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Investigation of a cryptic polyketide biosynthetic pathway from Rhodococcus erythropolis PR4
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Prasongpholchai, Panward (2021) Investigation of a cryptic polyketide biosynthetic pathway from Rhodococcus erythropolis PR4. PhD thesis, University of Warwick.
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WRAP_Theses_Prasongpholchai_2021.pdf - Submitted Version - Requires a PDF viewer. Download (38Mb) | Preview |
Official URL: http://webcat.warwick.ac.uk/record=b3736727~S15
Abstract
Polyketide natural products constitute an important family of medicinally relevant molecules comprising antibiotics, antifungal agents and immunosuppressants, as well as nutraceuticals such as de novo biosynthesised omega-3 and omega-6 fatty acids. The core structure of polyketide natural products is assembled in microorganisms and plants by large multifunctional enzymes known as polyketide synthases (PKSs). These catalyse multiple rounds of decarboxylative Claisen condensation of enzyme-bound acyl substrates to generate the natural product carbon skeleton, which is often further decorated by additional tailoring enzymes, mostly encoded for within the biosynthetic gene cluster, to yield the final bioactive compounds. Genes encoding for the production of polyketides are primarily found in microorganisms of the Streptomyces genus. However, advances in genome sequencing and bioinformatics have been unveiling the biosynthetic potential of many other microorganisms and plants, found in both common and exotic ecological niches, that were previously unknown or had been overlooked.
This PhD work focused on the investigation of a novel polyketide biosynthetic gene cluster (BGC) identified in Rhodococcus erythropolis PR4, a marine microorganism first isolated from the coast of Okinawa Island, Japan. In particular, the gene cluster encodes a type I iterative PKS housing six enzymatic domains: a ketosynthase (KS), an
acyltransferase (AT), two acyl carrier proteins (ACPs), a ketoreductase (KR) and a dehydratase (DH). Phosphopantetheinyl transferase (PPTase) and thioesterase (TE) domains encoded by a downstream gene were also identified and shown to act cooperatively to the core PKS to form polyketide products. In order to investigate the function of the putative PKS gene and other genes in the BGC, molecular biology, microbiology, chemical probes and analytical techniques were utilised. In particular, candidate genes were cloned from R. erythropolis PR4 and, following heterologous expression in E. coli, overproduced and purified proteins were tested for their function in vitro and in vivo. From these experiments, we were able to confirm the ability of the putative type I iterative PKS to synthesise linear polyene structures containing up to 9 conjugated double bonds. Furthermore, in an attempt to characterise the ultimate product of the BGC, direct cluster capture by transformation-associated recombination (TAR) technology and genetic manipulation of Rhodococcus erythropolis PR4 were also undertaken. Although, due to time constraints, we were unable to characterise the ultimate product and further work will be required to shed light on the full biosynthetic capability of this intriguing BGC, this work has laid the foundations for further research on iterative polyketide biosynthesis as well as the biosynthetic potential of understudied genera such as Rhodococcus.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QP Physiology Q Science > QR Microbiology |
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Library of Congress Subject Headings (LCSH): | Rhodococcus, Polyketides -- Synthesis, Biosynthesis | ||||
Official Date: | February 2021 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | Department of Chemistry | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Tosin, Manuela | ||||
Sponsors: | University of Warwick. Chancellor's International Scholarship | ||||
Extent: | xvi, 243 leaves : illustrations, charts | ||||
Language: | eng |
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