Preliminary Study Of The Antimicrobial Drug Screening Model Targeting Fabi

Enoyl-ACP reductase(FabI) is an essential enzyme in fatty acid synthesis pathway for most bacteria. Its a kind of NADH dependent single-function enzyme, does not exist in the eukaryotic cells. So it has been a target in the antibacterial research. This paper, based on the antisense RNA technology, constructed the engineered bacterium(E.coli/pHNF) that can suppress the FabI express effectively.Making use of the engineered bacteria, we established the supper-sensitive whole-cell model for screening the FabI-target directed inhibitors from fermentation samples. The effects of those antisense silence on the growth of E.coli/pHNF were investigated, Triclosan and ampicillin were used as the positive and negative control respectively, and the inhibitory activity was detected by the ELIASA. The optimized conditions of this model are as follows: concentration of IPTG is 37.5μmol/L, concentrations of triclosan as positive control are 25, 37.5, 50μg/mL, concentrations of ampicillin as negative control are 0.5, 2.5, 5μg/mL respectively, and the cultural time is 16 hours.Using this model,we screened the 5847 fermentation samples from endophytic fungus. The positive samples were 559, with the rate of 9.7%. Strain HCCB06314 with higher activity was further studied. According to the results of morphology, cultural characteristics, and ITS sequences, the strain HCCB06314 was identified as Fusarium sp.By means of silica gel chromatography and HPLC preparation, two metabolites were obtained from HCCB06314 sample. By MS and NMR analysis, the compounds HCCB06314-1 and HCCB06314-2 were identified as enniatin I and H respectively. By means of UPLC-MS, another seven enniatins could be found in 6314-10 part, and one of them probably was a new compound by searching the scinfinder according to its molecular weight.