From the first ambitious imagination of tropinone biosynthesis mechanism in plants published in 1917 to the de novo production of scopolamine in yeast realized in 2020, what did we learn from this long story of more than 100 years old?

The pyridine ring is a potent pharmacophore in alkaloid natural products. Nonetheless, its biosynthetic pathways are poorly understood. Rubrolones A and B are tropolone alkaloid natural products possessing a unique tetra-substituted pyridine moiety. Here, we report the gene cluster and propose a biosynthetic pathway for rubrolones, identifying a key intermediate that accumulates upon inactivation of sugar biosynthetic genes. Critically, this intermediate was converted to the aglycones of rubrolones by non-enzymatic condensation and cyclization with either ammonia or anthranilic acid to generate the respective pyridine rings. We propose that this non-enzymatic reaction occurs via hydrolysis of the key intermediate, which possesses a 1,5-dione moiety as an amine acceptor capable of cyclization. This study suggests that 1,5-dione moieties may represent a general strategy for pyridine ring biosynthesis, and more broadly highlights the utility of non-enzymatic diversification for exploring and expanding natural product chemical space.

The skeleton of tropane alkaloids is derived from ornithine-derived N-methylpyrrolinium and two malonyl-CoA units. The enzymatic mechanism that connects N-methylpyrrolinium and malonyl-CoA units remains unknown. Here, we report the characterization of three pyrrolidine ketide synthases (PYKS), AaPYKS, DsPYKS, and AbPYKS, from three different hyoscyamine- and scopolamine-producing plants. By examining the crystal structure and biochemical activity of AaPYKS, we show that the reaction mechanism involves PYKS-mediated malonyl-CoA condensation to generate a 3-oxo-glutaric acid intermediate that can undergo non-enzymatic Mannich-like condensation with N-methylpyrrolinium to yield the racemic 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid. This study therefore provides a long sought-after biosynthetic mechanism to explain condensation between N-methylpyrrolinium and acetate units and, more importantly, identifies an unusual plant type III polyketide synthase that can only catalyze one round of malonyl-CoA condensation.