Isolation&Purification Of Tetraene Macrolide Compounds And Antifungal Activity Against Phytopathogenic Fungi

Our project group isolated a strain of actinomycetes from the soil collected from Tianmu Mountain in Zhejiang Province,which has a high antibacterial activity against various plant pathogenic fungi;and it is identified as Streptomyces diastatochromogenes 1628 diastatochromogenes 1628.It was reported that the N-butanol extraction of the fermentation broth of this strain contains several tetraene macrolide compounds.In this study,we isolated and purified the N-butanol extraction of fermentation broth,and obtained three tetraene macrolide compounds;we examined their antibacterial activities and genetically engineered Streptomycetaceae diastatochromogenes 1628 by Ribosome Engineering to promote its productivity of antibiotics.The N-butanol extraction of fermentation fluid of Streptomycetaceae diastatochromogenes 1628 was purified by one-time AB-8 chromatography column filled with macroporous resin,two-time ODS chromatography column,and analysed by TLC and HPLC;three tetraene compounds were obtained at the end of the processes,numbered as ODS5-8,ODS5-7-2,and ODS5-7-4.Analysed by MS,NMR and compared with references,ODS5-8 is identified as Tetramycin A,ODS5-7-4 is proved as Tetrin B,and ODS5-7-2 is found that it is a new type of tetraene antibiotics,so it is named as Tetramycin P.A detective method,using HPLC,to study tetraene compounds was established in this project: mixture of compounds was purified by the Waters Sun Fire C18 chromatography column(250 mm × 4.6 mm,5 ?m),and eluted with methanol and water as mobile phase A and B respectively in gradient;flow rate: 1 ml/min;detection wavelength: 304 nm;injection volume: 5 ?L;Column temperature: 30 ?.HPLC standard curves of Tetramycin A,Tetramycin P,and Tetrin B were drawn within the linear concentration range: 25 ~ 500 mg / L.R2 were 0.9914,0.9862,and 0.9963,which stands for a good linear relation;and the average recoveries were 100.83%,100.15%,and 100.51%.RSD(%)were 4.57,4.12,and 7.89,that refers to the method is accurate and reliable.In summary,this quantitive method is simple and repeatable.We investigated the antibacterial activities of Tetramycin A and Tetramycin P to five common plant pathogenic fungi,including Rhizoctorzia solani,Rhizoctonia solani Kühn,Fusarium oxysporum f.sp.cucumerinm,Botrytis cinerea Pers,Alternaria solani,based on mycelium growth rate method and germination rate method.The results demonstrated that both Tetramycin A and Tetramycin P shows an apparent inhibition effect on mycelial growth of five plant pathogenic fungi.The EC50 of Tetramycin A inhibiting the mycelial growth of these five fungi are 3.93,2.13,3.27,5.36,5.99 mg/L respectively,and average inhibition rate reached 97.7% at 25 mg/L.The EC50 of Tetramycin P are 3.83,3.29,3.31,4.96,and 5.65 mg/L respectively;and average inhibition rate reached 97.3% at 30 mg/L.Both Tetramycin A and P can apparently decrease the spore germination rate of Fusarium oxysporum f.sp.cucumerinm and Botrytis cinerea Pers.Tetramycin A shows a better inhibition effect on spore germination of Botrytis cinerea Pers.when its concentration is 125 mg/L(spore germination rate is 0%);a weaker inhibition impact on Fusarium oxysporum f.sp.cucumerinum(spore germination rate is 4% but it is still significantly lower than that of control group).With the concentration of 75 mg/L,Tetramycin P shows a good inhibition of spore germination of Botrytis cinerea Pers.(spore germination rate is 0%)and Fusarium oxysporum f.sp.cucumerinum(5%),which is apparently lower than that of controls.The experiment results demonstrated that both Tetramycin A and Tetramycin P own an excellent antibacterial activity against these five common plant pathogenic fungi.S.diastatochromogenes 1628 can automatically synthesize four different antibiotics: Toyocamycin,Tetramycin A,Tetramycin P and Tetrin B.Six mutant strains of S.diastatochromogenes 1628,SD10,SD88,SD99,SD143,SD160,SD189,and SD218 were screened out through Ribosome Engineering technique,showing resistance to Streptomycin,Rifampin,Paromomycin,Erythromycin,Gentamicin,and Tetracycline respectively.The concentration of Toyocamycin and Tetramycin A produced by mutant strain SD160 induced by Erythromycin reached 678 and 1005 mg/L – 3.8 and 5.4 times higher than those of wild 1628 respectively.The production of Tetramycin P and Tetrin B reached 1015 and 583 mg/L by induced SD99 using Paromomycin,and those are 3.8 and 5.1 times higher than the production of wild 1628.All the results illustrated that Ribosome Engineering can effectively promote four antibiotics' production of S.diastatochromogenes 1628.Compared with the wild type,the productions of mutants increased three to five times,which laid a good foundation for industrial-scale production.