Background <p>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.</p> Results <p>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^M1 (Q270N) towards JI-20A-P is 1.74 times higher than that of GenB3^WT. Similarly, the activity of GenB3^M2 (L361C/A412T/Q270N) towards JI-20Ba-P is 1.34 times higher than that of GenB3^WT. The activity of GenB4^M1 (L356C) towards sisomicin is 1.51 times higher than that of GenB4^WT, while GenB4^M2 (L356C/A407T/Q265N) towards verdamicin C2a is 1.34 times higher than that of GenB4^WT. Furthermore, the beneficial effects of these mutants have been validated in engineered strains. Molecular dynamics simulations indicate that GenB3^M1 establishes a hydrogen bond network in the active center, while GenB4^M1 reduces the distance between K238 and the reaction center. It is also noted that the GenB3^M2 exhibits a synergistic effect specifically on JI-20Ba-P, as the C6’-CH₃ group stabilization restricts the movement of the substrate, which contrasts with JI-20A-P.</p> Conclusion <p>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 <i>M. echinospora</i> by further enhancing the dideoxygenation process.</p>