Point mutation of V252 in neomycin C epimerase enlarges substrate-binding pocket and improves neomycin B accumulation in Streptomyces fradiae

Neomycin, an aminoglycoside antibiotic with broad-spectrum antibacterial resistance, is widely used in pharmaceutical and agricultural fields. However, separation and purification of neomycin B as an active substance from Streptomyces fradiae are complicated. Although NeoN can catalyze conversion of neomycin C to neomycin B, the underlying catalytic mechanism is still unclear. In this study, the genomic information of high-yielding mutant S. fradiae SF-2 was elucidated using whole-genome sequencing. Subsequently, the mechanism of NeoN in catalyzing conversion of neomycin C to neomycin B was resolved based on NeoN–SAM–neomycin C ternary complex. Mutant NeoN V252A showed improved NeoN activity, and the recombinant strain SF-2-NeoN V252A accumulated 16,766.6 U/mL neomycin B, with a decrease in neomycin C ratio from 16.1% to 6.28%, when compared with the parental strain SF-2. In summary, this study analyzed the catalytic mechanism of NeoN, providing significant reference for rational design of NeoN to improve neomycin B production and weaken the proportion of neomycin C. Graphical Abstract