In general,the initiation or closure of antibiotic biosynthesis is determined by regulatory proteins,but most of their mechanisms of action remain unknown.The 2-deoxystreptamine-containing aminoglycosides(2-DOS AGs)fo...In general,the initiation or closure of antibiotic biosynthesis is determined by regulatory proteins,but most of their mechanisms of action remain unknown.The 2-deoxystreptamine-containing aminoglycosides(2-DOS AGs)form a unique category among antibiotics.Genomic analysis revealed that a group of hypothetical regulatory genes represented by neo I are widely distributed in the biosynthetic gene clusters(BGCs)of natural products from Streptomyces species,including several 2-DOS AGs.Only limited knowledge is available for the roles of Neo Itype regulators although neomycin and some of the related AGs have been developed as therapeutic drugs for decades.This study focuses on the functional determination of neo I and its homologues situated in the BGCs of six AGs.We found that the yield of neomycin in neo I disruption mutant(Δneo I)increased by 50%compared to the wild-type(WT)strain((420.6±44.1)mg L^(-1)),while it was partially restored by the complementation of neo I,demonstrating that Neo I acted as a repressor in neomycin biosynthesis.Further electrophoretic mobility shift assays(EMSAs)and DNase I footprinting assays indicated that Neo I could specifically bind to the promoter region between neo E and neo I with conserved nucleotides(5'-CVHYMRCHDKAGYGGACR-3'),as determined by site-directed mutagenesis.Interestingly,cross-bindings of the Neo I homologues from the six different BGCs to their corresponding DNA targets were manifested,and the five exogenous Neo I homologues could complement Neo I function of repressing neomycin biosynthesis.Our results suggested that Neo I-type regulators represent widespread and conservative regulatory characteristics in the biosynthesis of 2-DOS AGs,which would be significant for optimizing the biosynthetic pathways of valuable commercialized aminoglycoside antibiotics.展开更多
基金supported by the National Natural Science Foundation of China (32170043,82173720)the National Key Research and Development Program of China (2020YFA0907800)。
文摘In general,the initiation or closure of antibiotic biosynthesis is determined by regulatory proteins,but most of their mechanisms of action remain unknown.The 2-deoxystreptamine-containing aminoglycosides(2-DOS AGs)form a unique category among antibiotics.Genomic analysis revealed that a group of hypothetical regulatory genes represented by neo I are widely distributed in the biosynthetic gene clusters(BGCs)of natural products from Streptomyces species,including several 2-DOS AGs.Only limited knowledge is available for the roles of Neo Itype regulators although neomycin and some of the related AGs have been developed as therapeutic drugs for decades.This study focuses on the functional determination of neo I and its homologues situated in the BGCs of six AGs.We found that the yield of neomycin in neo I disruption mutant(Δneo I)increased by 50%compared to the wild-type(WT)strain((420.6±44.1)mg L^(-1)),while it was partially restored by the complementation of neo I,demonstrating that Neo I acted as a repressor in neomycin biosynthesis.Further electrophoretic mobility shift assays(EMSAs)and DNase I footprinting assays indicated that Neo I could specifically bind to the promoter region between neo E and neo I with conserved nucleotides(5'-CVHYMRCHDKAGYGGACR-3'),as determined by site-directed mutagenesis.Interestingly,cross-bindings of the Neo I homologues from the six different BGCs to their corresponding DNA targets were manifested,and the five exogenous Neo I homologues could complement Neo I function of repressing neomycin biosynthesis.Our results suggested that Neo I-type regulators represent widespread and conservative regulatory characteristics in the biosynthesis of 2-DOS AGs,which would be significant for optimizing the biosynthetic pathways of valuable commercialized aminoglycoside antibiotics.
