Biosynthesis And Bioactivity Of Mildiomycin Analogues As New Agrochemicals

Mildiomycin (MIL), a novel nucleoside antibiotic with a specific and strong inhibitory activity against powdery mildews, is the secondary metabolite of Streptoverticillium rimofaciens. The purpose of this work is to produce various mildomycin analogues by adding different precursors, such as structural analogues of hydroxymethyl cytosine, into the fermentation broth, from them, mildiomycin analogues with higher bioactivity may be identified. Two of them: Mildiomycin 5-flucytosine-substituted analogue (MIL-F) and mildiomycin cytosine-substituted analogue (MIL-C) were studied in detail, including the optimization of fermentation conditions and separation process, identification of molecular structure, evaluation of bioactivity and toxicity.1. After the addition of 5-flucytosin into the fermentation process of Streptoverticilliumrimofaciens, 5-flucytosine-substituted analogue (MIL-F) was obtained. The MIL-F yield reached 0.6 g/l after the optimization of fermentation conditions. The separation and purification process of MIL-F from fermentation broth was developed, and the purity of MIL-F product was above 95%. The molecular structure of MIL-F was examined by Infrared, MNR and MS etc. The bioactivity and toxicity of MIL-F were evaluated, and the results indicated that MIL-F should be a new biopesticide with higher efficiency, lower toxicity and less stimulation.2. After the addition of cytosin into the fermentation broth of Streptoverticillium rimofaciens,cytosine-substituted analogue (MIL-F) was obtained. It was found that by controlling the addition of cytosine, the biosynthesis of mildiomycin was inhibited and it is possible that the MIL-C was the only product. The yield of MIL-C was increased from 350 mg/l to 1336.5 mg/1 by the optimization of fermentation conditions with one-factor-in-one experiment, Plackett-Burman design and response surface method. Based on the ion-exchange characteristic of MIL-C from fermentation broth by DK110 weak cationic exchange resin, a separation technology was developed and the crude product of MIL-C with 70% purity was obtained. After decolor and further purification by a CM650 ion-exchange gel column, the purity of the final MIL-C product was higher than 95%. The molecular structure of MIL-C was also examined by UVS, NMR and MS.3. The bioactivity of MIL-C was investigated in vitro and in vivo including the greenhousetests and field tests in two different places in one year. The results showed that MIL-C was remarkable efficiency against the powdery mildew of cucumber and pumpkin, and the potency of drug can be kept stable. MIL-C was safe to cucumber and pumpkin without any negative effect on their flower and fruit growth, and it was also environmental friendly. The safety evaluation of MIL-Cshowed that it was regard as a safe agro-antibiotic with low toxicity and less motivation by the evaluation of toxicological tests.4. Some important parameters of MIL, MIL-C and MIL-F was analyzed and compared by the software of chemoffice. And the main pharmacophore was guessed as the guanazole group in the structure of MIL analogue, which means that destroying this group will make the MIL analogues deactivation.In this work, two kinds of mildiomycin analogues, MIL-F and MIL-C, were produced. The yields were improved greatly because of the optimization of both fermentation and separation processes. The bioactivity of these two MIL-analogues were higher than that of mildiomycin against powdery mildew, which indicated that it is possible to develop MIL-F and MIL-C into new biopesticides. The study in structure-function relation in mildiomycin analogues should be beneficial in discovering new biopesticides.