BACKGROUND: Aminoglycoside antibiotics continue to play an indispensable role in clinical antibacterial agents. However, the protection and deprotection procedures in the chemical pathways of semi-synthetic antibiotics are long, atom- and step-inefficient, which severely hampers the development of novel AGs. RESULTS: Here, GenB3 and GenB4 are employed to synthesize sisomicin, Oxo-verdamicin, Oxo-gentamicin C1a, and Oxo-gentamicin C2a. Subsequently, a semi-rational strategy is applied to enhance the activities of GenB3 and GenB4. The activity of GenB3 CONCLUSION: Our results not only lay the foundation for the mild and efficient synthesis of C6'-modified AGs analogues but also serve as a reference for synthesizing additional single components in M. echinospora by further enhancing the dideoxygenation process.