Total biosynthesis of the cyclic AMP booster forskolin from Coleus forskohlii

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. DOI: http://dx.doi.org/10.7554/eLife.23001.001
eLife digest Unlike animals, plants cannot move away from a herbivore or other threats. Instead, they have evolved to produce a vast array of chemical compounds to protect themselves. Some of these compounds are also important to humans, for example, as medicines or fragrances. Plants usually only produce small amounts of these compounds in mixtures with many other compounds, which makes it difficult to purify them. As a result, the methods of purifying the compounds may require huge amounts of plant material, or be expensive and not environmentally friendly. One solution to this would be to genetically engineer microbes like bacteria or yeast to produce the compounds instead. In order to do that, we need to understand exactly which enzymes the plant uses to make each compound and introduce them into suitable microbes. A compound called forskolin has been used since ancient times in traditional Indian medicine to treat conditions like high blood pressure, asthma and heart complications. Forskolin is found exclusively in the root of a plant called Coleus forskohlii, which is native to India and south-east Asia. It is stored inside cells within the bark of the root in structures called oil bodies, which are similar to oil drops. However, it is not known where forskolin is made, or which enzymes are involved. Pateraki, Andersen-Ranberg et al. set out to uncover how C. forskohlii produces this compound. The experiments show that forskolin is produced within the cells that contain the oil bodies. A technique called RNA sequencing was used to identify several genes that are highly active in these cells and encode enzymes that could potentially be involved in producing forskolin. Further experiments demonstrated that these enzymes drive a cascade of chemical reactions that convert a molecule called 13R-manoyl oxide into forskolin. Next, Pateraki, Andersen-Ranberg et al. inserted the genes into yeast cells that could already produce 13R-manoyl oxide, which allowed the yeast to produce relatively high amounts of forskolin. These findings show that it is possible to identify the genes involved in the production of medicinal compounds in a relatively short amount of time. This knowledge will aid the development of a method that can be used to produce forskolin and other similar compounds on a large scale without needing to harvest C. forskohlii plants. DOI: http://dx.doi.org/10.7554/eLife.23001.002