PhD defence for Dan Luo – Københavns Universitet

PhD defence for Dan Luo

Title: Biosynthesis of macrocyclic diterpenoids in Euphorbia lathyris L.

Summary

Species in the genus Euphorbia have a long    history of use as medicinal plants for traditional treatments. Their pharmaceutical bioactivity is largely due to the rich production of chemically diverse isoprenoids, the most characteristic of which are the    macrocyclic diterpenoids. This PhD thesis documents the investigation of the    biosynthetic pathways of macrocyclic diterpenoids known as Euphorbia factors    in Euphorbia lathyris L. (caper spurge). These macrocyclic diterpenoids are the current industrial source of ingenol mebutate, which is approved for the treatment of actinic keratosis, a precancerous skin condition.

Metabolite profiling of various tissues of E. lathyris L. revealed that the mature seeds constituted a highly specialized tissue for the biosynthesis of lathyrane and ingenane    diterpenoids. RNA–seq and transcriptome analysis of E. lathyris L. mature seeds followed by functional characterization of candidate genes identified two cytochrome P450s, CYP71D445 and CYP726A27, as regio-specific casbene monooxygenases. Co-expression of ADH1, together with the identified CYPs, in N. benthamiana resulted in an unconventional pathway from casbene to the lathyrane diterpenoid jolkinol C. Catalytic features of CYP71D445, CYP726A27, and ADH1 were further characterized by the combined in vitro    assays with microsomal fractions from yeast strains expressing the P450s and the recombinant ADH1 enzyme purified from E. coli. When coupled with these P450 enzymes, E. lathyris ADH1 catalyzes the cyclization of oxidized casbene to produce jolkinol C, a potential key intermediate in ingenol biosynthesis. Attempts to explore the downstream enzymatic steps in the biosynthetic pathways of ingenol mebutate resulted in the discovery of a new casbene monooxygenase, CYP726A29. Investigation of the catalytic functions of CYP726A29 revealed an alternative route from casbene to the monocyclic backbone cembrene. Furthermore, a novel norditerpene, with new-to-nature C18    backbone, was identified, through oxidative modifications of casbene.

Results of this thesis offer the possibility to identify the subsequent enzymatic steps and to ultimately reconstruct the biosynthetic pathways of pharmaceutically active diterpenoids and new bioactive intermediates for biotechnological production.

Supervisors

Associate Professor Irini Pateraki

Professor Birger Lindberg Møller

Assesment Committee

Professor Joerg Bohlmann, University of British Columbia, Canada

Managing Director Jørgen Hansen, Evolva A/S

Associate Professor Sotirios Kampranis (chair)

Reception

Reception afterwards in the 117 meeting rooms