TY - BOOK

T1 - Identification and characterization of biosynthetic components involved in Vitex agnus-castus diterpenes biosynthesis

AU - Sundram, Tamil Chelvan Meenakshi

PY - 2017

Y1 - 2017

N2 - Many plant terpenoids are commercially important compound, largely used in pharmaceuticals,nutraceuticals, cosmetics and fragrance industry. However, their low production levels inplanta still remain as the major challenge in meeting the industrial demand for continuoussupply of these valuable diterpenoids. Moreover, chemical synthesis of many of theseterpenoids is difficult due to their structural complexity. Recent advances in biotechnology andsynthetic biology have paved a way towards alternative and bio sustainable production ofbioactive terpenoids.In recent years, various synthetic biology and metabolic engineering tools have advanced theheterologous production of terpenoids. With regard to this, we reviewed the currentdevelopment in engineering bacteria E. coli; cyanobacteria Synechocystis sp. and Anabaenasp.; tobacco Nicotiana benthamiana and Nicotiana tabacum; and moss Physcomitrella patensas host platforms for biosynthesis of several high-value terpenoids (Chapter 2).Here, in this PhD project we initially investigated the biosynthesis of Vitex agnus-castusditerpenoids. Bioactive diterpenoids detected in Vitex agnus-castus were reported to bebeneficial for the treatment of menstrual related disorders. Using MALDI-MSI and LC-HRMSanalysis, diterpenoids were found mainly present in the glandular trichomes of V. agnus -castusleaves and fruits. Transcriptome sequencing of leaf glandular trichomes and whole leaveshelped in the identification of six Vitex agnus-castus diterpene synthases (diTPSs) enzymes(VacTPSs) – VacTPS1, VacTPS2, VacTPS3, VacTPS4, VacTPS5 and VacTPS6. Diterpenesynthases are the first dedicated enzymes for the synthesis of diterpenoids. Phylogeneticanalysis using other characterized Lamiaceae diTPSs grouped VacTPS1, VacTPS3 andVacTPS5 as members of class II diTPSs while VacTPS2, VacTPS4 and VacTPS6 as membersof class I diTPSs. Next, VacTPSs were functionally characterized in planta using Nicotianabenthamiana-Agrobacterium-mediated transient expression system. VacTPS1 was identifiedas peregrinol diphosphate while VacTPS3 as syn- copalyl diphosphate synthase. PairingVacTPS1 with VacTPS2 yielded vitexagnusin D and 9,13(R)-epoxy-labd-14-ene, while pairingVacTPS1 with VacTPS6 yielded labd-13(16),14-dien-9-ol. Coupling VacTPS3 with VacTPS2yielded vitexifolin A and coupling with VacTPS6 yielded two different products:dehydroabietadiene and syn-isopimara-7,15-diene. Expression of VacTPS5 alone resulted inthe production of kolavenyl and when paired with VacTPS2, catalysed the cyclization of GGPPinto kolavelool. In addition, a cytochrome P450, VacCYP76BK1, was identified as a highlyexpressed transcript in V. agnus -castus leaf and fruit trichomes. The activity ofVacCYP76BK1 was confirmed through Saccharomyces cerevisiae in vitro microsomal assaysas 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. Plastidialtargeting sequence truncated cDNAs expressing VacTPS1 and VacTPS3 enzymes (tVacTPS1and 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 anyadditional diterpene products when compared to in planta results. Coupling tVacTPS1 withVacTPS6 in in vitro assay yielded labd-13(16),14-dien-9-ol, consistent to the result obtainedin planta. Combination of tVacTPS3 with VacTPS6 afforded syn-isopimara-7,15-diene as asingle major product in in vitro assays, contrary to in planta expression which affordeddehydroabitadiene as major product. However, so far, the attempts of expressing a functionalVacTPS2 in E. coli cells were unsuccessful even after using full-length sequences and threedifferent variants of plastidial targeting sequence truncated cDNAs. We next attempted toengineer a green platform for V. agnus-castus diterpenes biosynthesis by expressing thecharacterized (VacTPSs) in Synechocystis sp. PCC 6803. To this extend, we only managed tosynthesize syn-copalyl diphosphate by expressing the VacTPS3 in Synechocystis cells(Chapter 4).In summary, through this study we discovered and characterized VacTPSs and P450 enzymesinvolved in the first steps of Vitex agnus-castus diterpenoids biosynthesis. Besides, weexplored the alternative possibility of producing V. agnus -castus diterpenes in Synechocystis.

