Cannabinoids are a unique class of secondary metabolites that are found exclusively in the plant species Cannabis sativa. They exhibit significant therapeutic properties and have been studied in detail due to their medical importance. Over 100 cannabinoids have been isolated from the cannabis plant. The two most prominent, due to their abundance in the plant and medicinal application, are tetrahydrocannabinol (THC) and cannabidiol (CBD). These cannabinoids have been shown to be effective in treating a myriad of diseases, from alleviating the side effects associated with chemotherapy to reducing intensity of muscle spasms in multiple sclerosis patients. Currently the primary source of cannabinoids is the cultivation of the cannabis plant, but this has major drawbacks including the resource requirements, inconsistent yields and difficulties in metabolite purification. Cultivation of cannabis also limits access to rarer, less abundant, cannabinoids, preventing the exploration of their potential therapeutic uses. Synthetic biology represents a solution to these problems. By repurposing and constructing artificial biological systems from normalised parts, synthetic biology can enable efficient production of high value compounds, such as cannabinoids. This thesis describes the evaluation of potential biocatalyst parts and their subsequent prototyping for use in an E. coli production platform for cannabinoids. Through structurally guided mutagenesis studies the mechanism of cyclisation discrimination by the first enzyme of the cannabinoid biosynthesis pathway, namely tetraketide synthase, and its relevance to the wider type III polyketide class of enzymes was investigated. By repurposing and engineering the non-native prenyl transferase AtaPT, a lysate production system and a whole cell production system of the cannabinoids cannabigerolic acid and cannabigerol, respectively, were constructed. Both of these compounds represent entry points to the wider cannabinoid chemical space. Finally, a series of methods were trialled to enable the expression of native C. sativa cannabinoid synthases in E. coli. Alongside this, a library of candidate homologs were screened in an effort to identify non-canonical cannabinoid synthases. The construction of the cannabinoid production systems and insight into the biocatalyst parts gained in this project has set the foundation of an E. coli cannabinoid production platform.
Date of Award | 1 Aug 2022 |
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Original language | English |
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Awarding Institution | - The University of Manchester
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Supervisor | Nigel Scrutton (Supervisor) & Eriko Takano (Supervisor) |
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Biocatalyst parts evaluation and pathway refactoring for synthetic drug manufacturing
Kearsey, L. (Author). 1 Aug 2022
Student thesis: Phd