Isepamicin is a weakly toxic but highly active aminoglycoside antibiotic derivative of gentamicin B. Gentamicin B is a naturally occurring minor component isolated from Micromonospora echinospora. 2ʹ-NH2-containing gentamicin C complex is a dominant compound produced by wild-type M. echinospora; by contrast, 2ʹ-OH-containing gentamicin B is produced as a minor component. However, the biosynthetic pathway of gentamicin B remains unclear. Considering that gentamicin B shares a unique C2ʹ hydroxyl group with kanamycin A, we aimed to design a new biosynthetic pathway of gentamicin B by combining twelve steps of gentamicin biosynthesis and two steps of kanamycin biosynthesis. We blocked the biosynthetic pathway of byproducts and generated a strain overproducing JI-20A, which was used as a precursor of gentamicin B biosynthesis, by disrupting genK and genP. The amount of JI-20A produced in M. echinospora ∆K∆P reached 911 μg/ml, which was 14-fold higher than that of M. echinospora ∆P. The enzymes KanJ and KanK necessary to convert 2ʹ-NH2 into 2ʹ-OH from the kanamycin biosynthetic pathway were heterologously expressed in M. echinospora ΔKΔP to transform JI-20A into gentamicin B. The strain with kanJK under PermE* could produce 80 μg/ml of gentamicin B, which was tenfold higher than that of the wild-type strain. To enhance gentamicin B production, we employed different promoters and gene integration combinations. When a PhrdB promoter was used and kanJ and kanK were integrated in the genome through gene replacement, gentamicin B was generated as the major product with a maximum yield of 880 μg/ml. We constructed a new biosynthetic pathway of high-level gentamicin B production; in this pathway, most byproducts were removed. This method also provided novel insights into the biosynthesis of secondary metabolites via the combinatorial biosynthesis.

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.

Both kanamycin A and kanamycin B, antibiotic components produced by Streptomyces kanamyceticus, have medical value. Two different pathways for kanamycin biosynthesis have been reported by two research groups. In this study, to obtain an optimal kanamycin A-producing strain and a kanamycin B-high-yield strain, we first examined the native kanamycin biosynthetic pathway in vivo. Based on the proposed parallel biosynthetic pathway, kanN disruption should lead to kanamycin A accumulation; however, the kanN-disruption strain produced neither kanamycin A nor kanamycin B. We then tested the function of kanJ and kanK. The main metabolite of the kanJ-disruption strain was identified as kanamycin B. These results clarified that kanamycin biosynthesis does not proceed through the parallel pathway and that synthesis of kanamycin A from kanamycin B is catalyzed by KanJ and KanK in S. kanamyceticus. As expected, the kanamycin B yield of the kanJ-disruption strain was 3268±255 μg/mL, 12-fold higher than that of the original strain. To improve the purity of kanamycin A and reduce the yield of kanamycin B in the fermentation broth, four different kanJ- and kanK-overexpressing strains were constructed through either homologous recombination or site-specific integration. The overexpressing strain containing three copies of kanJ and kanK in its genome exhibited the lowest kanamycin B yield (128±20 μg/mL), which was 54% lower than that of the original strain. Our experimental results demonstrate that kanamycin A is derived from KanJ-and-KanK-catalyzed conversion of kanamycin B in S. kanamyceticus. Moreover, based on the clarified biosynthetic pathway, we obtained a kanamycin B-high-yield strain and an optimized kanamycin A-producing strain with minimal byproduct.

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: Aminoglycosides (AGs) are one of the initial classes of antibiotics that have been used clinically and possess broad spectrum of activity. Nevertheless, their clinical utilization is restricted by safety issues associated with nephrotoxicity and ototoxicity.Methods: Glycosyltransferase (GT) KanM2 was introduced into M. echinospora to produce the gentamicin derivatives, in which a kanosamine sugar ring was introduced to replace the garosamine. The premature termination codon (PTC) readthrough activity of genkamicins (GKs) was compared using dual luciferase reporter assay. The toxicity of GK was assessed in vitro in HEK-293 and NCI-H1299 cells and determined based on cell viability calculated after 48 h of treatment with different concentrations of the compounds. The NCI-H1299 cells harbouring the R213X nonsense mutation were treated with different concentrations of the derivatives to compare their expression of p53 proteins. The expression of p53 and its downstream targets p21 and BAX was detected using Western blotting and qRT-PCR in NCI-H1299 cells containing the R213X nonsense mutation treated with different concentrations of GK-Ae and G418. Finally, immunofluorescence and flow cytometry were used to determine the subcellular localization of full-length p53 protein induced by GK-Ae treatment and its effect on apoptosis in cancer cells.Results: Eight gentamicin derivatives were obtained in this study. GK-Ae displayed similar PTC readthrough activity and reduced toxicity compared to natural aminoglycoside G418. Moreover, GK-Ae increased the levels of both p53 and its downstream targets p21 and BAX, and promoted apoptosis of cancer cells.Conclusion: These results demonstrate the potential of combinatorial biosynthesis to increase the diversity of structures of AGs and provide directions for the development of new AGs with low toxicity and high PTC readthrough activity.