Towards Elucidating Carnosic Acid Biosynthesis in Lamiaceae: Functional Characterization of the Three First Steps of the Pathway in Salvia fruticosa and Rosmarinus officinalis

Abstract

Carnosic acid (CA) is a phenolic diterpene with anti-tumour, anti-diabetic, antibacterial and neuroprotective properties that is produced by a number of species from several genera of the Lamiaceae family, including Salvia fruticosa (Cretan sage) and Rosmarinus officinalis (Rosemary). To elucidate CA biosynthesis, glandular trichome transcriptome data of S. fruticosa were mined for terpene synthase genes. Two putative diterpene synthase genes, namely SfCPS and SfKSL, showing similarities to copalyl diphosphate synthase and kaurene synthase-like genes, respectively, were isolated and functionally characterized. Recombinant expression in Escherichia coli followed by in vitro enzyme activity assays confirmed that SfCPS is a copalyl diphosphate synthase. Coupling of SfCPS with SfKSL, both in vitro and in yeast, resulted in the synthesis miltiradiene, as confirmed by 1D and 2D NMR analyses (1H, 13C, DEPT, COSY H-H, HMQC and HMBC). Coupled transient in vivo assays of SfCPS and SfKSL in Nicotiana benthamiana further confirmed production of miltiradiene in planta. To elucidate the subsequent biosynthetic step, RNA-Seq data of S. fruticosa and R. officinalis were searched for cytochrome P450 (CYP) encoding genes potentially involved in the synthesis of the first phenolic compound in the CA pathway, ferruginol. Three candidate genes were selected, SfFS, RoFS1 and RoFS2. Using yeast and N. benthamiana expression systems, all three where confirmed to be coding for ferruginol synthases, thus revealing the enzymatic activities responsible for the first three steps leading to CA in two Lamiaceae genera.

Total biosynthesis of the cyclic AMP booster forskolin from Coleus forskohlii

“…In a parallel series of experiments, CYP76AHs from rosemary and salvia known to accept miltiradiene as substrate were tested for their ability to use 13 R -manoyl oxide as a substrate. Transient expression of Ro CYP76AH4 (Zi and Peters, 2013), Ro FS1 and Sf FS (Božić et al, 2015) in N. benthamiana leaves able to synthesize 13 R -manoyl oxide demonstrated that Ro CYP76AH4 was able to efficiently convert 13 R -manoyl oxide to 11-oxo-13 R -manoyl, while Ro FS1 and Sf FS were able to produce 11-hydroxy manoyl oxide, in addition to 11-oxo-13 R -manoyl oxide (Figure 7).

10.7554/eLife.23001.017Figure 6.GC-MS analysis of miltiradiene-derived diterpenoids obtained by transient expression of CfCYP76AH15 in N. benthamiana  leaves.( A ) Total ion chromatograms (TIC) of extracts transiently expressing CfCXS, CfGGPPS, CfTPS1 and CfTPS3 (miltiradiene biosynthesis genes) in combination with water (-) or CfCYP76AH15 .

…”

“…Thus, it is likely that the ability of CYP76AHs to catalyze 11-oxo-13 R -manoyl oxide has evolved convergently in these plants (Figure 11).

10.7554/eLife.23001.023Figure 11.Phylogeny of known full-length CYP76AHs.The enzymes used are listed below with their accession numbers or source of publication: Cf CYP76AH15, KT382358; Cf CYP76AH17, KT382360; Cf CYP76AH8, KT382348; Cf CYP76AH11, KT382349; Cf CYP76AH16, KT382359; Cf CYP76AH9, KT382347; Cf CYP76AH10, KT382346; Cf CYP71D381, KT382342; Ro FS1, AJQ30187 (Božić et al, 2015); Sm CYP76AH3, KR140168 (Guo et al, 2016); Ro FS2, AJQ30188 (Božić et al, 2015); Sf FS, AJQ30186 (Božić et al, 2015); Ro CYP76AH4, (Zi and Peters, 2013); Ro CYP76AH5v1, (Zi and Peters, 2013); Ro CYP76AH5v2, (Zi and Peters, 2013); Ro CYP76AH6, (Zi and Peters, 2013); Ro CYP76AH7, (Zi and Peters, 2013); Sm CYP76AH1, AGN04215 (Guo et al, 2013); Sp CYP76AH24, ALM25796 (Ignea et al, 2016a). Coleus forskohlii enzymes are indicated by a solid black triangle.

…”

Synthetic biology for production of natural and new‐to‐nature terpenoids in photosynthetic organisms

“…Next to miltiradiene and abietatriene (a spontaneous oxidation product), monohydroxylated derivatives thereof were observed to accumulate (Figure b; Brückner et al ., ). Interestingly, none of these derivatives were reported in a more recent study, in which CPP synthase and miltiradiene synthase from Salvia fruticosa were combined with different P450s to produce ferruginol from miltiradiene, indicating that an additional biosynthetic step may outcompete the endogenous conversion (Božić et al ., ).…”

Combined metabolome and transcriptome profiling provides new insights into diterpene biosynthesis in S. pomifera glandular trichomes

“…Due to the biological significance of carnosol, carnosic acid and tanshinones, there is increasing need for improved or alternative methods of production of these metabolites. As a result, efforts aiming to elucidate the related biosynthetic pathways has led to the identification of several enzymatic steps, not only in S. miltiorrhiza [ 24 , 25 ] but also in other species [ 26 , 27 ]. Specifically, diterpene synthases catalyzing the same enzymatic reactions with SmCDS and SmKSL have been identified in Rosmarinus officinalis [ 26 ] and in Salvia fruticosa [ 27 ].…”

“…As a result, efforts aiming to elucidate the related biosynthetic pathways has led to the identification of several enzymatic steps, not only in S. miltiorrhiza [ 24 , 25 ] but also in other species [ 26 , 27 ]. Specifically, diterpene synthases catalyzing the same enzymatic reactions with SmCDS and SmKSL have been identified in Rosmarinus officinalis [ 26 ] and in Salvia fruticosa [ 27 ]. Identification of ferruginol synthase in S. miltiorrhiza , R. officinalis and S. fruticosa enabled the synthesis of ferruginol in yeast cells [ 22 , 27 ].…”