Background: New semi-synthetic aminoglycoside antibiotics generally use chemical modifications to avoid inactivity from pathogens. One of the most used modifications is 3′,4′-di-deoxygenation, which imitates the structure of gentamicin. However, the mechanism of di-deoxygenation has not been clearly elucidated. Results: Here, we report that the bifunctional enzyme, GenB4, catalyzes the last step of gentamicin 3′,4′-di-deoxygenation via reduction and transamination activities. Following disruption of genB4 in wild-type M. echinospora, its products accumulated in 6′-deamino-6′-oxoverdamicin (1), verdamicin C2a (2), and its epimer, verdamicin C2 (3). Following disruption of genB4 in M. echinospora ΔgenK, its products accumulated in sisomicin (4) and 6′-N-methylsisomicin (5, G-52). Following in vitro catalytic reactions, GenB4 transformed sisomicin (4) to gentamicin C1a (9) and transformed verdamicin C2a (2) and its epimer, verdamicin C2 (3), to gentamicin C2a (11) and gentamicin C2 (12), respectively. Conclusion: This finding indicated that in addition to its transamination activity, GenB4 exhibits specific 4′,5′ double-bond reducing activity and is responsible for the last step of gentamicin 3′,4′-di-deoxygenation. Taken together, we propose three new intermediates that may refine and supplement the specific biosynthetic pathway of gentamicin C components and lay the foundation for the complete elucidation of di-deoxygenation mechanisms.

Background: The C-3′,4′-dideoxygenation structure in gentamicin can prevent deactivation by aminoglycoside 3′-phosphotransferase (APH(3′)) in drug-resistant pathogens. However, the enzyme catalyzing the dideoxygenation step in the gentamicin biosynthesis pathway remains unknown. Results: Here, we report that GenP catalyzes 3′ phosphorylation of the gentamicin biosynthesis intermediates JI-20A, JI-20Ba, and JI-20B. We further demonstrate that the pyridoxal-5′-phosphate (PLP)-dependent enzyme GenB3 uses these phosphorylated substrates to form 3′,4′-dideoxy-4′,5′-ene-6′-oxo products. The following C-6′-transamination and the GenB4-catalyzed reduction of 4′,5′-olefin lead to the formation of gentamicin C. To the best of our knowledge, GenB3 is the first PLP-dependent enzyme catalyzing dideoxygenation in aminoglycoside biosynthesis. Conclusions: This discovery solves a long-standing puzzle in gentamicin biosynthesis and enriches our knowledge of the chemistry of PLP-dependent enzymes. Interestingly, these results demonstrate that to evade APH(3′) deactivation by pathogens, the gentamicin producers evolved a smart strategy, which utilized their own APH(3′) to activate hydroxyls as leaving groups for the 3′,4′-dideoxygenation in gentamicin biosynthesis.

Background: Tetramycin is a 26-member tetraene antibiotic used in agriculture. It has two components, tetramycin A and tetramycin B. Tetramycin B is obtained by the hydroxylation of tetramycin A on C4. This reaction is catalyzed by the cytochrome P450 monooxygenase TtmD. The two components of tetramycin have different antifungal activities against different pathogenic fungi. Therefore, the respective construction of high-yield strains of tetramycin A and tetramycin B is conducive to more targeted action on pathomycete and has a certain practical value. Results: Streptomyces ahygroscopicus S91 was used as the original strain to construct tetramycin A high-yield strains by blocking the precursor competitive biosynthetic gene cluster, disrupting tetramycin B biosynthesis, and overexpressing the tetramycin pathway regulator. Eventually, the yield of tetramycin A in the final strain was up to 1090.49 ± 136.65 mg·L^− 1. Subsequently, TtmD, which catalyzes the conversion from tetramycin A to tetramycin B, was overexpressed. Strains with 2, 3, and 4 copies of ttmD were constructed. The three strains had different drops in tetramycin A yield, with increases in tetramycin B. The strain with three copies of ttmD showed the most significant change in the ratio of the two components. Conclusions: A tetramycin A single-component producing strain was obtained, and the production of tetramycin A increased 236.84% ± 38.96% compared with the original strain. In addition, the content of tetramycin B in a high-yield strain with three copies of ttmD increased from 26.64% ± 1.97 to 51.63% ± 2.06%.

As a member of the first-generation aminoglycoside antibiotics, gentamicin was once widely used in clinical practice. However, due to its drug resistance, toxicity, and side effects and the development of novel antibiotics, gentamicin has gradually faded from the spotlight. However, in recent years, aminoglycoside antibiotics have regained significant attention from the medical community as a critical therapeutic option for severe infections caused by multidrug-resistant bacteria. This review provides a detailed overview of the complete biosynthetic pathway of gentamicin and its recent advances, including the biosynthesis of the core structure 2-deoxystreptamine (2-DOS), the biosynthesis of the first pseudotrisaccharide intermediate gentamicin A2, the key branch point intermediate X2, and its parallel pathways. The methylation network, the transamination–epimerization network, and the di-deoxygenation processes within the gentamicin biosynthetic system are highlighted. Recent progress in the engineering of the gentamicin biosynthetic pathway and the development of novel gentamicin derivatives as new aminoglycoside antibiotics are also summarized. Finally, this review offers perspectives on the future research directions and uses of gentamicin. In summary, a detailed dissection of the complex biosynthetic network of gentamicin is provided to lay the groundwork for targeted and random structural modifications of gentamicin using combinatorial biosynthetic and high-throughput screening technologies to develop new aminoglycoside antibiotics with lower host toxicity and greater activity against resistant strains.

Monoclonal antibodies (mAbs), as potent therapeutic agents, have been widely applied in the treatment of various major diseases, including infectious diseases, autoimmune disorders, cancers, and neurodegenerative diseases. However, early-generation mAbs were limited by high immunogenicity, short half-life, and insufficient affinity, which compromised their therapeutic efficacy. With technological advancements, novel approaches such as high-throughput screening and glycosyl modification have been introduced to improve the performance of mAbs. Furthermore, computer-aided design techniques—including molecular docking, molecular dynamics simulations, and artificial intelligence -based methods—are increasingly being employed to accelerate the optimization process. This review summarizes recent progress in the optimization of therapeutic mAbs, with a focus on technological breakthroughs and applications in affinity enhancement, development of broad-spectrum mAbs, specificity modulation, immunogenicity reduction, and stability improvement. Additionally, it discusses current challenges and future directions in antibody optimization. This review aims to provide insights and references for the development and optimization of next-generation antibody drugs, ultimately promoting the clinical application of safer and more effective mAb-based therapies.