The Preparation Of New Ramoplanin Derivatives And The Preliminary Research On The Biosynthesis Halogenase Gene

With the development of vancomycin resistance, ramoplanin became one of the promising drug-resistant bacteria drug candidates in phase â…¢ clinical studies. It was tempting to get better antimicrobial activity ramoplanin analogues through ramoplanin structure modification. Three ramoplanin genetic engineered strains were preserved in our laboratory, but the fermentation production was very low, hindering the follow-up studies.Three strains were screening: fermentation titer 489 ug/mL production of ramoplanin aglycon strain named SIPI-A.dorf29-2016, the fermentation titer 368 ug/mL production of deschlororamoplanin strain named SIPI-A.dorf20-2016 and the fermentation titer 476 ug/mL production of deschlororamoplanin aglycon strain named SIPI-A.dorf20-dorf29-2016. The ramoplanin fermentation titer of the three screened strains was nearly ten-fold higher than that of their start strains. The final fermentation unit increased by twice after a series optimization of fermentation medium and fermentation conditions.The preliminary treatment method of ramoplanin aglycon fermentation broth should be established at first: the effect of the variety of organic solvent, the concentration of the organic solvent, the volume of organic solvent, extraction time, extraction, pH, leaching times and ultrasonic impact on fermentation broth were studied respectively. Then the investigation on macroporous adsorption and desorption ability of resin NM100 and SP825 and pigment removal,it developed for ramoplanin aglycon. Then crude product separation technology of ramoplanin aglycon should be established: the adsorption capacity of macroporous resin NM100, separating capacity of SP825, removal capacity of. It was a good way to remove pigment, enrichment purity of ramoplanin aglycon was more than 90%, the overall yield of separation was around 55%. Low pressure liquid chromatography preparation conditions and high pressure liquid chromatography preparation conditions were optimized, a refined separation process of the ramoplanin aglycon was explored, which the liquid purity was more than 98%. The whole process is suitable for separation and purification to obtain deschlororamoplanin and deschlororamoplanin ramoplanin aglycon.The results of antibacterial activity test showed that there was no difference in bacteriostatic activity between the three kinds of ramoplanin analogues. The lack of chlorinecaused no impact for its activity. In order to further understand the ramoplanin biosynthesis mechanism, ramoplanin halogenase was heterologous expression in E. Coli BL21.After a series of the induced condition optimization, the soluble protein can be obtained in TransB(DE3) host after add membrane solubilizer benzyl alcohol before IPTG induced expression. Bioinformatics research showed that halogenase catalytic processes required the participation of FADH2, so the FAD reductase were constructed and expressed. With the optimization of induced conditions in E. ColiBL21-fre strain, abundant soluble protein can be obtained. The enzyme activity of crude enzyme was 6U/ml. This study laid a solid foundation for exploring for the diversity of new halogenated glycopeptide antibiotics.