbuehelia atrocaerulea was the basis for comparative proteomic and transcriptomic work in this thesis, enabling the identification of a putative pleurotin producing biosynthetic gene cluster (BGC).
Saccharomyces cerevisiae and Aspergillus oryzae strains were selected as heterologous hosts for the putative BGC. Having faster culturing times, simpler growth requirements, and having GRAS status (generally regarded as safe). The putative BGC was expressed heterologously to discover biosynthetic intermediates to pleurotin and to gather further evidence to contribute to the eventual characterisation of the entire pleurotin pathway, while developing a base strain for eventual optimisation for pleurotin production in the future.
Metabolites from these strains have been detected, and NMR spectroscopy conducted on one of these which was purified (3-farnesyl-4-hydroxy-benzoic acid), a known precursor to pleurotin, not present in untransformed heterologous hosts.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QH Natural history > QH426 Genetics Q Science > QK Botany Q Science > QP Physiology Q Science > QR Microbiology |
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Library of Congress Subject Headings (LCSH): | Fungi -- Genetics, Biosynthesis, Genomes -- Analysis, Nucleotide sequence, Antibiotics -- Synthesis | ||||
Official Date: | June 2023 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | School of Life Sciences | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Alberti, Fabrizio ; Corre, Christophe | ||||
Format of File: | |||||
Extent: | xiii, 200 pages : illustrations | ||||
Language: | eng |
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