Zhai Hang, Yang Lihua, Ye Qi, Kong Zhijun, Pei Jiye, Ji Yuan, Liu Botong, Chen Xiaotang, Tian Tingting, Ni Xianpu, Xia Huanzhang, Zhang Shumin
School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, No.103 Wenhua Road, Shenyang, Liaoning, China.
Microb Cell Fact. 2025 Feb 27;24(1):49. doi: 10.1186/s12934-025-02678-0.
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.
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.
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.
氨基糖苷类抗生素在临床抗菌药物中继续发挥着不可或缺的作用。然而，半合成抗生素化学途径中的保护和脱保护过程冗长，原子和步骤效率低下，这严重阻碍了新型氨基糖苷类药物的开发。
在此，利用GenB3和GenB4合成西索米星、氧代弗氏霉素、氧代庆大霉素C1a和氧代庆大霉素C2a。随后，应用半理性策略增强GenB3和GenB4的活性。GenB3（Q270N）对JI-20A-P的活性比GenB3高1.74倍。同样，GenB3（L361C/A412T/Q270N）对JI-20Ba-P的活性比GenB3高1.34倍。GenB4（L356C）对西索米星的活性比GenB4高1.51倍，而GenB4（L356C/A407T/Q265N）对弗氏霉素C2a的活性比GenB4高1.34倍。此外，这些突变体的有益效果已在工程菌株中得到验证。分子动力学模拟表明，GenB3在活性中心建立了氢键网络，而GenB4缩短了K238与反应中心之间的距离。还应注意的是，GenB3对JI-20Ba-P表现出协同效应，因为C6'-CH基团的稳定限制了底物的移动，这与JI-20A-P形成对比。
我们的结果不仅为温和高效地合成C6'-修饰的氨基糖苷类类似物奠定了基础，也为通过进一步增强双脱氧过程在棘孢小单孢菌中合成其他单一组分提供了参考。
