Combinatorial biosynthesis in yeast leads to over 200 diterpenoids.

Diterpenoids form a diverse group of natural products, many of which are or could become pharmaceuticals or industrial chemicals. The modular character of diterpene biosynthesis and the promiscuity of the enzymes involved make combinatorial biosynthesis a promising approach to generate libraries of diverse diterpenoids. Here, we report on the combinatorial assembly in yeast of ten diterpene synthases producing (+)-copalyl diphosphate-derived backbones and four cytochrome P450 oxygenases (CYPs) in diverse combinations. This resulted in the production of over 200 diterpenoids. Based on literature and chemical database searches, 162 of these compounds can be considered new-to-Nature. The CYPs accepted most substrates they were given but remained regioselective with few exceptions. Our results provide the basis for the systematic exploration of the diterpenoid chemical space in yeast using sequence databases. Golden Gate assembly of 10 diterpene synthases (diTPS) and 4 cytochrome CYP oxygenases (CYPs) from diverse plant species in the yeast Saccharomyces cerevisiae leads to the production of more than 200 distinct diterpenoids. 132 of these diterpenoids are new-to-Nature indicating that combinatorial assembly of diTPS and CYPs in yeast is a promising approach to generate diverse libraries of diterpenoids in high-throughput. [Display omitted] • The biosynthesis of diterpenoids is modular. • Modularity allows the combinatorial assembly of diterpene synthases and cytochrome P450 oxygenases. • 10 diterpene synthases and 4 CYPs from different plants were combined using Golden Gate cloning and expressed in yeast. • In total more than 200 diterpenoids could be detected. • 162 of these diterpenoids are new-to-Nature. [ABSTRACT FROM AUTHOR]