Abstract
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.
Highlights
Plants synthesize an impressive diversity of specialized metabolites enabling them to communicate and adapt to environmental challenges (Mithofer and Boland, 2012; Woldemariam et al, 2011)
Discovery of multifunctional cytochromes P450 in Coleus forskohlii producing a multitude of 13R-manoyl oxide-derived diterpenoids and identification of a biosynthetic pathway for forskolin
The transcriptome was queried for transcripts encoding cytochrome P450s (CYPs) belonging to the CYP71 clan, based on their established role in monooxygenation reactions in the biosynthesis of specialized metabolites (Nelson, 2013; Werck-Reichhart and Feyereisen, 2000)
Summary
Plants synthesize an impressive diversity of specialized metabolites enabling them to communicate and adapt to environmental challenges (Mithofer and Boland, 2012; Woldemariam et al, 2011). Specialized plant metabolites and direct derivatives thereof still constitute more than a third of approved pharmaceuticals (Cragg and Newman, 2013; David et al, 2015). With over 50,000 known structures according to the ‘Dictionary of natural products’ (http:// dnp.chemnetbase.com/), terpenoids are the largest class of plant specialized metabolites and constitute a vast repository of bio-active natural products including many structurally complex compounds (Pateraki et al, 2015). Traditional chemical synthesis of plant-derived diterpenoid pharmaceuticals remains economically challenging, despite recent examples of elegant strategies mimicking natural routes (Appendino, 2014; Kawamura et al, 2016; Yuan et al, 2016).
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