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Title: Chemical probing of polyketide bio-assemblies
Author: Jenkins, Robert
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2019
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Polyketides constitute an invaluable source of products for the agrochemical, pharmaceutical and biotechnology industries. Due to the enzyme-bound covalent nature of their intermediates, polyketide biosynthesis is challenging to study. The use of chemical probes offers a complementary approach to molecular biology and enzymology to characterise the nature of biosynthetic intermediates. My PhD work involved the synthesis and the use of novel chemical probes to investigate biosynthetic pathways leading to several polyketide compounds of biomedical interest, including thiolactomycin and tetronasin. The importance of these compounds is introduced in Chapter 1, together with an overview of the current knowledge of enzyme mechanisms involved in polyketide assembly. Chapter 2 focuses on the use of chemical probes for the investigation of thiolactomycin bio-assembly. Thiolactomycin is a thiotetronate natural product produced by a soil bacterium discovered in the early 1980’s. Its antibiotic, antimalarial and antituberculotic activities have been extensively studied and associated to type II fatty acid synthase inhibition, however its polyketide biosynthetic origins have only been recently unveiled. The chemical probes utilised to investigate thiolactomycin assembly were designed to mimic malonate building blocks utilised in polyketide biosynthesis, and were found to intercept putative early stage intermediates, which were characterised by LC-HRMS. The species in question were isolated from in vivo (live microorganisms) and vitro (recombinant enzymes) experiments; parallel investigations with other thiotetronate producing organisms corroborated our early results. In order to shed light on the late stages of intermediate processing, synthetic substrates mimicking established biosynthetic intermediates were prepared as N-acetyl cysteamine thioesters (SNAcs). Their processing and conversion to thiolactomycin by the enzyme machinery was demonstrated in vivo by supplementation of mutant bacterial cultures. Unnatural substrate mimics were also prepared and were shown to produce unnatural analogues of thiolactomycin by the same machinery to a small extent. Further investigations in vitro involved the expression of recombinant proteins and their incubation with synthetic substrates in order to monitor specific individual transformations. Overall some insights into the final biosynthetic steps were gathered, but future work will be required to underpin the exact mechanisms of sulphur insertion and cyclisation leading to thiotetronate formation. Chapter 3 focuses on biosynthetic investigations on the complex polyether tetronasin, a natural product of polyketide origin used as an additive in ruminal feedstock exerting antibiotic activity as an ionophore. The structure of tetronasin contains numerous chiral centres and several rings including tetrahydrofuran, pyran and cyclohexane moieties of unclear biosynthetic origin. Chemical probes mimicking polyketide malonate building blocks were synthesised and supplemented to cultures of the tetronasin producer S. longisporoflavus and of two mutant strains with inactivated genes shown to be essential for tetronasin production in the hope of capturing biosynthetic intermediates for detection by LC-HRMS. A few highly abundant and advanced species were detected and partially characterised; these provided key snapshots on the timing of the THF ring formation and other transformations in tetronasin bio-assembly, including O-methylation. Additional novel substrates were also prepared to probe the mechanism of tetronate ring formation; in preliminary in vivo experiments they did not prove fruitful, however they are currently being utilised for in vitro experiments concerning enzyme activity and crystallisation. Chapter 4 entails the synthesis of new chemical probes bearing structural variations on the original ‘chain termination’ probes devised by the Tosin group; in particular, variations involving the replacement of an amide moiety with isosteric moieties were devised and implemented. The ultimate aim of this task was to investigate the tolerance of PKSs towards unnatural substrates throughout the polyketide assembly process. The new probes were tested on a variety of in vitro and in vivo PKS systems and their efficiency in capturing polyketide biosynthetic intermediates evaluated. Limited success was observed across most systems which appeared to be strongly linked to either probe toxicity or poor in vivo hydrolysis of the probes methyl ester precursors. To overcome the latter issue, probes bearing photo-cleavable esters for a more controlled and efficient in situ generation of active carboxylate probes were synthesised. Unfortunately, this had little effect on the abundance and array of polyketide intermediates detected in vitro an in vivo, leading to the conclusion that changing of the probe amide functionality has a clear negative effect on its use for PKS species capture. Chapter 5 consists of conclusions and suggestions for future work for the different areas of investigation discussed in the three previous chapters. Following this, Chapter 6 reports experimental details of chemical synthesis, biological experiments methods and analytical methods utilised in this work. Chapter 7 and 8 are the appendix and bibliography respectively.
Supervisor: Not available Sponsor: Engineering and Physical Sciences Research Counsil
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QD Chemistry ; QP Physiology