Discovery and Biosynthesis of Farnesyl Pyrophosphate-Derived Noncanonical C ₁₇ Terpenes from <i>Pseudomonas</i> Species

Terpenes make up a structurally and functionally highly diverse class of natural products found across all living organisms. <i>Pseudomonas</i> species possess significant potential for the biosynthetic assembly of farnesyl pyrophosphate (FPP)-derived terpenes with unusual carbon skeletons. However, their structural and biosynthetic diversity has been largely unexplored. Here, we report the discovery of grimophan, a C₁₇ terpene featuring a rare deltacyclane skeleton. The compound was accessed by heterologous expression of the <i>pgr</i> biosynthetic gene cluster from <i>Pseudomonas grimontii</i> DSM 17515 in <i>E. coli</i>. The roles of the enzymes involved in grimophan biosynthesis were elucidated through <i>in vitro</i> reconstitution of the entire biosynthetic pathway. In-depth functional studies on the involved SAM-dependent methyltransferases led to the discovery of methyltransferase-like enzymes that do not perform functional group transfer, but rather significantly enhance the production titer of noncanonical terpenes. These enzymes thus constitute valuable tools for enhancing biotechnological production levels of noncanonical C₁₆ and C₁₇ terpenes. The function of the cognate terpene synthase PgrE was evaluated by targeted point mutations within the observed Asp-rich motif D₉₂DMPLG₉₇ to map the 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 noncanonical C₁₇ terpenes and provides a basis for their targeted discovery by genome mining and heterologous expression and for engineering C₁₇ terpene structural frameworks.