AB - Many plant terpenoids are commercially important compound, largely used in pharmaceuticals,nutraceuticals, cosmetics and fragrance industry. However, their low production levels inplanta still remain as the major challenge in meeting the industrial demand for continuoussupply of these valuable diterpenoids. Moreover, chemical synthesis of many of theseterpenoids is difficult due to their structural complexity. Recent advances in biotechnology andsynthetic biology have paved a way towards alternative and bio sustainable production ofbioactive terpenoids.In recent years, various synthetic biology and metabolic engineering tools have advanced theheterologous production of terpenoids. With regard to this, we reviewed the currentdevelopment in engineering bacteria E. coli; cyanobacteria Synechocystis sp. and Anabaenasp.; tobacco Nicotiana benthamiana and Nicotiana tabacum; and moss Physcomitrella patensas host platforms for biosynthesis of several high-value terpenoids (Chapter 2).Here, in this PhD project we initially investigated the biosynthesis of Vitex agnus-castusditerpenoids. Bioactive diterpenoids detected in Vitex agnus-castus were reported to bebeneficial for the treatment of menstrual related disorders. Using MALDI-MSI and LC-HRMSanalysis, diterpenoids were found mainly present in the glandular trichomes of V. agnus -castusleaves and fruits. Transcriptome sequencing of leaf glandular trichomes and whole leaveshelped in the identification of six Vitex agnus-castus diterpene synthases (diTPSs) enzymes(VacTPSs) – VacTPS1, VacTPS2, VacTPS3, VacTPS4, VacTPS5 and VacTPS6. Diterpenesynthases are the first dedicated enzymes for the synthesis of diterpenoids. Phylogeneticanalysis using other characterized Lamiaceae diTPSs grouped VacTPS1, VacTPS3 andVacTPS5 as members of class II diTPSs while VacTPS2, VacTPS4 and VacTPS6 as membersof class I diTPSs. Next, VacTPSs were functionally characterized in planta using Nicotianabenthamiana-Agrobacterium-mediated transient expression system. VacTPS1 was identifiedas peregrinol diphosphate while VacTPS3 as syn- copalyl diphosphate synthase. PairingVacTPS1 with VacTPS2 yielded vitexagnusin D and 9,13(R)-epoxy-labd-14-ene, while pairingVacTPS1 with VacTPS6 yielded labd-13(16),14-dien-9-ol. Coupling VacTPS3 with VacTPS2yielded vitexifolin A and coupling with VacTPS6 yielded two different products:dehydroabietadiene and syn-isopimara-7,15-diene. Expression of VacTPS5 alone resulted inthe production of kolavenyl and when paired with VacTPS2, catalysed the cyclization of GGPPinto kolavelool. In addition, a cytochrome P450, VacCYP76BK1, was identified as a highlyexpressed transcript in V. agnus -castus leaf and fruit trichomes. The activity ofVacCYP76BK1 was confirmed through Saccharomyces cerevisiae in vitro microsomal assaysas 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. Plastidialtargeting sequence truncated cDNAs expressing VacTPS1 and VacTPS3 enzymes (tVacTPS1and 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 anyadditional diterpene products when compared to in planta results. Coupling tVacTPS1 withVacTPS6 in in vitro assay yielded labd-13(16),14-dien-9-ol, consistent to the result obtainedin planta. Combination of tVacTPS3 with VacTPS6 afforded syn-isopimara-7,15-diene as asingle major product in in vitro assays, contrary to in planta expression which affordeddehydroabitadiene as major product. However, so far, the attempts of expressing a functionalVacTPS2 in E. coli cells were unsuccessful even after using full-length sequences and threedifferent variants of plastidial targeting sequence truncated cDNAs. We next attempted toengineer a green platform for V. agnus-castus diterpenes biosynthesis by expressing thecharacterized (VacTPSs) in Synechocystis sp. PCC 6803. To this extend, we only managed tosynthesize syn-copalyl diphosphate by expressing the VacTPS3 in Synechocystis cells(Chapter 4).In summary, through this study we discovered and characterized VacTPSs and P450 enzymesinvolved in the first steps of Vitex agnus-castus diterpenoids biosynthesis. Besides, weexplored the alternative possibility of producing V. agnus -castus diterpenes in Synechocystis.

UR - https://soeg.kb.dk/permalink/45KBDK_KGL/fbp0ps/alma99122780036805763

M3 - Ph.D. thesis

BT - Identification and characterization of biosynthetic components involved in Vitex agnus-castus diterpenes biosynthesis

PB - Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen

ER -