Nikolaj Hansen

Additional file 5: Figure S4. (A) In vivo functional characterisation of Eremophila CPTs. GC-MS chromatograms of hexane extracts of E. coli cultures treated with alkaline phosphatase. (B) Mass spectra of prenyl alcohols: nerylnerol (a), (2Z,6Z)-farnesol (b) and nerol (c).

Additional file 1: Table S1 Transcriptome sequencing statistics. Table S2. MVA and MEP pathway analysis. Table S3. List of candidate terpene synthases (TPSs) and cis-prenyl transferases (CPTs) studied in this work. E. lucida = El, E. drummondii = Ed and E. denticulata subsp. trisulcata = Edt. DeepLoc-1.0 was used to predict protein subcellular localisation [57]. Table S4. Protein sequences used in the terpene synthase phylogenetic analysis. Table S5. 1H (600 MHz) and 13C (150 MHz) data of (3Z,7Z,11Z)-cembratrien-15-ol (6) in CDCl3. Table S6. 1H (600 MHz) and 13C (150 MHz) data of 5-hydroxyviscidane (8) in CDCl3. Table S7. 1H (600 MHz) and 13C (150 MHz) data of serrulat-14-ene (12) in CDCl3. Table S8. 1H (600 MHz) and 13C (150 MHz) data of 8,9-dihydroserrulat-14-ene (11) in CDCl3. Table S9. Protein sequences used in the cis-prenyl transferase phylogenetic analysis. Table S10. List of primers used in this study.

Additional file 1: Table S1. Clone and primer list. Table S2. Yeast expression constructs. Table S3. Assigned 1C and 13C NMR peaks for compound 5 and 6. Figure S1. Comparison of the mass spectra of TMS derivatized TwOSC products from tobacco leaf co-infiltration experiments, with TMS derivatized authentic standards. Figure S2. CYP712K1 and CYP712K2 oxygenate the triterpene products of TwOSC2 and TwOSC3 in tobacco. Two leaf discs (Ø3 cm) of leaves co-expressing the indicated enzymes were extracted in 1 mL ethyl acetate. A 0.15 mL aliquot of this extract was dried, TMS derivatized and analyzed by GC–MS. None of the novel peaks 7-9 had mass spectra or retention times matching any of the analyzed authentic standards (betulinic acid, hederagenin, or oleanolic acid; not shown). Figure S3. Comparison of LC–ESI–MS and LC-APCI-MS (base peak chromatograms) for the reliable quantification of friedelin and polpunonic acid. Authentic standards of friedelin and polpunonic acid (30 µg/mL) were ana...

Background Eremophila R.Br. (Scrophulariaceae) is a diverse genus of plants with species distributed across semi-arid and arid Australia. It is an ecologically important genus that also holds cultural significance for many Indigenous Australians who traditionally use several species as sources of medicines. Structurally unusual diterpenoids, particularly serrulatane and viscidane-types, feature prominently in the chemical profile of many species and recent studies indicate that these compounds are responsible for much of the reported bioactivity. We have investigated the biosynthesis of diterpenoids in three species: Eremophila lucida, Eremophila drummondii and Eremophila denticulata subsp. trisulcata. Results In all studied species diterpenoids were localised to the leaf surface and associated with the occurrence of glandular trichomes. Trichome-enriched transcriptome databases were generated and mined for candidate terpene synthases (TPS). Four TPSs with diterpene biosynthesis act...

Background Celastrol is a promising anti-obesity agent that acts as a sensitizer of the protein hormone leptin. Despite its potent activity, a sustainable source of celastrol and celastrol derivatives for further pharmacological studies is lacking. Results To elucidate the celastrol biosynthetic pathway and reconstruct it in Saccharomyces cerevisiae, we mined a root-transcriptome of Tripterygium wilfordii and identified four oxidosqualene cyclases and 49 cytochrome P450s as candidates to be involved in the early steps of celastrol biosynthesis. Using functional screening of the candidate genes in Nicotiana benthamiana, TwOSC4 was characterized as a novel oxidosqualene cyclase that produces friedelin, the presumed triterpenoid backbone of celastrol. In addition, three P450s (CYP712K1, CYP712K2, and CYP712K3) that act downstream of TwOSC4 were found to effectively oxidize friedelin and form the likely celastrol biosynthesis intermediates 29-hydroxy-friedelin and polpunonic acid. To fa...

Tripterygium wilfordii (Celastraceae) is a medicinal plant with anti-inflammatory and immunosuppressive properties. Identification of a vast array of unusual sesquiterpenoids, diterpenoids and triterpenoids in T. wilfordii has spurred investigations of their pharmacological properties. The tri-epoxide lactone triptolide was the first of many diterpenoids identified, attracting interest due to the spectrum of bioactivities. To probe the genetic underpinning of diterpenoid diversity, an expansion of the class II diterpene synthase (diTPS) family was recently identified in a leaf transcriptome. Following detection of triptolide and simple diterpene scaffolds in the root, we sequenced and mined the root transcriptome. This allowed identification of the root-specific complement of TPSs and an expansion in the class I diTPS family. Functional characterization of the class II diTPSs established their activities in the formation of four C-20 diphosphate intermediates, precursors of both gen...