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  • (2019) Soeriyadi, Angela
    Microorganisms are a valuable source of natural products with medically and industrially relevant activities. Cyanobacteria are one of the most chemically diverse microbial phyla, but have been largely underexplored due to their slow growth and intractability to genetic engineering. In recent years, genomic and metagenomic investigations of the aquatic environment have uncovered the untapped diversity of cyanobacterial nonribosomal peptide and polyketide biosynthetic gene clusters. This dissertation describes the application of emerging synthetic biology techniques using Escherichia coli as a heterologous host, focussing on translating the bioactive potential of cyanobacteria into industrial applications, while simultaneously characterising and tailoring this biochemical capacity. E. coli GB05-MtaA was previously shown to be a suitable host for the relatively simple cyanobacterial nonribosomal peptide synthetase (NRPS) pathway lyngbyatoxin (LTX). A synthetic biology approach for characterising and expanding lyngbyatoxin chemical diversity was explored. This thesis reports an in vitro investigation of wild-type LtxA NRPS activity that unravelled the multispecificity of the first adenylation domain to L-Val related amino acids, which correlates with the formation of novel lyngbyatoxin analogues in vivo. Efficient site-directed mutagenesis of the adenylation domain to modulate substrate specificities was performed through Red/ET-based recombineering, resulting in a library of mutated LTX pathways. Investigation of in vitro and in vivo pathway activity revealed the complexity of LTX biosynthesis, dictated by tailoring enzymes. The benefits of using this approach to probe LTX biosynthesis motivated the use of a similar application for the directed production of neosaxitoxin (neoSTX). NeoSTX is a paralytic shellfish toxin (PST) which has medically-relevant activities. The polyketide synthase (PKS)-like enzyme SxtA, which initiates PST biosynthesis, was expressed in E. coli to assess the suitability of this host. Efficient production of PST intermediates by heterologously expressed SxtA paved the way for the expression of an engineered neoSTX biosynthetic pathway in E. coli. Several variants of E. coli strains were successfully constructed to produce neoSTX. This synthetic biology process, with an E. coli expression system, is a feasible strategy to facilitate the commercial production of neoSTX.

  • (2018) Hawtrey, Tom
    This thesis describes the development of small molecules inhibitors of the CLK, DYRK and SRPK splicing kinases. Chapter 1 outlines the importance of alternative splicing in controlling cellular function and highlights the value of small molecule inhibitors of the splicing kinases as chemical probes or therapeutic agents. Prior work in the Morris group on the development of novel inhibitors was summarised and set the stage for the work to be done. Chapter 2 describes the development of the pyrrolo[1,2-c]pyrimidine scaffold which has previously been shown to inhibit the CLK and DYRK kinases. In this work, synthetic access to this scaffold was optimised, with the two lead compounds synthesised in improved yields of 18% over 9 and 7 steps respectively. This enabled 17 new analogues which varied the C2’, C5 and C1 positions to be prepared. Key analogues were highly potent, with benzylamine 2.36 a 5 nM inhibitor and pyrimidine 2.60 an 8 nM inhibitor of CLK1, both with modest selectivity over DYRK1A. N,N-Dimethylaminoethylamine 2.23 showed enhanced 10-fold selectivity for CLK1 over DYRK1A. Crystal structures revealed that the C2’ substituent occupies an additional portion of the binding site, with these new interactions contributing to the ability to modulate the potency and selectivity. Chapters 3 and 4 detail the investigation of a phenylcarboxamide scaffold, previously shown to inhibit SRPK1. An efficient and divergent synthetic route that allowed the preparation of analogues containing variations at several key substituents in 4-5 steps and good yields was developed. Varying the head group enabled the potent and selective inhibition the CLK kinases, with N-methylpyrazole 3.6 a 6 nM inhibitor of CLK1 with >30-fold selectivity over both DYRK1A and SRPK1. Varying the saturated heterocycle revealed that 2-furanylmethylpiperazino analogues show improved potency but decreased selectivity, while N,N-dimethylaminoethylpiperazino analogues showed improved selectivity for CLK. Varying the carboxylate side chain led to quinoline 3.49 being identified as a highly potent but non-selective DYRK1A inhibitor (IC50=14 nM). X-ray crystallography revealed the importance of these groups for controlling selectivity. The conclusions and future directions of the work are given in Chapter 5 and full experimental procedures have been provided in Chapter 6.