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A large number of diterpenes have been isolated from Euphorbiaceae plants, many of which are of interest due to toxicity or potential therapeutic activity. Specific Euphorbiaceae diterpenes of medical interest include the latent HIV-1 activator prostratin (and related 12-deoxyphorbol esters), the analgesic resiniferatoxin, and the anticancer drug candidate ingenol 3-angelate. In spite of the large number of diterpenes isolated from these plants and the similarity of their core structures, there is little known about their biosynthetic pathways. Other than the enzymes involved in gibberellin biosynthesis, the only diterpene synthase isolated to date from the Euphorbiaceae has been casbene synthase, responsible for biosynthesis of a macrocyclic diterpene in the castor bean (Ricinus communis). Here, we have selected five Euphorbiaceae species in which to investigate terpene biosynthesis and report on the distribution of diterpene synthases within this family. We have discovered genes encoding putative casbene synthases in all of our selected Euphorbiaceae species and have demonstrated high-level casbene production through expression of four of these genes in a metabolically engineered strain of Saccharomyces cerevisiae. The only other diterpene synthase found among the five plants was a neocembrene synthase from R. communis (this being the first report of a neocembrene synthase gene). Based on the prevalence of casbene synthases, the lack of other candidates, and the structure of the casbene skeleton, we consider it likely that casbene is the precursor to a large number of Euphorbiaceae diterpenes. Casbene production levels of 31 mg/L were achieved in S. cerevisiae and we discuss strategies to further increase production by maximizing flux through the mevalonate pathway.

Marine invertebrates have proven to be a rich source of secondary metabolites. The growing recognition that marine microorganisms associated with invertebrate hosts are involved in the biosynthesis of secondary metabolites offers new alternatives for the discovery and development of marine natural products. However, the discovery of microorganisms producing secondary metabolites previously attributed to an invertebrate host poses a significant challenge. This study describes an efficient chemical screening method utilizing a 96-well plate-based bacterial cultivation strategy to identify and isolate microbial producers of marine invertebrate-associated metabolites.

Synthetic biology depends on the ability to rapidly produce strains with improved phenotypes but is limited by the ability to rapidly produce strain collections with directed mutations. Here, we present a system capable of overcoming this limitation through automated P1-phage transductions of Escherichia coli. By combining the Keio collection of single-gene deletion E. coli mutants with P1-phage, it is possible to generate an engineered host-strain collection consisting of every possible gene deletion mutant. This strategy was tested by transducing 355 genetic markers from the Keio collection into five different host strains, and it achieved a 98% success rate. This method offers an improved mechanism for rapidly engineering collections of microbes and provides one method for rapidly deploying a broader synthetic biology effort.

We have developed a novel method to clone terpene synthase genes. This method relies on the inherent toxicity of the prenyl diphosphate precursors to terpenes, which resulted in a reduced-growth phenotype. When these precursors were consumed by a terpene synthase, normal growth was restored. We have demonstrated that this method is capable of enriching a population of engineered Escherichia coli for those clones that express the sesquiterpene-producing amorphadiene synthase. In addition, we enriched a library of genomic DNA from the isoprene-producing bacterium Bacillus subtilis strain 6051 in E. coli engineered to produce elevated levels of isopentenyl diphosphate and dimethylallyl diphosphate. The selection resulted in the discovery of two genes ( yhfR and nudF ) whose protein products acted directly on the prenyl diphosphate precursors and produced isopentenol. Expression of nudF in E. coli engineered with the mevalonate-based isopentenyl pyrophosphate biosynthetic pathway resulted in the production of isopentenol.

Drug-resistant strains of the malaria parasite are widespread, and as a result mortality due to malaria has increased significantly in recent years. Artemisinin, isolated from the herb Artemisia annua (sweet wormwood), is one drug that shows a high efficacy in killing multi-resistant strains of the parasite. The drug is extremely expensive, and high demand has led to a shortage of artemisinin, available only by extraction from the plant source. Ro et al. now report the development of a yeast strain engineered to carry a cytochrome P450 monooxygenase from A. annua that can produce the drug precursor, artemisinic acid. Artemisinin can be synthesized from this precursor. If the efficiency of this process can be improved, this engineered yeast strain has the potential to alleviate the drug shortage. Through the bio-engineering of Saccharomyces cerevisiae high titres of artemisinic acid were produced using a novel cytochrome P450 monooxygenase. Optimization of this process on an industrial scale may significantly reduce the cost of artemisinin, which could then be used to combat malaria in resource-poor settings. Malaria is a global health problem that threatens 300–500 million people and kills more than one million people annually1. Disease control is hampered by the occurrence of multi-drug-resistant strains of the malaria parasite Plasmodium falciparum2,3. Synthetic antimalarial drugs and malarial vaccines are currently being developed, but their efficacy against malaria awaits rigorous clinical testing4,5. Artemisinin, a sesquiterpene lactone endoperoxide extracted from Artemisia annua L (family Asteraceae; commonly known as sweet wormwood), is highly effective against multi-drug-resistant Plasmodium spp., but is in short supply and unaffordable to most malaria sufferers6. Although total synthesis of artemisinin is difficult and costly7, the semi-synthesis of artemisinin or any derivative from microbially sourced artemisinic acid, its immediate precursor, could be a cost-effective, environmentally friendly, high-quality and reliable source of artemisinin8,9. Here we report the engineering of Saccharomyces cerevisiae to produce high titres (up to 100 mg l-1) of artemisinic acid using an engineered mevalonate pathway, amorphadiene synthase, and a novel cytochrome P450 monooxygenase (CYP71AV1) from A. annua that performs a three-step oxidation of amorpha-4,11-diene to artemisinic acid. The synthesized artemisinic acid is transported out and retained on the outside of the engineered yeast, meaning that a simple and inexpensive purification process can be used to obtain the desired product. Although the engineered yeast is already capable of producing artemisinic acid at a significantly higher specific productivity than A. annua, yield optimization and industrial scale-up will be required to raise artemisinic acid production to a level high enough to reduce artemisinin combination therapies to significantly below their current prices.