Two Cryptic Self‐Resistance Mechanisms in Streptomyces tenebrarius Reveal Insights into the Biosynthesis of Apramycin.

Apramycin is a clinically promising aminoglycoside antibiotic (AGA). To date, mechanisms underlying the biosynthesis and self‐resistance of apramycin remain largely unknown. Here we report that apramycin biosynthesis proceeds through unexpected phosphorylation, deacetylation, and dephosphorylation steps, in which a novel aminoglycoside phosphotransferase (AprU), a putative creatinine amidohydrolase (AprP), and an alkaline phosphatase (AprZ) are involved. Biochemical characterization revealed that AprU specifically phosphorylates 5‐OH of a pseudotrisaccharide intermediate, whose N‐7′ acetyl group is subsequently hydrolyzed by AprP. AprZ is located extracellularly where it removes the phosphate group from a pseudotetrasaccharide intermediate, leading to the maturation of apramycin. Intriguingly, 7′‐N‐acetylated and 5‐O‐phosphorylated apramycin that were accumulated in ΔaprU and ΔaprZ respectively exhibited significantly reduced antibacterial activities, implying Streptomyces tenebrarius employs C‐5 phosphorylation and N‐7′ acetylation as two strategies to avoid auto‐toxicity. Significantly, this study provides insight into the design of new generation AGAs to circumvent the emergence of drug‐resistant pathogens. [ABSTRACT FROM AUTHOR]