Discovery and Biosynthesis of FPP-Derived Non-Canonical C17 Terpenes from Pseudomonas Species

Terpenes are a structurally and functionally highly diverse class of natural products found across all living organisms. Pseudomonas species possess significant potential for the biosynthetic assembly of farnesyl pyrophosphate (FPP)-derived terpenes with unusual carbon skeletons. However, their structural and biosynthetic diversity is largely unexplored. Here, we report the discovery of grimophan, an unprecedented C17 terpene featuring a deltacyclane skeleton. The compound was accessed by heterologous expression of the pgr biosynthetic gene cluster from Pseudomonas grimontii DSM 17515 in E. coli. The roles of the enzymes involved in grimophan biosynthesis were elucidated through in vitro reconstitution of the entire biosynthetic pathway. In-depth functional studies on the involved SAM-dependent methyltransferases led to the discovery of novel methyltransferase-like enzymes that do not perform functional group transfer, but rather significantly enhance the production titer of non-canonical terpenes. These enzymes thus constitute valuable tools for increasing biotechnological production levels of terpenes. The function of the cognate terpene synthase PgrE was evaluated by targeted point mutations within the conserved Asp-rich motif D92DMPLG97 to map effects of active-site residues on product formation. These investigations facilitated redirecting product selectivity, leading to alternative products. Overall, our work sheds light on the general biosynthetic logic leading to non-canonical C17 terpenes, provides a basis for their targeted discovery by genome mining and heterologous expression, and for engineering C17 terpene structural frameworks.