Forskolin is a unique structurally complex labdane-type diterpenoid used in the treatment of glaucoma and heart failure based on its activity as a cyclic AMP booster. Commercial production of forskolin relies exclusively on extraction from its only known natural source, the plant Coleus forskohlii, in which forskolin accumulates in the root cork. Here, we report the discovery of five cytochrome P450s and two acetyltransferases which catalyze a cascade of reactions converting the forskolin precursor 13R-manoyl oxide into forskolin and a diverse array of additional labdane-type diterpenoids. A minimal set of three P450s in combination with a single acetyl transferase was identified that catalyzes the conversion of 13R-manoyl oxide into forskolin as demonstrated by transient expression in Nicotiana benthamiana. The entire pathway for forskolin production from glucose encompassing expression of nine genes was stably integrated into Saccharomyces cerevisiae and afforded forskolin titers of 40 mg/L.

The ginkgo tree (Ginkgo biloba) is considered a living fossil due to its 200 million year’s history under morphological stasis. Its resilience is partly attributed to its unique set of specialized metabolites, in particular, ginkgolides and bilobalide, which are chemically complex terpene trilactones. Here, we use a gene cluster-guided mining approach in combination with co-expression analysis to reveal the primary steps in ginkgolide biosynthesis. We show that five multifunctional cytochrome P450s with atypical catalytic activities generate the tert-butyl group and one of the lactone rings, characteristic of all G. biloba trilactone terpenoids. The reactions include scarless C–C bond cleavage as well as carbon skeleton rearrangement (NIH shift) occurring on a previously unsuspected intermediate. The cytochrome P450s belong to CYP families that diversifies in pre-seed plants and gymnosperms, but are not preserved in angiosperms. Our work uncovers the early ginkgolide pathway and offers a glance into the biosynthesis of terpenoids of the Mesozoic Era.

Forskolin is a high-value diterpenoid produced exclusively by the Lamiaceae plant Coleus forskohlii. Today forskolin is used pharmaceutically for its adenyl-cyclase activating properties. The limited availability of pure forskolin is currently hindering its full utilization, thus a new environmentally friendly, scalable and sustainable strategy is needed for forskolin production. Recently, the entire biosynthetic pathway leading to forskolin was elucidated. The key steps of the pathway are catalyzed by cytochrome P450 enzymes (CYPs), which have been shown to be the limiting steps of the pathway. Here we study whether protein engineering of the substrate recognition sites (SRSs) of CYPs can improve their efficiency towards forskolin biosynthesis in yeast. As a proof of concept, we engineered the enzyme responsible for the first putative oxygenation step of the forskolin pathway: the conversion of 13R-manoyl oxide to 11-oxo-13R-manoyl oxide, catalyzed by the CYP76AH15. Four CYP76AH15 variants—engineered in the SRS regions—yielded at least a twofold increase of 11-oxo-13R-manoyl oxide when expressed in yeast cells grown in microtiter plates. The highest titers (5.6-fold increase) were observed with the variant A99I, mutated in the SRS1 region. Double or triple CYP76AH15 mutant variants resulted in additional enzymes with optimized performances. Moreover, in planta CYP76AH15 can synthesize ferruginol from miltiradiene. In this work, we showed that the mutants affecting 11-oxo-13R-manoyl oxide synthesis, do not affect ferruginol production, and vice versa. The best performing variant, A99I, was utilized to reconstruct the forskolin biosynthetic pathway in yeast cells. Although these strains showed increased 11-oxo-manoyl oxide production and higher accumulation of other pathway intermediates compared to the native CYP76AH15, lower production of forskolin was observed. As demonstrated for CYP76AH15, site-directed mutagenesis of SRS regions of plant CYPs may be an efficient and targeted approach to increase the performance of these enzymes. Although in this work we have managed to achieve higher efficiency and specificity of the first CYP of the pathway, further work is necessary in order to increase the overall production of forskolin in yeast cells.

The development of medical applications exploiting the broad bioactivities of the diterpene therapeutic triptolide from Tripterygium wilfordii is limited by low extraction yields from the native plant. Furthermore, the extraordinarily high structural complexity prevents an economically attractive enantioselective total synthesis. An alternative production route of triptolide through engineered Saccharomyces cerevisiae (yeast) could provide a sustainable source of triptolide. A potential intermediate in the unknown biosynthetic route to triptolide is the diterpene dehydroabietic acid. Here, we report a biosynthetic route to dehydroabietic acid by transient expression of enzymes from T. wilfordii and Sitka spruce (Picea sitchensis) in Nicotiana benthamiana. The combination of diterpene synthases TwTPS9, TwTPS27, and cytochromes P450 PsCYP720B4 yielded dehydroabietic acid and a novel analog, tentatively identified as ‘miltiradienic acid’. This biosynthetic pathway was reassembled in a yeast strain engineered for increased yields of the pathway intermediates, the diterpene olefins miltiradiene and dehydroabietadiene. Introduction in that strain of PsCYP720B4 in combination with two alternative NADPH-dependent cytochrome P450 reductases resulted in scalable in vivo production of dehydroabietic acid and its analog from glucose. Approaching future elucidation of the remaining biosynthetic steps to triptolide, our findings may provide an independent platform for testing of additional recombinant candidate genes, and ultimately pave the way to biotechnological production of the high value diterpenoid therapeutic.