UBC Theses and Dissertations
Evaluating E. coli as a chassis for the production of capsaicinoids and capsinoids Haghighat Kashani, Amir
Capsaicinoids are a class of compounds natively produced by pepper plants as secondary metabolites. They possess therapeutic properties and have been used for treating medical conditions such as chronic pain, overactive bladder and diabetes. Capsaicin, a well-known chemical in this class, is responsible for the spicy taste of chili peppers. The precursors for capsaicinoids include vanillylamine and an acyl-CoA. Capsinoids, a group of capsaicinoid analogues, use vanillyl alcohol as a precursor instead of vanillylamine, making them less pungent. Here, we describe preliminary work towards the development of an E. coli strain for the production of capsaicinoids and capsinoids using vanillin and glycerol as precursors. Successful implementation of this system could allow targeted production of compound libraries or individual compounds if required. Such flexibility can be achieved through the mixing and matching of different enzymes within the host system. This platform can lead to production of high-value low-volume chemical products that have pharmaceutical applications. However, a sufficiently productive platform could also provide a less resource intensive source of capsaicinoids, currently gathered from pepper plants at less than 1% yield. In this work, we evaluate E. coli as a host for the synthesis of capsaicinoids and capsinoids. Initially, we created a pathway and successfully demonstrated synthesis of C6 to C12 straight and branched-chain fatty acids. We then focused on candidate enzymes for acyl-CoA synthesis to activate the aforementioned fatty acids, and a number of potential candidates were identified based on literature review. Furthermore, we investigated vanillylamine and vanillyl alcohol production. Previous publications have demonstrated conversion of vanillin to vanillylamine in E. coli using transaminases. Vanillyl alcohol on the other hand can be synthesized from vanillin using the endogenous reductases in E. coli. Lastly, we attempted expression of capsaicin synthase in E. coli with and without the SUMO tag, however results were inconclusive. More investigation needs to be performed to determine whether capsaicin synthase, a key enzyme on our pathway, can be expressed in its active form.
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