Biosynthesis of capsaicinoids by recombinant Saccharomyces cerevisiae

Abstract: Throughout history, people have used products from different microorganisms and plants for many medicinal and nutritional applications. Chilli peppers have been used for spicing up food, but also for their pharmacological properties. They contain alkaloid molecules called capsaicinoids, which have been shown to activate and desensitise the heat receptor - Transient Receptor Potential Vanilloid type 1 (TRPV1) cation channel. The TRPV1 receptor is a target for pain relief treatments for a range of health conditions, therefore capsaicinoids are important drug candidates.Capsaicinoids can be manufactured by extraction directly from the chili pepper,which typically results in relatively small amounts and therefore requires large areas of land. Furthermore, capsaicinoids yield can be affected by environmental and genotypical factors. Another way of production can be through the use of synthetic biology and metabolic engineering strategies to introduce enzymatic reactions in a model microorganism. This way, plant-derived or novel capsaicinoids with potentially improved therapeutic properties can be produced from given precursors, simple sugars or renewable raw materials.The aim of this work was to engineer baker’s yeast Saccharomyces cerevisiae for a whole-cell biocatalytic production of capsaicinoids. The structure of thesecompounds is comprised of a vanilloid moiety and a fatty acyl chain joined by anamide bond. The last step for synthesis of these compounds in the plant is through condensation of the precursors vanillylamine (derived from vanillin) and a fatty acyl-CoA forming the amide bond. Amide forming reactions are very important in chemical synthesis and research has been directed towards finding efficient enzymes to perform them. Furthermore, yeast as a model microorganism has been modified to produce vanillin from glucose; however, reductive amination of vanillin to vanillylamine, or production of capsaicinoids in yeast has not been achieved previously.This thesis addresses how capsaicinoids can be produced, the challenges for in vivo reductive amination of vanillin to vanillylamine and the identification andcharacterisation of several heterologous N-acyltransferase (NAT) and CoA-ligase(CL) amide-forming enzymes. Vanillylamine was produced from vanillin, byoverexpressing a vanillin aminotransferase. The transamination reaction wasimproved by the co-expression of an alanine dehydrogenase, which also removed the need for amine donor supplementation. Cultivation conditions were shown to have an effect on the transamination, with anaerobic conditions and ethanol as a cosubstrate leading to decrease in by-product formation and improved reductive amination of vanillin to vanillylamine. Moreover, combinations of NAT and CL enzymes were evaluated for production of nonivamide (a model capsaicinoid) from vanillylamine and nonanoic acid. Finally, nonivamide, was successfully produced in vivo from precursors vanillin and nonanoic acid, after implementing strain engineering strategies and evaluating reaction conditions. Even though the NAT and CL enzyme cascade effectively produced nonivamide, the titres and yields of the process can be further improved, indicating that the amidation step is the current bottleneck in the production and requires further optimisation.Lastly, the potential of S. cerevisiae as a biosensor was evaluated by overexpressing the TRPV1 receptor and modulating its activation. This was done as a first step towards the goal of using yeast as a screening platform for strains producing capsaicinoids with activity on the TRPV1 receptor.Transferring the capsaicinoid enzyme cascade in a cell host is not straightforward and requires optimal expression of several heterologous enzymes, adjustments of reaction conditions and strategies in reducing inhibitory effects and by-products. However, biosynthesis in whole-cells may offer advantages due to simple cell derived generation of enzymes, intermediates and co-factors, positively affecting the cost and performing the reaction in aqueous environment as a more environmentally sustainable way of production.