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Investigation and utilisation of the promiscuity of the sesquiterpene synthase amorpha-4,11-diene synthase

Davies, Heulwen 2021. Investigation and utilisation of the promiscuity of the sesquiterpene synthase amorpha-4,11-diene synthase. PhD Thesis, Cardiff University.
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Abstract

Terpenoids are a vast family of natural products utilised by humans as insect repellents, medicines and fragrances. The sesquiterpenoid (15-carbon terpenoid) artemisinin, a secondary metabolite of Artemisia annua, is the key antimalarial used to treat the devastating illness of malaria which kills around three million people a year. It is naturally generated by oxidative metabolism of the sesquiterpene amorpha-4,11-diene, produced from farnesyl diphosphate (FDP) by amorpha-4,11-diene synthase (ADS). Currently artemisinin is predominantly sourced by extraction from A. annua, which is converted from FDP in six steps, but more stable and cost-effective alternative sources are still desired. The first part of this thesis details an investigation into which amino acid residues are key in facilitating conversion of achiral FDP into bicyclic amorpha-4,11 diene by ADS, probing 12 residues. Variants of ADS were created using site-directed mutagenesis and changes to the product profile analysed by GC-MS. The variants G401L and G401Y, in the G helix of ADS, generated two compounds which were identified as selina-4(15),7(11)-diene and germacrene B; never before seen products of ADS or its variants. This work shows that active site selectivity of ADS is finely tuned and furthers our understanding for its application in biocatalytic production of artemisinin. 12-Hydroxy farnesyl diphosphate (12-OH FDP), an analogue of FDP, can be converted to dihydroartemisinic aldehyde (DHAAl) in one step by ADS, compared to the four from FDP to DHAAl occurring in A. annua. The investigation of new chemoenzymatic routes to these key intermediates is described. A novel synthesis of DHAAl was developed using prenol and isoprenol, a chemical oxidation, two kinases, a prenyltransferase and ADS. Although less laborious than previous chemical synthesis, both kinases used were inefficient, and work to improve their activity was unsuccessful. Improving upon this, a simpler synthesis was developed from the diphosphorylation of 12‑hydroxyfarnesol using two kinases to make 12- OH-FDP with ~99% enzymatic conversion achieved compared to the maximum 52% reported for the previous chemical method. This was subsequently converted by ADS to DHAAl with 1:1 epimeric selectivity for the desired 11-R epimer, compared to 7:2 reported previously.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Chemistry
Date of First Compliant Deposit: 11 July 2022
Last Modified: 11 Jul 2022 13:29
URI: https://orca.cardiff.ac.uk/id/eprint/151190

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