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Title: Genome mining for actinomycete biosynthetic gene clusters
Author: Dudbridge, Frederic Henry
ISNI:       0000 0004 7230 4855
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2018
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Whole-genome sequencing has shown that the large (8-12 Mbp) genomes of Streptomyces and allied genera of Gram-positive filamentous bacteria house a rich and previously underestimated repertoire of gene clusters for biosynthesis of specialised metabolites, including antibiotics, immunosuppressants and anticancer compounds. Many of these clusters remain uncharacterised because they are not expressed under the culture conditions used. Even for strains from which a specific compound has been identified, the challenge remains to link the compound to its gene cluster, and to develop procedures for analysing and manipulating the biosynthetic pathway. In this work, three strains have been studied that address different aspects of this challenge. Streptomyces sp. DSM4137 is a genetically amenable strain and a notably prolific producer of diverse natural products, but also has multiple biosynthetic gene clusters that remain uncharacterised. In an attempt to differentiate those clusters where the genes are expressed from those that are essentially silent, the transcriptome of DSM4137 was analysed using total RNASeq and the results were used to inform analysis of HPLC/MS data of extracts under the same conditions. There was shown to be good correlation between the RNASeq results and the pattern of metabolites produced, suggesting that RNASeq may be a useful complement in the search for novel gene clusters. In contrast, Saccharopolyspora spinosa, producer of the valuable insecticidal spinosyns, is not genetically amenable. A new technique has been developed for the mobilisation of an entire biosynthetic gene cluster and refactoring attempted to increase the production of spinosyns in a heterologous strain. Total transcriptome was analysed by RNAseq to give an insight into the regulation of the WT strain, helping identify future methods for strain manipulation for increase yields.
Supervisor: Leadlay, Peter Sponsor: BBSRC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Streptomyces ; genome-mining ; biosynthesis