Improving activity of GenB3 and GenB4 in gentamicin dideoxygenation biosynthesis by semi-rational engineering.

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 (Q270N) towards JI-20A-P is 1.74 times higher than that of GenB3. Similarly, the activity of GenB3 (L361C/A412T/Q270N) towards JI-20Ba-P is 1.34 times higher than that of GenB3. The activity of GenB4 (L356C) towards sisomicin is 1.51 times higher than that of GenB4, while GenB4 (L356C/A407T/Q265N) towards verdamicin C2a is 1.34 times higher than that of GenB4. Furthermore, the beneficial effects of these mutants have been validated in engineered strains. Molecular dynamics simulations indicate that GenB3 establishes a hydrogen bond network in the active center, while GenB4 reduces the distance between K238 and the reaction center. It is also noted that the GenB3 exhibits a synergistic effect specifically on JI-20Ba-P, as the C6'-CH group stabilization restricts the movement of the substrate, which contrasts with JI-20A-P.

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.