| dc.description.abstract | 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. | en |
