Nerylneryl diphosphate is the precursor of serrulatane, viscidane and cembrane-type diterpenoids in Eremophila species

Abstract

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 activity were identified: ElTPS31 and ElTPS3 from E. lucida were found to produce (3Z,7Z,11Z)-cembratrien-15-ol and 5-hydroxyviscidane, respectively, and EdTPS22 and EdtTPS4, from E. drummondii and E. denticulata subsp. trisulcata, respectively, were found to produce 8,9-dihydroserrulat-14-ene which readily aromatized to serrulat-14-ene. In all cases, the identified TPSs used the cisoid substrate, nerylneryl diphosphate (NNPP), to form the observed products. Subsequently, cis-prenyl transferases (CPTs) capable of making NNPP were identified in each species.

Conclusions: We have elucidated two biosynthetic steps towards three of the major diterpene backbones found in this genus. Serrulatane and viscidane-type diterpenoids are promising candidates for new drug leads. The identification of an enzymatic route to their synthesis opens up the possibility of biotechnological production, making accessible a ready source of scaffolds for further modification and bioactivity testing.

Keywords: Bioactive diterpenoids; Eremophila; Plant natural products; Serrulatanes; Terpene synthase; Viscidanes; cis-prenyltransferase.

Fig. 1

a Examples of diterpenoids reported from E. denticulata subsp. trisulcata: 8,17-dihydroxyserrulat-14-en-19-oic acid (1) [52]; E. drummondii: 7,8-dihydroxy-16-caffeoyloxyserrulat-19-oic acid (2), 7,8-dihydroxyserrulat-14-en-19-oic acid (3) [19]; E. lucida: 5-hydroxyviscida-3,14-dien-20-oic acid (4), (3Z, 7E, 11Z)-15-hydroxycembra-3,7,11-trien-19-oic acid (5) [18]. Bright field images of Eremophila spp. leaf cross sections: b E. denticulata subsp. trisulcata, (c) E. drummondii and (d) E. lucida. Arrows indicate resin layer coating the leaf surface, filled arrowheads indicate glandular trichomes, empty arrowheads indicate raised stomata and stars indicate internal oil glands. Scale bar = 100 μm

Fig. 2

Phylogenetic analysis of Eremophila TPSs. Maximum likelihood tree of TPSs based on aligned protein sequences calculated using MEGA 7 [56]. Tree is drawn to scale, with branch lengths representing the number of substitutions per site. Filled circles on branches indicate bootstrap support of above 75% based on 1000 repetitions. Genbank accession numbers are listed in Tables S3 and S4 (Additional file 1). Subcellular localisation predicted using DeepLoc-1.0 [57]

Fig. 3

In vivo functional characterisation of Eremophila TPSs. (A-C) GC-MS chromatograms of hexane extracts of E. coli cultures expressing ElTPS31, ElTPS3, EdtTPS4 and EdTPS22 in combination with either a GGPP synthase (AgGGPPS) or a NNPP synthase (SlCPT2). d, f and h Mass spectra of major TPS products. e, g, i and j Chemical structures of (3Z,7Z,11Z)-cembratrien-15-ol (6), 5-hydroxyviscidane (8), 8,9-dihydroserrulat-14-ene (11) and serrulat-14-ene (12)

Fig. 4

a Phylogenetic analysis of Eremophila CPTs. Maximum likelihood tree of CPTs based on aligned protein sequences calculated using MEGA 7 [56]. Tree is drawn to scale, with branch lengths representing the number of substitutions per site. Filled circles on branches indicate bootstrap support above 75% based on 1000 repetitions. Genbank accession numbers are listed in Tables S3 and S9 (Additional file 1). Eremophila CPTs in blue box were functionally characterised. NP = no main product detected. Subcellular localisation predicted using DeepLoc-1.0 [57]. b In vivo functional characterisation of Eremophila CPTs. GC-MS chromatograms of hexane extracts of E. coli cultures expressing Eremophila CPTs in combination with ElTPS31