The iridoids comprise a large family of distinctive bicyclic monoterpenes that possess a wide range of pharmacological activities, including anticancer, anti-inflammatory, antifungal and antibacterial activities. Additionally, certain iridoids are used as sex pheromones in agriculturally important species of aphids, a fact that has underpinned innovative and integrated pest management strategies. To harness the biotechnological potential of this natural product class, the enzymes involved in the biosynthetic pathway must be elucidated. Here we report the discovery of iridoid synthase, a plant-derived enzyme that generates the iridoid ring scaffold, as evidenced by biochemical assays, gene silencing, co-expression analysis and localization studies. In contrast to all known monoterpene cyclases, which use geranyl diphosphate as substrate and invoke a cationic intermediate, iridoid synthase uses the linear monoterpene 10-oxogeranial as substrate and probably couples an initial NAD(P)H-dependent reduction step with a subsequent cyclization step via a Diels-Alder cycloaddition or a Michael addition. Our results illustrate how a short-chain reductase was recruited as cyclase for the production of iridoids in medicinal plants. Furthermore, we highlight the prospects of using unrelated reductases to generate artificial cyclic scaffolds. Beyond the recognition of an alternative biochemical mechanism for the biosynthesis of cyclic terpenes, we anticipate that our work will enable the large-scale heterologous production of iridoids in plants and microorganisms for agricultural and pharmaceutical applications.

The natural diversity of plant metabolism has long been a source for human medicines. One group of plant-derived compounds, the monoterpene indole alkaloids (MIAs), includes well-documented therapeutic agents used in the treatment of cancer (vinblastine, vincristine, camptothecin), hypertension (reserpine, ajmalicine), malaria (quinine), and as analgesics (7-hydroxymitragynine). Our understanding of the biochemical pathways that synthesize these commercially relevant compounds is incomplete due in part to a lack of molecular, genetic, and genomic resources for the identification of the genes involved in these specialized metabolic pathways. To address these limitations, we generated large-scale transcriptome sequence and expression profiles for three species of Asterids that produce medicinally important MIAs: Camptotheca acuminata, Catharanthus roseus, and Rauvolfia serpentina. Using next generation sequencing technology, we sampled the transcriptomes of these species across a diverse set of developmental tissues, and in the case of C. roseus, in cultured cells and roots following elicitor treatment. Through an iterative assembly process, we generated robust transcriptome assemblies for all three species with a substantial number of the assembled transcripts being full or near-full length. The majority of transcripts had a related sequence in either UniRef100, the Arabidopsis thaliana predicted proteome, or the Pfam protein domain database; however, we also identified transcripts that lacked similarity with entries in either database and thereby lack a known function. Representation of known genes within the MIA biosynthetic pathway was robust. As a diverse set of tissues and treatments were surveyed, expression abundances of transcripts in the three species could be estimated to reveal transcripts associated with development and response to elicitor treatment. Together, these transcriptomes and expression abundance matrices provide a rich resource for understanding plant specialized metabolism, and promotes realization of innovative production systems for plant-derived pharmaceuticals.

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In developing and developed nations, plant cell culture systems are used to supply desirable compounds in lieu of chemical synthesis or natural extraction. When plant cell culture systems are unable to meet commercial demand, metabolic engineering offers a method to increase yields. However, to benefit from metabolic engineering approaches, effective transient transformation methods are required to rapidly identify and characterize key regulatory genes before intensive, time-consuming stable transformation efforts can proceed. This paper describes a particle bombardment-mediated transient transformation system for Taxus spp. in cell culture. Optimal parameters were established for the T. cuspidata cell line P991 and the T. canadensis cell line CO93D, resulting in reliable, efficient, transient expression of the firefly luciferase gene under control of the constitutive CaMV 35S promoter. Multiple bombardments and larger gold microcarriers (1.6 vs 1.0 microm in diameter) were particularly effective in increasing luciferase activity and in reducing variation among replicates. This particle bombardment-mediated transformation system was also shown to be capable of transiently expressing the DsRed and beta-glucuronidase reporter genes under the control of the maize ubiquitin and CaMV 35S promoters, respectively. With the ability to transiently transform Taxus spp. cell cultures using a variety of promoters and reporters, characterization of genes related to paclitaxel accumulation in culture can now proceed.

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