Anisomycin (1), a pyrrolidine antibiotic, exhibits diverse biological and pharmacologic activities. The biosynthetic gene cluster of 1 has been identified previously and the multistep assembly of the core benzylpyrrolidine scaffold was characterized. However, enzymatic modifications, such as acylation involved in 1 biosynthesis are unknown. In this study, the genetic manipulation of aniI proved that it encoded indispensable acetyltransferase for 1 biosynthesis. Bioinformatics analysis suggested AniI as a member of LbH-MAT-GAT sugar O-acetyltransferase, but the biochemical assay identified that its target site was the hydroxyl group of the pyrrolidine ring. AniI was found to be tolerant of acyl donors with different chain length for the biosynthesis of 1 and derivatives 12 and 13 with butyryl and isovaleryl groups, respectively. Meanwhile, it showed comparable activity towards biosynthetic intermediates and synthesized analogues, suggesting promiscuity to the pyrrolidine ring structure of 1. These data may inspire new viable synthetic routes for the construction of more complex pyrrolidine ring scaffolds in 1. Finally, the overexpression of aniI under the control of strong promoters contributed to the higher productivities of 1 and its analogues. These findings reported here not only improved the understanding of anisomycin biosynthesis but also expand the substrate scope of O-acetyltransferase working on the pyrrolidine ring and pave the way for future metabolic engineering construction of high-yield strain.
IMPORTANCE
Acylation is an important tailoring reaction during natural products biosynthesis. Acylation could increase the structural diversity, affect the chemical stability, volatility, biological activity and even the cellular localization of specialized compounds. Many acetyltransferases have been reported in natural product biosynthesis. The typical example of LbH-MAT-GAT sugar O-acetyltransferase subfamily was reported to catalyze the CoA-dependent acetylation of the 6-hydroxyl group of sugars. However, no protein of this family has been characterized to acetylate non-sugar secondary metabolic product. Here, AniI was found to catalyze the acylation of the hydroxyl group of the pyrrolidine ring and be tolerant of diverse acyl donors and acceptors, which made the biosynthesis more efficient and exclusive for 1 and its derivatives biosynthesis. Moreover, the overexpression of aniI serves as a successful example of genetic manipulation of a modification gene for the high production of final products and might set the stage for future metabolic engineering.