Studies On The Bioactive Agents From Halogenase Positive Marine-derived Streptomycetes

Marine actinomycetes are producers for many novel natural products with diverse functions which are associated with the special marine environments, and have been received increasing attention as important sources for drug discovery. Modification of natural products by halogenation is important for the activities of many compounds, and halogenase gene-based screening can speed the discovery of new halogenated antibiotics. Studies on the catalysis mechanisms of halogenase benefit modification of active compounds and improve activities of natural products.Four positive strains were identified by halogenase gene-based screening, which was numbered as L131, S077, S104and S187. The animo acid sequences of the halogenase of strain L131and S077showed the highest similiarity (71%) to the halogenae in vancomycin biosynthetic gene cluster, and inhibitory activities against Candida albicans and Fulvia fulva were revealed. Analysis of16S rRNA sequences showed that L131had the highest similarity (99%) to Streptomyces canescens. However, some morphological and physiochemical properties distinguished L131with the type strain of S. canescens. L131has white, ruffled colonies and soluble faint yellow pigments on Bennett agar, and cannot use arabinose and fructose as sole carbon sources. Strain L131showed better adaptation to marine environment, which can tolerate up to110g/L NaCl and high pH up to11. The optimized fermentation medium compositions (corn flour15.0g/L, soybean powder15.0g/L, yeast1.5g/L, CaCO30.5g/L, glycerol0.2%) was obtained, and better activity against C. albicans was obtained by72h cultivation. The active compounds against C. albicans are stable at pH5-10, with stronger polarity than ethyl acetate. The purification strategy was determined. To obtain crude antifungal components, culture broth was first absorbed by AB-8resin and purified by washing with40%methanol, followed by desorbing with100%methanol. The minimum inhibitory concentrations to both C. albicans and F. fulva of the crude extract were32.0μg/mL. The cell membrane and spore were injured and ROS was accumulated in tomato leaf pathogen F. fulva by the crude extract.The animo acid sequences of the halogenase of S187(which was named as Streptomyces xinghaiensis in subsequent studies) showed the highest similiarity (87%) to halogenae StaI and StaK in teicoplanin biosynthetic gene cluster. In order to obtain the soluble protein, SinH of S187was heterologous expressed in Escherichia coli and the conditions of soluble protein were studied. Expression vector pET28a-sinH was constructed and insoluble protein was obtained. Expression vector pHUIE-sinH with ubiquitin tag and intein was constructed and soluble protein SinH was obtained, however, low purity SinH was purified. Subsequently, coexpression system BL21/pET28a-sinH/pETcoco-pL1SL2was constructed and soluble protein SinH was obtained and purified by Ni-NTA affinity chromatography. At the same time, flavin reductase Fre was obtained using the transformants containing pET28a-fre and was purified uisng similar procedure. The enzyme activity test of halogense showed that L-Tyr, D-Tyr, L-Trp, D-Trp,4-hydroxy-D-phenylglycine were not substrates of SinH. It was thus proposed that SinH functions were in the later steps of biosynthesis.The function of halogenase SinH was further studied using gene disruption. The halogenase disruptant AsinH was obtained by λ-Red mediated homologous recombination. Unexpectedly, no difference of anticomplementary activity of knockout mutant AsinH was observed in4d-culture broth comparing with the wild type, whereas higher anticomplementary activity of AsinH was revealed at6d and8d. At the same time, the antibacterial activities of AsinH against Staphylo coccus aureus and Bacillus subtilis were also much higher than the wild type strain. It was deduced that novel dehalogenated analogues with stronger antimicrobial activity were obtained which was inconsistent with the previous reports of improved activity by halogenated compounds. Analysis of the HPLC profiling identified a different peak from that of the wild type, the peak at7.842min in wild type strain was lost in AsinH and a new peak of at7.356min appeared instead. The maximum absorption wavelength of the new peak is slightly shorter than the lost one, which we deduced that the new peak was corresponding to the dehalogenated compound.The studies presented in this work provide basis for further utilization of marine actinomycetes to produce antimicrobial drugs. Studies on the function of the halogenase SinH promote in-depth clarification of biosynthesis mechanisms of active compounds in S. xinghaiensis.