Rosemary extracts containing the phenolic diterpenes carnosic acid and its derivative carnosol are approved food additives used in an increasingly wide range of products to enhance shelf-life, thanks to their high anti-oxidant activity. We describe here the elucidation of the complete biosynthetic pathway of carnosic acid and its reconstitution in yeast cells. Cytochrome P450 oxygenases (CYP76AH22-24) from Rosmarinus officinalis and Salvia fruticosa already characterized as ferruginol synthases are also able to produce 11-hydroxyferruginol. Modelling-based mutagenesis of three amino acids in the related ferruginol synthase (CYP76AH1) from S. miltiorrhiza is sufficient to convert it to a 11-hydroxyferruginol synthase (HFS). The three sequential C20 oxidations for the conversion of 11-hydroxyferruginol to carnosic acid are catalysed by the related CYP76AK6-8. The availability of the genes for the biosynthesis of carnosic acid opens opportunities for the metabolic engineering of phenolic diterpenes, a class of compounds with potent anti-oxidant, anti-inflammatory and anti-tumour activities.

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The family Cistaceae (Angiosperm, Malvales) consists of 8 genera and 180 species, with 5 genera native to the Mediterranean area (Cistus, Fumara, Halimium, Helianthemum, and Tuberaria). Traditionally, a number of Cistus species have been used in Mediterranean folk medicine as herbal tea infusions for healing digestive problems and colds, as extracts for the treatment of diseases, and as fragrances. The resin, ladano, secreted by the glandular trichomes of certain Cistus species contains a number of phytochemicals with antioxidant, antibacterial, antifungal, and anticancer properties. Furthermore, total leaf aqueous extracts possess anti-influenza virus activity. All these properties have been attributed to phytochemicals such as terpenoids, including diterpenes, labdane-type diterpenes and clerodanes, phenylpropanoids, including flavonoids and ellagitannins, several groups of alkaloids and other types of secondary metabolites. In the past 20 years, research on Cistus involved chemical, biological and phylogenetic analyses but recent investigations have involved genomic and molecular approaches. Our lab is exploring the biosynthetic machinery that generates terpenoids and phenylpropanoids, with a goal to harness their numerous properties that have applications in the pharmaceutical, chemical and aromatic industries. This review focuses on the systematics, botanical characteristics, geographic distribution, chemical analyses, biological function and biosynthesis of major compounds, as well as genomic analyses and biotechnological approaches of the main Cistus species found in the Mediterranean basin, namely C. albidus, C. creticus, C. crispus, C. parviflorus, C. monspeliensis, C. populifolius, C. salviifolius, C. ladanifer, C. laurifolius, and C. clusii.

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.

Many cyanobacteria commonly identified as belonging to the genus Nostoc are well-known cyanobionts (symbionts) of a wide variety of plants and fungi. They form symbioses with bryophytes, pteridophytes, gymnosperms and angiosperms that are considerably different in the type of reciprocal interaction between the host and the cyanobiont. The phylogenetic and taxonomic relationships among cyanobionts isolated from different hosts and Nostoc strains isolated from free-living conditions are still not well understood. We compared phylogeny and morphology of symbiotic cyanobacteria originating from different host plants (genera Gunnera, Azolla, Cycas, Dioon, Encephalartos, Macrozamia and Anthoceros) with free-living Nostoc isolates originating from different habitats. After preliminary clustering with ARDRA (amplified rDNA restriction analysis), phylogeny was reconstructed on the basis of 16S rRNA gene sequences and compared with morphological characterization, obtaining several supported clusters. Two main Nostoc clusters harboured almost all cyanobionts of Gunnera, Anthoceros and of several cycads, together with free-living strains of the species Nostoc muscorum, Nostoc calcicola, Nostoc edaphicum, Nostoc ellipsosporum and strains related to Nostoc commune. We suggest that the frequent occurrence of symbiotic strains within these clusters is explained by the intensive hormogonia production that was observed in many of the strains studied. However, no evidence for discrimination between symbiotic and free-living strains, either by molecular or morphological approaches, could be found. Sequences of Azolla cyanobiont filaments, taken directly from leaf cavities, clustered tightly with sequences from the planktic cyanobacterium Cylindrospermopsis raciborskii, from the benthic Anabaena cylindrica 133 and from Anabaena oscillarioides HINDAK 1984/43, with high bootstrap values. The phylogenetic analysis showed that two distinct patterns of evolution of symbiotic behaviour might exist for the nostocacean cyanobacteria, one leading to symbioses of Nostoc species with a wide variety of plants, the other leading to the association of a unique cyanobacterial type with the water fern Azolla.