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.

Fig 1. Structure and biosynthesis of labdane-type diterpenes.

A) Chemical structure of three phenolic-type diterpenes from S. fruticosa. 1) carnosic acid. 2) carnosol. 3) rosmanol. B) The proposed biosynthetic pathway from GGDP to carnosic acid.

Fig 2. Accumulation of phenolic diterpenes in S. fruticosa leaves and trichomes.

A) Total phenolic diterpenes (PDs) (carnosic acid + carnosol) contents in three populations of S. fruticosa extracted from whole leaves of all developmental stages. B) Accumulation of total PDs (carnosic acid + carnosol) in young and old leaves of the genotype Kavoussi. C) Carnosic acid and carnosol contents in trichomes and leaves without trichomes of the genotype Kavoussi collected from very young leaves (up to 1cm long). Each bar represents the average of three independent biological samples ± SD. Asterisks denote significant differences between two indicated values (*p < 0.05; **p < 0.01; ***p < 0.001), based on Student’s t-test.

Fig 3. Phylogenetic analysis of SfCPS and SfKSL representing their relatedness to other plant labdane-type diterpenes.

The neighbour-joining tree was generated using MEGA version 5 [52] from amino acid sequence alignment. Protein abbreviations and accession numbers are listed in S1 Table. The tree was rooted using Physcomitrella patens PpCPS/KS as outgroup.

Fig 4. Quantitative expression analysis (qPCR) of SfCPS (A) and SfKSL (B) in trichomes and leaves without trichomes of young and old leaves.

Data bars represent the mean expression levels from three biological replicates ± SE. For each gene, values marked with a different letter are significantly different at p < 0.05, according to Student’s t-test. Transcript levels were normalized to elf4a gene (endogenous control).

Fig 5. The functional characterization of SfCPS and SfKSL in E. coli.

A) SfCPS characterization. (a) GC-MS profile (275 m/z extracted ion chromatograms) of the dephosphorylated product of mature SfCPS incubated with GGDP. (b) GC-MS of the control reaction- substrate GGDP omitted. (c) Mass spectrum of the product peak- copalol. B) SfKSL characterization. (a) GC-MS profile (272 m/z extracted ion chromatograms of the product (miltiradiene) of coupled reaction of SfCPS and SfKSL with GGDP as substrate, b) GC-MS profile of the enzymatic assay with SfCPS omitted. c) Mass spectrum of the reaction product—miltiradiene.

Fig 6. Miltiradiene production in yeast and agro-infiltrated Nicotiana benthamiana leaves.

A) Yeast expression assays. (a) GC-MS profile of a hexane fraction obtained by silica gel flash chromatography containing the purified products (peak 1 and 2) from yeast transformed with SfCPS and SfKSL. (b) GC-MS profile of non-transformed yeast strain AM104. B) N. benthamiana transient co-expression: (a) GC-MS profile of N. benthamiana leaves infiltrated with SfCPS and SfKSL (showing peaks 1 and 2). (b) GC-MS profile of N. benthamiana leaves infiltrated with empty vector as a control. C) Mass spectrum for: (a) miltiradiene (peak 1); (b) abietatriene (peak 2).

Fig 7. Diagnostic HMBC correlations for miltiradiene.

The correlations between carbons and protons via ² J _CH and ³ J _CH couplings are shown. In the case of cyclohexadiene ring correlations are observed also via ⁴ J _CH couplings through the double bonds.

Fig 8. Phylogenetic analysis of SfFS, RoFS1 and RoFS2 representing their relatedness to other plant cytochrome P450s.

The neighbour-joining tree was generated using MEGA version 5 [52] from amino acid sequence alignment. Protein abbreviations and accession numbers are listed in S2 Table.

Fig 9. High-throughput qPCR analysis of SfCPS (a), SfKSL (b) and SfFS (c) genes in trichomes and leaves without trichomes performed on Biomark system.

A) response of the genes 3 hours (3h) and 6 hours (6h) after mechanical wounding in planta. Zero hour (0h): non-wounded plants control. Data bars represent the mean expression levels from two biological replicates ± SE. B) expression profile of the genes in three different developmental stages of leaves (stages 1, 4 and 7, with 1 being the first expanded pair of leaves). Data bars are the mean values from three biological replicates ± SE. Transcript levels were normalized to phosphatase 2A (PP2A) gene (endogenous control). Asterisks and the hash symbol denote significant differences between two indicated values (^#p < 0.075; *p < 0.05; **p < 0.01), based on Student’s t-test.

Fig 10. Heterologous expression of ferruginol synthase genes in yeast (S. cerevisiae).

A) GC-MS chromatograms of novel products secreted into culture media of yeast co-expressing SfCPS, SfKSL and either SfFS or the empty vector (EV) pWTDH3myc. B), GC-MS chromatograms of novel products secreted into culture media of yeast co-expressing RoCPS1m, RoKSL1f and either RoFS1, RoFS2 and empty vector (EV) pWTDH3myc. F, peak corresponding to ferruginol. C), Mass spectrum of peak F.

Fig 11. Production of ferruginol in leaves of Nicotiana benthamiana p19 transgenic plants.

A) Total ion chromatograms of hexane extracts obtained from N. benthamiana leaves (a) untreated, (b) infiltrated with non-transformed A. tumefaciens strain GV3101, (c) infiltrated with A. tumefaciens strain GV3101 transformed with SfCPS, SfKSL, AtCPR1, SfFS, (d) infiltrated with A. tumefaciens strain GV3101 transformed with SfCPS, SfKSL, AtCPR1, RoFS1, (e) infiltrated with A. tumefaciens strain GV3101 transformed with SfCPS, SfKSL, AtCPR1, RoFS2. B) Ferruginol detection in hexane extracts of trichomes isolated from (a) S. fruticosa leaves and (b) R. officinalis leaves. C) Mass spectrum for ferruginol.