The Regulatory Mechanism Invovled In The Biosynthesis Of Muraymycin

Muraymycin, a potent translocase I inhibitor, of which the cellular targetis MraY in the peptide biosynthesis，that contribute to the antibioticsexcellent antimicrobial activity against G-positive bacteria. The antibioticsown their a narrow antibacterial spectrum, a particular target site and theunique mechanism of action, until now there wasn’t MraY inhibitors beingapplied in clinical, and few cross resistance would be produced when beingused with other types of antibiotics. It is the best choice for new, low toxity,and narrow spectrum of drugs with the uridyl peptide antibiotics, e.g.muraymycin. In2001, muraymycin was discovered, until in2011thebiosynthetic gene cluster of muraymycin was cloned. There are lots of othersimilar antibiotics, pacidamycin, caprazamycin, sansanmycin andnapsamycin.In the task, researches focus on the regulatory functions of Mur34, Mur33and Mur32invovled in the biosynthesis of muraymycin from different levels. The mutations of mur34and mur32were constructed by PCR-targeting firstly,and the mur34mutated18F3was successfully heterologous expressed in ahost of S. lividans TK24. The flanking genes on muraymycin gene cluster ofpJTU5030were mutated by PCR-targeting, and the minimal biosyntheticgene cluster of muraymycin was confined to a large genome fragment frommur12to mur33by heterologous expression. The mutation of mur34ongenomic DNA of S. sp. NRRL30471led to improvements in muraymycinproduction, growth inhibition activity on Bacillus subtilis and30-foldtranscription of muraymycin gene cluster. The complementation of mur34restored the genetic characteristics to the wild type producing strain. Resultsshowed that mur33and mur32making up one operon, the trancription startpoint (TSP) of mur33-mur32locates at the58bp upstream of the translationinitiation code (TIC) detected by5’RACE. The his-tagged Mur34wasexpressed in a host of BL21(DE3) and prepared by Ni-affinitychromatography. By EMSA and DNase I footprinting assay, it’s found thatMur34specifically bound to the regions between the TIC and TSP of mur33,implied that Mur34inhibit the transcription of mur33-mur32by preventingthe movement of RNA polymerase complex. Results of catecholdioxygenase activity assays confirmed the negative regulation of Mur34tomur33-mur32, and showed a more important function of-10than-35region for the promoter. Besides, results of the inhibition activity to B. subtilis,LC-MS and real-time PCR assays showed that the mur32mutation almostcouldn’t influence the fermentative characteristic of the producing strain.Mur33is a homologous protein of a positive regulator SsaA in siliconanalysis, especially at C-terminal. The homologous arms of mur33obainedby high fidelity PCR were ligated with pOJ446to construct mur33inframe-deleted mutation vector pJTU5020, which was conjugated into S. sp.NRRL30471to construct mur33mutation on chromosome DNA. The mur33mutation abolished the ability of muraymycin production and inhibitionactivity on B. subtilis of S. sp. NRRL30471, and made a larger decrease inthe transcription of muraymycin gene cluster. The complementation of mur33restored the genetic characteristics to the wild type producing strain.By utilizing autoinduction medium ZYM5052and NusA-tagged vector,the soluble Mur33was obtained successfully. Two strict negative controlsand the non-special poly dI-dC and competetive probes were applied inEMSAs for the preciseness, and a more special binding activity of Mur33tothe promoter region of mur12-mur31on muraymycin gene cluster wasdiscovered, which implied that Mur33is an positive regulator formuraymycin biosynthesis, also based on the information that all structuralgenes are under the control of mur12-mur31promoter. In summary, the regulatory network of muraymycin biosynthesis waspreliminary studied, Mur34negatively regulates the biosynthesis ofmuraymycin by inhibiting the transctription of mur33, which encodes apositive regulator. The discussions open the door to regulatory networkresearches on nucleoside antibiotics biosynthesis, especially the uridylpeptide antibiotics. The researches point out the way to rational improvementon muraymycin production and the directional engineering.