Acyltransferase AniI, a Tailoring Enzyme with Broad Substrate Tolerance for High-Level Production of Anisomycin
| Autor: | Zixin Deng, Dashan Zhang, Jinjin Wang, Jufang Shen, Yongjian Qiao, Xiaoqing Zheng, Lingxin Kong, Qing Wang, Delin You, Junbo Wang |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Stereochemistry
Coenzyme A Acylation 01 natural sciences Applied Microbiology and Biotechnology Pyrrolidine Metabolic engineering 03 medical and health sciences chemistry.chemical_compound Biosynthesis Acetyl Coenzyme A Acetyltransferases Escherichia coli Enzymology and Protein Engineering Cellular localization 030304 developmental biology 0303 health sciences Ecology 010405 organic chemistry 0104 chemical sciences Anti-Bacterial Agents chemistry Acyltransferase Anisomycin Food Science Biotechnology |
| Zdroj: | Appl Environ Microbiol |
| Popis: | Anisomycin (compound 1), a pyrrolidine antibiotic, exhibits diverse biological and pharmacologic activities. The biosynthetic gene cluster of compound 1 has been identified previously, and the multistep assembly of the core benzylpyrrolidine scaffold was characterized. However, enzymatic modifications, such as acylation, involved in compound 1 biosynthesis are unknown. In this study, the genetic manipulation of aniI proved that it encoded an indispensable acetyltransferase for compound 1 biosynthesis. Bioinformatics analysis suggested AniI as a member of maltose (MAT) and galactoside O-acetyltransferases (GAT) with C-terminal left-handed parallel beta-helix (LbH) subdomain, which were referred to as LbH-MAT-GAT sugar O-acetyltransferases. However, 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 lengths for the biosynthesis of compound 1 and derivatives 12 and 13 with butyryl and isovaleryl groups, respectively. Meanwhile, it showed comparable activity toward biosynthetic intermediates and synthesized analogues, suggesting promiscuity to the pyrrolidine ring structure of compound 1. These data may inspire new viable synthetic routes for the construction of more complex pyrrolidine ring scaffolds in compound 1. Finally, the overexpression of aniI under the control of strong promoters contributed to the higher productivities of compound 1 and its analogues. These findings reported here not only improve 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 strains. IMPORTANCE Acylation is an important tailoring reaction during natural product biosynthesis. Acylation could increase the structural diversity and 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 the LbH-MAT-GAT sugar O-acetyltransferase subfamily was reported to catalyze the coenzyme A (CoA)-dependent acetylation of the 6-hydroxyl group of sugars. However, no protein of this family has been characterized to acetylate a nonsugar 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 biosynthesis of compound 1 and its derivatives. 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. |
| Databáze: | OpenAIRE |
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