, Strains I03a-09005 Separation And Purification, Structural Identification Of Its Biological Activity Of Two Drug Screening, Targeting Mycobacterium Tuberculosis Cell Wall,

My work focus on two parts: First is the the isolation and purification the fermentation products of anti-tuberculosis microorganism I03A-09005. Macroporous rosin adsorption process, silica gel column chromatography and HPLC were used to prepare the pure samples from its fermentation products. An active compound, named 9005C, was purified, and the MIC to H37Rv is 0.25μg/ml～0.5μg/ml.The structure was identified by ESI-MS, H1-NMR, C-NMR, HMBC, HMQC and Q-COSY. The compound was actinomycin D.A new target of actinomycin D was found to be pantothenate synthase, which was confirmed by high-throughput screening, fluorescence quenching Spectra and defined by CD spectroscopy. After using virtual docking software to find the active group, we choose the groups with H-bond binding to the residues as screening factor to search the compound library. The screening result supply us two compounds with high inhibition and one with anti-mycobacterial activity.Second, When parts of cell wall are disrupted, the bacteria can not maintain normal osmotic pressure, which cause it low growth rate, while add sorbitol to culture will complement this effect. Based on this, we used C. glutamicum as the test strain to construct the model. Using this high-throughput screening model, we can obtain all inhibitors targeting the biosynthesis and assembly of cell wall. At the same time, we will construct 2 mutant which deleteΔaftA andΔaftB respectively, and overexpress some key enzymes to confirm the specific targets of positive samples.Then we clone and overexpress an enzyme IspD which is a key enzyme in cell wall biosynthesis. We use it to construct a screening model to screen and confirm the target of some positive hits.