Microbial Biosynthesis of Grapevine Specialised Metabolites

Abstract

Plants are capable of producing a wide variety of molecules, and these fall broadly into two categories: primary metabolites and specialised metabolites. Primary metabolites are critical for growth and survival. Specialised metabolites, which are present in some plant species but not others, do not have a role in the central metabolism of all plants. Specialised metabolites can play many roles to allow plants to fill specific ecological niches, such as chemical defence or attraction of pollinators. Specialised metabolites may also be useful to humans as drugs, flavours or fragrances. The research described in this thesis investigates the potential of applying microbial biosynthesis techniques to produce grapevine (Vitis vinifera) specialised metabolites. Two classes of specialised metabolites were focussed on: tartaric acid and its biosynthetic intermediates, and sesquiterpenoids. Tartaric acid accumulates in grape berries during development and is essential to the winemaking process as it lowers the pH of the wine must, preventing discolouration and microbial spoilage. Sesquiterpenoids are a large class of compounds, many of which have unique aromas, and some have been found to contribute significantly to the character of wine. Bioinformatics were used to identify candidate 2-keto-L-gulonate reductases that may be involved in the biosynthesis of tartaric acid. Two candidate enzymes were expressed and purified for in vitro characterisation. It was found that these candidates are capable of utilising 2-keto-L-gulonate as a substrate but do not produce L-idonate, the next step in the tartaric acid pathway. The candidate enzymes exhibited low-level, broad-spectrum reductase activity. A previously identified 2-keto-L-gulonate reductase was used in the development of an E. coli cell factory for the whole cell biocatalysis of 2-keto-L-gulonate to L-idonate. A yeast strain specially engineered to overproduce the sesquiterpene precursor farnesyl pyrophosphate (FPP) was used for the synthesis of a range of grapevine sesquiterpenoid compounds. Six grapevine sesquiterpene synthases were expressed by the engineered yeast strain alongside a promiscuous P450 monooxygenase to produce potential grapevine sesquiterpenoid products in vivo. The compounds were identified by gas-chromatography-mass-spectrometry. A total of seventeen unique compounds were confidently identified, with a number of uncharacterised compounds also produced. These compounds were found to possess a variety of aromas, and some compounds have beneficial health effects, which may be relevant for wine making and marketing. iv Finally, homology modelling and docking studies were used to compare three grapevine P450 monooxygenases and investigate the mechanism of the formation of the sesquiterpenoid (-)-rotundone, the pepper aroma compound found in some wines. Computational docking studies of (-)-rotundone inside the P450 binding sites suggests the oxidation of α-guaiene to (-)-rotundone is due to the unique shape of the binding site of V. vinifera Sesquiterpene Oxidase 2 (VvSTO2). Two other P450s, VvSTO4 and VvSTO6 are able to accept α-guaiene as a substrate but do not form rotundone, due to different positioning of α-guaiene within the P450 active sites. The investigation of these specialised metabolites may provide new insights into the biosynthesis of tartaric acid and sesquiterpenoids in grapevine, and assist in the identification and production of novel wine aromas and flavours.