Production of Plant Triterpenoids in the Yeast Saccharomyces cerevisiae

The triterpenoids comprise a diverse family of plant natural products with potential \napplications in many sectors, including medicine, food, agriculture, and home and \npersonal care. Triterpenoids are derived from the cyclisation of 2,3-oxidosqualene by \nan oxidosqualene cyclase (OSC), to generate a triterpene, which can then be oxidised \nby cytochromes P450 to produce triterpenoids, and glycosylated by UDP-glycosyltransferases \n(UGTs) to produce triterpenoid saponins. More than 150 \ntriterpene structures have been identified to date, which can be modified in a myriad \nof ways, resulting in the huge diversity of triterpenoids found in nature. The \nmodifications are often crucial for bioactivity. Numerous saponins have industrial \npotential as vaccine adjuvants, feeding deterrents, detergents and gelling agents, \ntheir amphipathic properties being critical to this activity. Meanwhile, many \ntriterpenoid aglycones have potential as therapeutics (including anti-cancer, anti-HIV \nand hepatoprotective drugs) and insecticides. Many of these compounds are derived \nfrom oleanolic acid, which is produced from the triterpene β-amyrin by oxidation of \ncarbon-28 (C-28; a methyl group) to a carboxyl group. \n \nDespite this great potential, it is currently challenging to obtain triterpenoids in the \nquantities required for industrial exploitation. Plants typically accumulate triterpenoids \nin low abundance and under specific conditions, and many triterpenoids are produced \nby non-crop plants that are difficult to cultivate. Furthermore, plants often produce \nnumerous structurally similar triterpenoids, making it difficult to purify the desired \ncompound(s). Meanwhile, the complexity of triterpenoids, which contain multiple \nchiral centres and often undergo stereo- and regiospecific oxygenations, makes their \nchemical synthesis challenging and economically prohibitive. Triterpenoid production \nin the budding yeast Saccharomyces cerevisiae could be a means to address the \nshortfall in production. S. cerevisiae is a genetically tractable and well characterised \nmicroorganism that naturally produces 2,3-oxidosqualene, and is widely used in \nindustrial fermentations for a variety of products. The present work focuses on the \nproduction of oleanane triterpenoids and saponins (i.e. derived from β-amyrin) in S. \ncerevisiae. \n \nIn Chapter 3, a gas chromatography-mass spectrometry (GC-MS) method to monitor \nand quantify the production of triterpenoids in yeast is presented. \nIn Chapter 4, twelve β-amyrin synthase (BAS) homologues are systematically \ncompared for productivity in yeast, and a difference in β-amyrin production of > 10- \nfold was observed. The homologues from Artemisia annua (AaBAS) and \nChenopodium quinoa (CqBAS1) were the most productive, each yielding 10.6 mg/L \nβ-amyrin. Expression of most BAS homologues resulted in considerably slower \ngrowth indicative of metabolic burden. However, a BAS from Avena strigosa (AsBAS) \nhad a negligible effect on growth while still producing a relatively high amount of β- \namyrin (8.8 mg/L). \n \nIn Chapter 5, sixteen C-28 oxidase P450s (co-expressed with AaBAS and the \ncytochrome P450 reductase ATR2) are compared for the production of oleanolic acid. \nAll strains grew slowly compared with a control strain carrying an empty vector. \nProduct profiles varied considerably, and a 6.8-fold difference in oleanolic acid titre \nwas observed. The CYP716AL1 enzyme from Catharanthus roseus produced the \nmost oleanolic acid in the initial screen (14.1 mg/L), but also accumulated substantial \namounts of β-amyrin (7.1 mg/L) and the intermediate compounds erythrodiol (6.7 \nmg/L) and oleanolic aldehyde (titre undetermined) compared with the other BASs. Co-expression \nof CYP716AL1 with AsBAS resulted in faster growth and the production \nof mainly oleanolic acid, with very little β-amyrin, erythrodiol, or oleanolic aldehyde \naccumulating. \n \nFinally, in Chapter 6, saponins derived from β-amyrin and oleanolic acid are produced \nin yeast through the additional expression of UGT enzymes. This study identified \nglycosylations at different positions on the triterpenoid backbone, and is the first \nreported production of an oleanane diglycoside saponin in yeast.