Study On The Regulation Of DptR2in Daptomycin Biosynthesis In Streptomyces Roseosporus SW0702

Daptomycin, a novel cyclic lipopeptide antibiotic against Gram-positive bacteria, is produced by Streptomyces roseosporus. It has potent anti-bacterial activity against most Gram-positive pathogens in vitro, including methicillin-resistant S. aureus (MRSA) and other antibiotic resistant strains. Until now, daptomycin structure, physicochemical property, action mechanism, and biosynthetic mechanism have been reported. Furthermore, chemical, chemo-enzymetic and combinational biosynthetic methods have been explored to obtain daptomycin derivatives to search for new antibiotics with higher anti-microbial activities. Finally, some strategies to improve daptomycin yields are also introduced, including the ribosome engineering, random mutagenesis and introduction of extra copies of some dpt genes. However, little is understood about its production regulation at the transcriptional levels.Here we first established the genetic manipulation system for daptomycin industrial producer S. roseosporus SW0702. The results showed that R5medium was the most suitable medium for S. roseosporus SW0702to produce spores. Besides, this strain was sensitive to the antibiotics tested in this study, including Apra, Kana, Chl, etc. Furthermore, we established a system of conjugation which was suitable for S. roseosporus SW0702. Both mycelium and spores (heated at30℃for10min) could be used as the receptors, and MS medium was the most effective medium for conjugation. The medium is best covered with antibiotics and nalidixic acid after cultivated at30℃for16h. At last, the condition of HPLC to detect daptomycin production was confirmed and YEME medium containing4%glucose was the best fermentation medium for S. roseosporus SW0702to produce daptomycin.After the genetic manipulation system was established, we studied the the regulation of DptR2, a DeoR-type regulator in daptomycin biosynthesis for the first time. Here we reported that in S. roseosporus SW0702, dptR2, located close to daptomycin biosynthetic gene cluster, was required for daptomycin production, but not for the expression of daptomycin gene cluster, suggesting DptR2was not a pathway-specific regulator. Furthermore, EMSA and qRT-PCR analysis suggested that DptR2was positively auto-regulated by binding to its own promoter. Meanwhile, the binding sites on dptR2promoter were determined by a DNase I footprinting assay, and the essentiality of the inverted complementary sequences in the protected region for DptR2binding was assessed by mutation analysis. Moreover, we found that DptR2DBD could bind the inter-genic sequence between two genes, encoding Na+/galactose co-transporter and the gal operon, respectively. But the two genes could not explain the mechanism of DptR2in daptomycin biosynthesis regulation. At last, we predicted that DptR2may affect glucose metabolism, resulting in the lack of precursor supplication for daptomycin biosynthesis. Our results for the first time reported the regulation of daptomycin production at the transcriptional level in S. roseosporus.