Many plant terpenoids are commercially important compound, largely used in pharmaceuticals,
nutraceuticals, cosmetics and fragrance industry. However, their low production levels in
planta still remain as the major challenge in meeting the industrial demand for continuous
supply of these valuable diterpenoids. Moreover, chemical synthesis of many of these
terpenoids is difficult due to their structural complexity. Recent advances in biotechnology and
synthetic biology have paved a way towards alternative and bio sustainable production of
bioactive terpenoids.
In recent years, various synthetic biology and metabolic engineering tools have advanced the
heterologous production of terpenoids. With regard to this, we reviewed the current
development in engineering bacteria E. coli; cyanobacteria Synechocystis sp. and Anabaena
sp.; tobacco Nicotiana benthamiana and Nicotiana tabacum; and moss Physcomitrella patens
as host platforms for biosynthesis of several high-value terpenoids (Chapter 2).
Here, in this PhD project we initially investigated the biosynthesis of Vitex agnus-castus
diterpenoids. Bioactive diterpenoids detected in Vitex agnus-castus were reported to be
beneficial for the treatment of menstrual related disorders. Using MALDI-MSI and LC-HRMS
analysis, diterpenoids were found mainly present in the glandular trichomes of V. agnus -castus
leaves and fruits. Transcriptome sequencing of leaf glandular trichomes and whole leaves
helped in the identification of six Vitex agnus-castus diterpene synthases (diTPSs) enzymes
(VacTPSs) – VacTPS1, VacTPS2, VacTPS3, VacTPS4, VacTPS5 and VacTPS6. Diterpene
synthases are the first dedicated enzymes for the synthesis of diterpenoids. Phylogenetic
analysis using other characterized Lamiaceae diTPSs grouped VacTPS1, VacTPS3 and
VacTPS5 as members of class II diTPSs while VacTPS2, VacTPS4 and VacTPS6 as members
of class I diTPSs. Next, VacTPSs were functionally characterized in planta using Nicotiana
benthamiana-Agrobacterium-mediated transient expression system. VacTPS1 was identified
as peregrinol diphosphate while VacTPS3 as syn- copalyl diphosphate synthase. Pairing
VacTPS1 with VacTPS2 yielded vitexagnusin D and 9,13(R)-epoxy-labd-14-ene, while pairing
VacTPS1 with VacTPS6 yielded labd-13(16),14-dien-9-ol. Coupling VacTPS3 with VacTPS2
yielded vitexifolin A and coupling with VacTPS6 yielded two different products:
dehydroabietadiene and syn-isopimara-7,15-diene. Expression of VacTPS5 alone resulted in
the production of kolavenyl and when paired with VacTPS2, catalysed the cyclization of GGPP
into kolavelool. In addition, a cytochrome P450, VacCYP76BK1, was identified as a highly
expressed transcript in V. agnus -castus leaf and fruit trichomes. The activity of
VacCYP76BK1 was confirmed through Saccharomyces cerevisiae in vitro microsomal assays
as well as yeast in vivo assays, and showed that it can convert peregrinol into labd-13Z-ene-
9,15,16-triol (Chapter 3).
In vitro diTPS enzymatic assays, using recombinant enzymes produced in E. coli BL-21DE3-
C41 cells were employed to verify the activities of VacTPSs observed in planta. Plastidial
targeting sequence truncated cDNAs expressing VacTPS1 and VacTPS3 enzymes (tVacTPS1
and tVacTPS3) and the full-length VacTPS6 enzymes were produced through expression in E.
coli. In vitro assays of the single class II VacTPSs (tVacTPS1 and tVacTPS3) did not show any
additional diterpene products when compared to in planta results. Coupling tVacTPS1 with
VacTPS6 in in vitro assay yielded labd-13(16),14-dien-9-ol, consistent to the result obtained
in planta. Combination of tVacTPS3 with VacTPS6 afforded syn-isopimara-7,15-diene as a
single major product in in vitro assays, contrary to in planta expression which afforded
dehydroabitadiene as major product. However, so far, the attempts of expressing a functional
VacTPS2 in E. coli cells were unsuccessful even after using full-length sequences and three
different variants of plastidial targeting sequence truncated cDNAs. We next attempted to
engineer a green platform for V. agnus-castus diterpenes biosynthesis by expressing the
characterized (VacTPSs) in Synechocystis sp. PCC 6803. To this extend, we only managed to
synthesize syn-copalyl diphosphate by expressing the VacTPS3 in Synechocystis cells
(Chapter 4).
In summary, through this study we discovered and characterized VacTPSs and P450 enzymes
involved in the first steps of Vitex agnus-castus diterpenoids biosynthesis. Besides, we
explored the alternative possibility of producing V. agnus -castus diterpenes in Synechocystis.