Biosynthetic Lego: reprogramming RiPP biosynthesis

Eyles, Tom (2018) Biosynthetic Lego: reprogramming RiPP biosynthesis. Doctoral thesis, University of East Anglia.

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Abstract

Ribosomally synthesised and post translationally modified peptides (RiPPs) are a diverse class of industrially-important and clinically-relevant natural products. Reprogramming the biosynthesis of RiPPs can provide an understanding of their biosynthesis, increases in their yield, and compound derivatives. In this thesis, two RiPP biosynthetic pathways are reprogrammed to achieve these aims.

Bottromycin is a potent antibiotic RiPP, however it is produced in low yields by its native producer and it is rapidly hydrolysed in blood plasma. It was hypothesised that the bottromycin gene cluster could be reprogrammed to increase the production of bottromycin and to derivatise it. Synthetic biology techniques available at the start of the project were deemed inappropriate for use in reprogramming the bottromycin gene cluster as they lacked the ability to conduct refactoring, produce gene insertions/deletions, and make targeted mutations in single steps in the high-GC bottromycin gene cluster. Here, a one-step yeast-based method that enables efficient and flexible modifications to the bottromycin gene cluster is presented. Multiple modifications are showcased, including refactoring, gene deletions and targeted mutations. This facilitated the construction of an inducible, riboswitch-controlled pathway that achieved a 120-fold increase in pathway productivity in a heterologous host. Additionally, an unexpected biosynthetic bottleneck resulted in the production of a suite of new bottromycin-related metabolites.

Thiostreptamide S4 is part of a family of promising antitumor RiPPs, the thioviridamide-like molecules. The gene cluster responsible for thiostreptamide S4 production has been identified, yet the biosynthesis has not been elucidated. It was hypothesised that reprogramming the thiostreptamide S4 gene cluster could provide insights into its biosynthesis. These modified clusters were constructed, and in-depth metabolomics enabled an understanding of the biosynthetic pathway. This biosynthetic understanding could pave the way for future engineering projects and allow key biosynthetic steps to be identified for use in genome mining.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Gillian Aldus
Date Deposited: 15 Jan 2019 10:58
Last Modified: 15 Jan 2019 10:58
URI: https://ueaeprints.uea.ac.uk/id/eprint/69571
DOI:

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