Neomycin,a crucial aminoglycoside antibiotic,is primarily biosynthesized by Streptomyces fradiae through fermentation.Its widespread applications encompass disease management in crops,treatment of bacterial infections...Neomycin,a crucial aminoglycoside antibiotic,is primarily biosynthesized by Streptomyces fradiae through fermentation.Its widespread applications encompass disease management in crops,treatment of bacterial infections in respiratory and gastrointestinal tracts,among other domains,leading to substantial market demand.Increasing evidence underscores the pivotal role of transcription factors in microbial metabolic regulation,orchestrating the coordinated expression of multiple genes in specific pathways.This orchestration holds the potential to enhance engineered microbial strains,thereby facilitating the precise and efficient synthesis of neomycin.Leveraging transcriptomic analyses of the wild-type strain SF-1 and the mutation-derived high-yield strain SF-2,this study identified significant variations in the expression levels of seven transcription factors.By constructing recombinant strains overexpressing these seven transcription factors,the optimal factor NecR,influencing neomycin production,was pinpointed.Further,promoter optimization was employed to augment neomycin synthesis.Under shaken flask cultivation conditions,the titer of neomycin B reached 11,546 U/mL,marking a 23%enhancement over the mutation-derived high-yield strain SF-2.The in vivo fluorescence reporter gene characterization using EMSA binding revealed that NecR can bind to the promoter region of neoS,thereby enhancing the transcriptional levels of neoS,subsequently promoting neomycin synthesis.This investigation not only furnishes pivotal insights for the construction of high-yield neomycin-producing strains but also elucidates the central role of transcription factors in microbial metabolic regulation.This revelation is poised to offer novel avenues and strategies in the realm of microbial metabolic engineering,holding promise for significant breakthroughs in antibiotic production and other bioproduct synthesis domains.展开更多
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financially supported by National Natural Science Foundations of China(No.32300059)Key research and development projects and achievement transformation projects of Wuhu(Grant No.2023YF096)+1 种基金Scientific Research Start-up Fund for Introduced Talents of Anhui Polytechnic University(2022YOO068)Natural Science Research Project of Colleges and Universities in Anhui Province(Grant 2022AH050971)。
文摘Neomycin,a crucial aminoglycoside antibiotic,is primarily biosynthesized by Streptomyces fradiae through fermentation.Its widespread applications encompass disease management in crops,treatment of bacterial infections in respiratory and gastrointestinal tracts,among other domains,leading to substantial market demand.Increasing evidence underscores the pivotal role of transcription factors in microbial metabolic regulation,orchestrating the coordinated expression of multiple genes in specific pathways.This orchestration holds the potential to enhance engineered microbial strains,thereby facilitating the precise and efficient synthesis of neomycin.Leveraging transcriptomic analyses of the wild-type strain SF-1 and the mutation-derived high-yield strain SF-2,this study identified significant variations in the expression levels of seven transcription factors.By constructing recombinant strains overexpressing these seven transcription factors,the optimal factor NecR,influencing neomycin production,was pinpointed.Further,promoter optimization was employed to augment neomycin synthesis.Under shaken flask cultivation conditions,the titer of neomycin B reached 11,546 U/mL,marking a 23%enhancement over the mutation-derived high-yield strain SF-2.The in vivo fluorescence reporter gene characterization using EMSA binding revealed that NecR can bind to the promoter region of neoS,thereby enhancing the transcriptional levels of neoS,subsequently promoting neomycin synthesis.This investigation not only furnishes pivotal insights for the construction of high-yield neomycin-producing strains but also elucidates the central role of transcription factors in microbial metabolic regulation.This revelation is poised to offer novel avenues and strategies in the realm of microbial metabolic engineering,holding promise for significant breakthroughs in antibiotic production and other bioproduct synthesis domains.
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.
