Purification, Identification And Cultural Optimization Of Phenazine Derivatives Produced By A Streptomyces Griseoluteus P510

Streptomyces griseoluteus P510is isolated from soil. In previous studies,griseoluteic acid （GA） has already been isolated as an extracellular productfrom the strain. However, we found other active ingredients from extracellularcompounds produced by S. griseoluteus P510, and also from the intracellularproducts. Therefore, we research other active components from the fermenta-tion products of S.griseoluteus P510. Besides, we study here roughly the me-dium on the P510product diversity as well.Fristly, based on the investigation of inhibition activity and other charac-teristics, such as color pigment and UV-Vis spectrum, of each fraction afterevery purification step, the active substance was separated and purified frombroth and mycelia. Through extraction by ethyl acetate at pH2.0,2.1g crudewas obtained from10L culture broth. In addition, extraction by acetone,79.3mg crude was obtained from the mycelial cake. Parts with antimicrobial activ-ity were obtained from crude after silica gel column chromatography, using astepwise gradient solvent system consisting of CHCl3and MeOH as eluationbuffer. Through reversed-phase C18high performance liquid chromatogram phy/HPLC （methanol-0.1%acetic acid water55:45, as the mobile phase）, in-hibitive substance A （3.2mg） and griseolutic acid （9.1mg）was further purified.The structures of the isolated compounds were elucidated by means of spec-troscopic methods including UV, ESI-MS、UPLC-MS-MS and¹H-NMR. Itwas confirmed to be phenazine-1-carboxylic acid （A）. Substance IX （6.5mg）was purified, which was identified to be1-hydroxyphenazine.Secondly, we research the inhibition activity of the above three products.The results have showed that the three subtrances have the good inhibition ac-tivity against several pathogens of plant disease （Fusarium graminearum、Phyricularia grisea、Pathogen of Stevia leaf spot disease、Alternaria solani、Fusarium oxysporium、 Sclerotinia sclerotiorum）. Especially, phenazine-1-carboxylic acid （MIC<1μg/mL）、 griseolutic acid （MIC=2μg/mL）and1-hydroxyphenazine （MIC=2μg/mL） showed the best inhibition activity inFusarium oxysporium.Finally, in order to investigate the influence of the medium on the synthe-sis of the above three products purified from S. griseoluteus P510, the experi-ments were carried out through changing the ingredient of medium and theculture time. The positive effect of glucose, yeast extract and MgCl₂·6H₂O onthe synthesis of GA and PCA was found in S. griseoluteus P510. Also, the pos-itive effect of glycerol, soybean meal and MgCl₂·6H₂O on the synthesis of1-OH-PHZ was found. Besides, the positive effect of carbon nitrogen ratio（1:1） on the synthesis of GA and1-OH-PHZ was proved in S. griseoluteus P510, while for PCA, the condition of carbon nitrogen ratio （4:1） was moreappropriate and the possible reason was dicussed in the text.As biocontrol agents, the phenazine antibiotics are known for their anti-microbial and antifungal activities. Streptomyces is one of main sources forphenazine derivatives. Fractionation of the extract from S. griseoluteus P510as directed by the antibacterial activity firstly delivered the known phenazineantibiotics phenazine-1-carboxylic acid,1-hydroxyphnazine. In addition, theknown compounds griseoluteic acid was isolated. We report here roughly themedium on the S. griseoluteus P510product diversity as well, which couldfurther confirm the generate-conversion mechanism of griseolutein and lay thefoundation for follow-up directed to improve yield of the target product.