Publications

• Towards elucidating carnosic acid biosynthesis in Lamiaceae: Functional characterization of the three first steps of the pathway in Salvia fruticosa and Rosmarinus officinalis.

  Bozic D., Papaefthimiou D., Brueckner K., de Vos R.C., Tsoleridis C.A., Katsarou D., Papanikolaou A., Pateraki I., Chatzopoulou F.M., Dimitriadou E., Kostas S., Manzano D., Scheler U., Ferrer A., Tissier A., Makris A.M., Kampranis S.C., Kanellis A.K.

  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 ... >> More

  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. << Less

  PLoS ONE 10:e0124106-e0124106(2015) [PubMed] [EuropePMC]

• Characterization of CYP76AH4 clarifies phenolic diterpenoid biosynthesis in the Lamiaceae.

  Zi J., Peters R.J.

  Miltiradiene (1) is the precursor of phenolic diterpenoids such as ferruginol (2), requiring aromatization and hydroxylation. While this has been attributed to a single cytochrome P450 (CYP76AH1), characterization of the rosemary ortholog CYP76AH4 led to the discovery that these CYPs simply hydrox ... >> More

  Miltiradiene (1) is the precursor of phenolic diterpenoids such as ferruginol (2), requiring aromatization and hydroxylation. While this has been attributed to a single cytochrome P450 (CYP76AH1), characterization of the rosemary ortholog CYP76AH4 led to the discovery that these CYPs simply hydroxylate the facilely oxidized aromatic intermediate abietatriene (3). << Less

  Org. Biomol. Chem. 11:7650-7652(2013) [PubMed] [EuropePMC]

• CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts.

  Guo J., Zhou Y.J., Hillwig M.L., Shen Y., Yang L., Wang Y., Zhang X., Liu W., Peters R.J., Chen X., Zhao Z.K., Huang L.

  Cytochrome P450 enzymes (CYPs) play major roles in generating highly functionalized terpenoids, but identifying the exact biotransformation step(s) catalyzed by plant CYP in terpenoid biosynthesis is extremely challenging. Tanshinones are abietane-type norditerpenoid naphthoquinones that are the m ... >> More

  Cytochrome P450 enzymes (CYPs) play major roles in generating highly functionalized terpenoids, but identifying the exact biotransformation step(s) catalyzed by plant CYP in terpenoid biosynthesis is extremely challenging. Tanshinones are abietane-type norditerpenoid naphthoquinones that are the main lipophilic bioactive components of the Chinese medicinal herb danshen (Salvia miltiorrhiza). Whereas the diterpene synthases responsible for the conversion of (E,E,E)-geranylgeranyl diphosphate into the abietane miltiradiene, a potential precursor to tanshinones, have been recently described, molecular characterization of further transformation of miltiradiene remains unavailable. Here we report stable-isotope labeling results that demonstrate the intermediacy of miltiradiene in tanshinone biosynthesis. We further use a next-generation sequencing approach to identify six candidate CYP genes being coregulated with the diterpene synthase genes in both the rhizome and danshen hairy roots, and demonstrate that one of these, CYP76AH1, catalyzes a unique four-electron oxidation cascade on miltiradiene to produce ferruginol both in vitro and in vivo. We then build upon the previous establishment of miltiradiene production in Saccharomyces cerevisiae, with incorporation of CYP76AH1 and phyto-CYP reductase genes leading to heterologous production of ferruginol at 10.5 mg/L. As ferruginol has been found in many plants including danshen, the results and the approaches that were described here provide a solid foundation to further elucidate the biosynthesis of tanshinones and related diterpenoids. Moreover, these results should facilitate the construction of microbial cell factories for the production of phytoterpenoids. << Less

  Proc. Natl. Acad. Sci. U.S.A. 110:12108-12113(2013) [PubMed] [EuropePMC]