Discovery Of Active Natural Products In Streptomyces Hyalinus NBRC13850 And Functional Study Of Key Transcription Factors Involved In Nybomycin Biosynthesis

Streptomyces hyalinus NBRC13850 was an actinomycete isolated from Okinawa soil and producing strain of Nybomycin(NM).Nybomycin comprised a unique heterocyclic class of compound that contain a linearly fused pyridoquinolone ring system and an angularly fused oxazoline unit.Nybomycin had antibacterial and antibacterial activity.In this study,to obtain new active natural products and to elucidate the regulatory mechanisms in the nybomycin biosynthesis pathway,we have intensively explored Streptomyces hyalinus NBRC13850 and discovered new active natural products.We identified the nybomycin biosynthetic gene cluster from the strain Streptomyces hyalinus NBRC13850 by genome sequencing.The function of key transcription factors was verified in the nybomycin biosynthesis gene cluster by bioinformatics analysis and in vivo gene inactivation.According to the strategy of“One strain-many compound”,we screened the culture medium of nybomycin producing strain Streptomyces hyalinus NBRC13850,and finally selected A2 medium for amplification fermentation.Five compounds were isolated and identified,including three new compounds.Compound(±)1 was a pair of enantiomers of benzazole,Specific optical rotations were[?]^D₂₅-12.8(c 0.11,Me OH)and[?]^D₂₅+13.1(c 0.10,Me OH).Compound 2 was a demethyldeoxynybomycin,compound 3 was a nybomycin,and compound 4 was deoxynybmycin.By measuring the minimum inhibitory concentration of the compound,it was found that compound(-)1 with a specific optical rotation of[?]^D₂₅-12.8(c 0.11,Me OH)can be used as synergist of deoxynybmycin to improve antibacterial activity against methicillin-resistant Staphylococcus aureus(MRSA)and Mycobacterium smegmatis MC²155.Compound 2,compound 3,compound 4 had significant biological activity against methicillin-resistant Staphylococcus aureus(MRSA),Mycobacterium smegmatis MC²155,multidrug-resistant Pseudomonas aeruginosa Cl1.The nybmycin biosynthetic gene cluster was obtained by sequencing the Streptomyces hyalinus NBRC13850 genome.Bioinformatics analysis showed that dnm R1 and dnm R2 were gene regulatory genes in biosynthetic gene cluster.The gene deletion mutants of dnm R1 and dnm R2 were constructed by PCR-targeting and conjugative transfer,and the functions of dnm R1 and dnm R2 were verified.dnm R1was a repressor,and its gene deletion mutant can increases the yield of nybmycin;dnm R1 was a activator,and its gene deletion mutant cannot produce nybmycin.In summary,five natural products were obtained in this study,and three were new compounds.The bioactivity assay showed good antibacterial activity,These data establish a basis for lead compounds.We identified the nybomycin biosynthetic gene cluster.Two key transcription factors were functionally verified and provided a basis for the study of secondary metabolism of nybmycin.