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Title: Production of plant triterpenoids in the yeast Saccharomyces cerevisiae
Author: Dale, Matthew Peter
ISNI:       0000 0004 7969 127X
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2019
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The triterpenoids comprise a diverse family of plant natural products with potential applications in many sectors, including medicine, food, agriculture, and home and personal care. Triterpenoids are derived from the cyclisation of 2,3-oxidosqualene by an oxidosqualene cyclase (OSC), to generate a triterpene, which can then be oxidised by cytochromes P450 to produce triterpenoids, and glycosylated by UDP-glycosyltransferases (UGTs) to produce triterpenoid saponins. More than 150 triterpene structures have been identified to date, which can be modified in a myriad of ways, resulting in the huge diversity of triterpenoids found in nature. The modifications are often crucial for bioactivity. Numerous saponins have industrial potential as vaccine adjuvants, feeding deterrents, detergents and gelling agents, their amphipathic properties being critical to this activity. Meanwhile, many triterpenoid aglycones have potential as therapeutics (including anti-cancer, anti-HIV and hepatoprotective drugs) and insecticides. Many of these compounds are derived from oleanolic acid, which is produced from the triterpene β-amyrin by oxidation of carbon-28 (C-28; a methyl group) to a carboxyl group. Despite this great potential, it is currently challenging to obtain triterpenoids in the quantities required for industrial exploitation. Plants typically accumulate triterpenoids in low abundance and under specific conditions, and many triterpenoids are produced by non-crop plants that are difficult to cultivate. Furthermore, plants often produce numerous structurally similar triterpenoids, making it difficult to purify the desired compound(s). Meanwhile, the complexity of triterpenoids, which contain multiple chiral centres and often undergo stereo- and regiospecific oxygenations, makes their chemical synthesis challenging and economically prohibitive. Triterpenoid production in the budding yeast Saccharomyces cerevisiae could be a means to address the shortfall in production. S. cerevisiae is a genetically tractable and well characterised microorganism that naturally produces 2,3-oxidosqualene, and is widely used in industrial fermentations for a variety of products. The present work focuses on the production of oleanane triterpenoids and saponins (i.e. derived from β-amyrin) in S. cerevisiae. In Chapter 3, a gas chromatography-mass spectrometry (GC-MS) method to monitor and quantify the production of triterpenoids in yeast is presented. In Chapter 4, twelve β-amyrin synthase (BAS) homologues are systematically compared for productivity in yeast, and a difference in β-amyrin production of > 10- fold was observed. The homologues from Artemisia annua (AaBAS) and Chenopodium quinoa (CqBAS1) were the most productive, each yielding 10.6 mg/L β-amyrin. Expression of most BAS homologues resulted in considerably slower growth indicative of metabolic burden. However, a BAS from Avena strigosa (AsBAS) had a negligible effect on growth while still producing a relatively high amount of β- amyrin (8.8 mg/L). In Chapter 5, sixteen C-28 oxidase P450s (co-expressed with AaBAS and the cytochrome P450 reductase ATR2) are compared for the production of oleanolic acid. All strains grew slowly compared with a control strain carrying an empty vector. Product profiles varied considerably, and a 6.8-fold difference in oleanolic acid titre was observed. The CYP716AL1 enzyme from Catharanthus roseus produced the most oleanolic acid in the initial screen (14.1 mg/L), but also accumulated substantial amounts of β-amyrin (7.1 mg/L) and the intermediate compounds erythrodiol (6.7 mg/L) and oleanolic aldehyde (titre undetermined) compared with the other BASs. Co-expression of CYP716AL1 with AsBAS resulted in faster growth and the production of mainly oleanolic acid, with very little β-amyrin, erythrodiol, or oleanolic aldehyde accumulating. Finally, in Chapter 6, saponins derived from β-amyrin and oleanolic acid are produced in yeast through the additional expression of UGT enzymes. This study identified glycosylations at different positions on the triterpenoid backbone, and is the first reported production of an oleanane diglycoside saponin in yeast.
Supervisor: Rosser, Susan ; French, Chris Sponsor: Biotechnology and Biological Sciences Research Council (BBSRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: triterpenoids ; baker's yeast ; cyclisation of 2,3-oxidosqualene ; oxidosqualene cyclase ; OSC ; Saccharomyces cerevisiae