Streptomyces Roseosporus Pleiotropic Regulatory Gene WhiB4Influences Morphogenesis And Daptomycin Production

BackgroundDaptomycin is an acidic cyclic lipopeptide antibiotic which produced by Streptomyces roseosporus. It was approved by the FDA in2003for the treatment of skin and skin structure infections caused by Gram-positive pathogens and for the treatment of bacteremia and right-sided endocarditis caused by Staphylococcus aureus strains and methicillin-resistant Staphylococcus aureus(MRSA) in2006.Daptomycin has potent in vitro bactericidal activity against Gram-positive pathogens, including MRSA, penicillin-resistant Streptococcus pneumoniae (PRSP), vancomycin-resistant enterococci (VRE), and vancomycin-resistant S. aureus (VRSA) and other antibiotic resistant strains. Besides linezolid, daptomycin is the only novel antibiotic structural class to be approved in the last four decades. It has become the best alternative to vancomycin and has a broad development prospects.But daptomycin production is very low, there is considerable interest in daptomycin-producing strain improvement via different approaches.The whiB-like genes(wbl) are exclusive to the actinomycetes and are absent from all other organisms studied so far. In the previous work, a whiB-like putative transcription factor (wblAsco) was identified as a global antibiotic down-regulator in S.coelicolor. Simultaneously wblAsco was also identified as an essential gene for sporulation of aerial hyphae in many Streptomyces. so wbl appears to play a negative role in the control of antibiotic biosynthesis and is essential for sporulation. ObjectiveTo understand the influence of whiB4on cell growth and the biosynthesis of daptomycin in Streptomyces roseosporus NRRL15998.Methods1. We optimized the condition of a genetic transformation system which mediated by Escherichia coli.2. To find the gene whiB4in Streptomyces roseosporus NRRL15998genome and clone the gene. Try to construct the whiB4knockout and overexpression plasmids.3. By the intergeneric conjugal transfer system,we got the whiB4knockout and overexpression mutants. The daptomycin production and the growth of wild type and mutants were measured through bioassay and measurement of cell dry weight. RT-qPCR was used to detect the transcription modulation for dptE.4. By scanning electron microscopy(SEM) and cultured on solid AS-I medium, we observed the phenotype of wild type and mutants.Results1.In this study, we optimized the conjugal transfer system. AS-1medium was the preferred medium for conjugation. The efficiencies of conjugation [10-6/10-5transconjugants/recipient colony forming units (CFU)] were obtained when spores of S.roseosporus were heat treated at50℃for10min prior to mixing with E.coli ET12567/pUZ8002. In addition, the best coverage time on antibiotics was16to18hours.2. whiB4was cloned from Streptomyces roseosporus NRRL15998genome. We successfully constructed whiB4knockout and overexpression vectors. Using the conjugal transfer system, we successfully got whiB4delation and overexpression mutants.3. Gene disruption of whiB4from Streptomyces roseosporus NRRL15998resulted in an approximately43%increase in daptomycin productivity. Overexpression of whiB4in the wild type strain led to a decrease of55%in daptomycin production. RT-qPCR analysis showed that the transcription level of dptE was relatively high in whiB4delation mutants.4. Deletion of whiB4blocked aerial mycelium sporulation. However it did not significantly affect cell growth in the fermentation.ConclusionThe whiB4gene of Streptomyces roseosporus NRRL15998appears to play a negative role in the control of daptomycin biosynthesis and is essential for sporulation.