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Exploring epoxyketone synthases and their biosynthetic potential
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Rothe, Marlene Luise (2022) Exploring epoxyketone synthases and their biosynthetic potential. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b3883978
Abstract
Epoxyketone proteasome inhibitors derived from natural products are invaluable for the treatment of multiple myeloma and are currently in clinical trials against autoimmune disorders. Epoxyketone metabolites are produced by various bacterial phyla, including Actinobacteria, Cyanobacteria, and Proteobacteria. Their epoxyketone pharmacophore is assembled by acyl-CoA dehydrogenase-like enzymes called epoxyketone synthases. These enzymes use a-dimethyl-b-keto carboxylic acid substrates assembled by a combination of non-ribosomal peptide synthetases, polyketide synthases, and fatty acid synthases. To explore whether it is possible to replace chemically challenging and unsustainable methods for epoxyketone formation in synthetic proteasome inhibitor production with an enzyme catalysed procedure, the substrate tolerance of epoxyketone synthases was investigated. Chemically stable methyl ester analogues of the substrate for epoxyketone synthase EpnF were synthesised with an alanine, leucine, cyclopentenylalanine, phenylalanine, and tryptophan residue in place of the native 4,5-dehydroleucine residue. To probe the substrate tolerance of EpnF, coupled enzyme assays were performed using pig liver esterase, to generate the carboxylic acid substrate in situ, and EpnF. While the alanine, leucine, cyclopentenylalanine, and phenylalanine derived substrates were tolerated by EpnF, no epoxyketone formation was observed for the tryptophan-derived substrate analogue. Although the phenylalanine derived analogue was accepted by EpnF, a precursor of the proteasome inhibitor oprozomib, which has a phenylalanine residue in the same position, was not converted to the corresponding epoxyketone. A bioinformatics approach was developed to identify and analyse novel epoxyketone biosynthetic gene clusters in silico. A cryptic gene cluster hypothesised to direct the production of the tryptophan derived epoxyketone tryptopeptin A was identified in Streptomyces sparsogenes ATCC 25498. Comparison of the metabolite profiles with that of known tryptopeptin A producer Streptomyces sp. maeda85 indicated that S. sparsogenes ATCC 25498 does not produce tryptopeptin A. However, collaborative work to clone the gene cluster and express it in a heterologous host showed that it directs the production of several tryptopeptin related metabolites.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QD Chemistry R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer) R Medicine > RS Pharmacy and materia medica |
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Library of Congress Subject Headings (LCSH): | Ketones, Antineoplastic agents, Streptomyces, Protease inhibitors, Biosynthesis, Drug development, Multiple myeloma | ||||
Official Date: | September 2022 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | Department of Chemistry | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Challis, Gregory L. ; Alberti, Fabrizio | ||||
Sponsors: | Synbio Technologies | ||||
Format of File: | |||||
Extent: | xxvii, 178 pages : colour illustrations | ||||
Language: | eng |
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