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Cell-free expression for natural product research
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Capel, Patrick (2022) Cell-free expression for natural product research. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b3821840
Abstract
Cell-Free Expression (CFE) is the reconstitution of protein biosynthesis in vitro. CFE reactions are ran by incubating three components together; a `crude extract', including the biological ma- chinery required for protein synthesis isolated from an organism of choice, a genetic program and a master mix containing small molecules required for transcription and translation. Amongst other factors, proteins can be produced quickly using CFE, thus, CFE has seen widespread application in synthetic biology. A promising application is the investigation of natural prod- uct biosynthesis. Many natural products routinely used in agriculture and medicine have been isolated from Streptomyces. Investigating their biosynthesis in E. coli remains problematic due long gene lengths and high GC content, problems that are expected to remain when utilising E. coli CFE platforms. Thus, the aim of this thesis is to develop and optimise Streptomyces CFE platforms, to characterise libraires of promoters and to demonstrate the production of natural products. Six strains of Streptomyces were selected to produce crude extract for CFE. Methods to produce extract were developed, along with optimisation of the master mix for greater activ- ity and compatibility with liquid chromatography-mass spectrometry. Due to the complexity of natural product biosynthetic pathways, and the need to control the relative levels of biosyn- thetic enzymes in the pathway to ensure good ux, two promoter libraries were characterised in the extracts of the selected strains. The application of these Streptomyces based CFE platforms to natural product research was demonstrated by expressing a three gene operon containing the early stages of the haem biosynthetic pathway, producing uroporphyrinogen III and an unknown pigment. To conclude, Streptomyces crude extract was di_cult to reliably produce from a range of strains. However, by utilizing functional batches of extract, it was possible to characterise promoters of different strengths from libraries as well as synthesise compounds from multi-enzyme systems.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QD Chemistry Q Science > QH Natural history |
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Library of Congress Subject Headings (LCSH): | Proteins -- Synthesis, Escherichia coli, Streptomyces, Natural products | ||||
Official Date: | January 2022 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | School of Life Sciences | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Corre, Christophe ; de los Santos, Emzo | ||||
Sponsors: | Syngenta UK (Firm), sponsor. Engineering and Physical Sciences Research Council, sponsor. Biotechnology and Biological Sciences Research Council (Great Britain) | ||||
Format of File: | |||||
Extent: | xx, 181 leaves : illustrations, charts | ||||
Language: | eng |
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