The Biosynthesis of Aescin

Jan-Smith, Eliot (2022) The Biosynthesis of Aescin. Doctoral thesis, University of East Anglia.

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Abstract

Aescin is a mixture of triterpenoid saponins found in horse chestnuts (Aesculus spp.) which has elicited interest from the chemical industry for a range of applications, from medicinal use to inclusion in household cleaning products. Wider development of products containing aescin would be greatly aided by cheaper, more environmentally-friendly supplies – one potential means to achieve this could be production through heterologous expression of biosynthetic enzymes. This work therefore seeks to elucidate the biosynthetic pathway to aescin and reconstitute it in a heterologous plant host. To this end a biosynthetic pathway was predicted by database mining, and previously reported triterpenoid biosynthetic enzymes from other species were used to reconstitute early steps by transient expression in Nicotiana benthamiana. An existing transcriptome assembly for A. pavia was used to identify the first active enzyme from the aescin pathway, a betaamyrin C-21β hydroxylase. Together with enzymes from other species, this was used to isolate a product not previously obtained in N. benthamiana: kudzusapogenol C.

Novel transcriptomic resources for A. pavia and A. hippocastanum were subsequently generated and used to discover further aescin pathway enzymes involved in formation and oxidation of the aescin beta-amyrin aglycone: AhOSC1, AhCYP1, AhCYP2, and AhCYP3. The activities of the cytochrome P450s (CYPs) identified have been validated by scale-up, purification and NMR, allowing isolation of novel aescin intermediates up to 21β,22α,24,28-OH beta-amyrin. Identification of a cellulose synthase-like enzyme (AhCSL1) and a UDP-dependent sugar transferase (AhUGT1) capable of adding D-glucuronic acid and D-galactose, respectively, along with partial characterisation of the activities of a BAHD and a second UGT, led to detection of intermediates only two transformations removed from aescin. The enzymes identified through this work therefore provide an excellent platform for further work on bioengineering of aescin and analogues thereof. The novel activities of some of the enzymes characterised here may also enable production of new-to-nature acylated and glycosylated triterpenoids.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Chris White
Date Deposited: 29 Sep 2022 10:33
Last Modified: 29 Sep 2022 10:33
URI: https://ueaeprints.uea.ac.uk/id/eprint/88716
DOI:

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