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.

The present study was conducted to examine the influence of a pathogen Sclerotinia sclerotiorum (Lib.) de Bary on the actinobacterial community associated with the soybean roots. A total of 70 endophytic actinobacteria were isolated from the surface-sterilized roots of either healthy or diseased soybeans, and they were distributed under 14 genera. Some rare genera, including Rhodococcus, Kribbella, Glycomyces, Saccharothrix, Streptosporangium and Cellulosimicrobium, were endemic to the diseased samples, and the actinobacterial community was more diverse in the diseased samples compared with that in the heathy samples. Culture-independent analysis of root-associated actinobacterial community using the high-throughput sequencing approach also showed similar results. Four Streptomyces strains that were significantly abundant in the diseased samples exhibited strong antagonistic activity with the inhibition percentage of 54.1–87.6%. A bioactivity-guided approach was then employed to isolate and determine the chemical identity of antifungal constituents derived from the four strains. One new maremycin analogue, together with eight known compounds, were detected. All compounds showed significantly antifungal activity against S. sclerotiorum with the 50% inhibition (EC₅₀) values of 49.14–0.21 mg/L. The higher actinobacterial diversity and more antifungal strains associated with roots of diseased plants indicate a possible role of the root-associated actinobacteria in natural defense against phytopathogens. Furthermore, these results also suggest that the root of diseased plant may be a potential reservoir of actinobacteria producing new agroactive compounds.