Point mutation of V252 in Neomycin C epimerase enlarges substrate binding pocket and improve the accumulation of Neomycin B in Streptomyces fradiae

Abstract Neomycin, aminoglycoside antibiotics with broad-spectrum antibacterial resistance, is widely used in pharmaceutical and agricultural fields. However, the separation and purification process of neomycin B as an active substance is complicated in Streptomyces fradiae. Although NeoN can catalyze neomycin C to generate neomycin B, the catalytic mechanism of NeoN is still unclear. In this study, whole genomics sequencing clarified the genomic information of high-yielding mutant SF-2. Subsequently, the mechanism of NeoN in catalyzing neomycin C synthesis of neomycin B was resolved based on the NeoN-SAM-neomycin C ternary complex. Finally, mutant NeoNV252A improved the activity of NeoN and the recombinant strain SF-2-NeoNV252A accumulated neomycin B 16766.6 U/mL and the ratio of neomycin C decreased from 16.1–6.28% relative to the starting strain SF-2. In summary, this work had analyzed the catalytic mechanism of NeoN, which had certain reference significance for rationally design NeoN to improve the production of neomycin B and weaken the proportion of neomycin C.