Isolation And Purification Of Phenazine Derivatives Produced By A Streptomyces P510 Isolated From Soil

As biocontrol agents, the phenazine antibiotics are known for their antimicrobial and antifungal activities. Streptomyces is one of main sources for phenazine derivatives.The 16S rDNA sequence analysis and culture characteristic studies indicate that Streptomyces P510, which was isolated from the rhizosphere soil, belongs to Streptomyces griseoluteus, which can produce phenazine derivatives.In this research, systemic study was done on P510 strain and its culture broth to identify efficiently inhibitive phenazine compounds, including biosynthetic gene analysis and inhibitive activity investigation.Analogical sequences (3 kb in total) of phenazine biosynthetic gene ephzCDE in Streptomyces DSM1042 and phzCDE in Pseudomonas PAO1 were found from S. griseoluteus P510. Compared to the phenazine biosynthetic gene in Streptomyces and Pseudomonas, the similarity was 71-74%. The culture broth showed high inhibitory activity against bacteria (B. Subtilis and B.Aureus) as well as several pathogens of plant disease (Rhizoctonia solani, Pythiumultimum, Sclerotinia sclerotiorum and Pathogen of Stevia Leaf Spot Disease). Phenazine biosynthesis gene found in S. griseoluteus P510 as well as the presence of secondary metabolites with antimicrobial activity in cluture broth demonstrate the possibility production of phenazine derivatives by S. griseoluteus P510.Based on the investigation of inhibition activity against B.Subtilis and other characteristics, such as color pigment and UV-Vis spectrum, of each fraction after every purification step, the active substance was separated and purified from broth. Through extraction by ethyl acetate at pH 2.0, 0.5g crude was obtained from 10 L culture broth. Two partsⅡ(116.2mg) andⅤ(71.4mg) with antimicrobial activity were obtained from crude after silica gel column chromatography, using ethyl acetate as eluation buffer. Through reversed-phase C18 high performance liquid chromatography/HPLC (50% methanol as the mobile phase), inhibitive substance C (8 mg) was further purified from partⅤ, with purity by area normalization method is 95.92%. It was confirmed to be griseoluteic acid,a derivative of phenazine-1-carbonate. Substance B (13.7mg) was purified from partⅡ, which was identified to be 4-hydroxide-phenyl-formaldehyde. Another inhibitve substance A (2mg) is separated from partⅡ. However, the structure is still unknown.In order to improve the production of griseoluteic acid (substance C), the optimization of experiments were carried out through changing the ingredient of medium and fermentation conditions. The optimized medium ingredients contains glucose 2g/L, yeast extract 8g/L, 10g glass beads in 50 mL culture broth. Under optimized experimental conditions, the biomass increased 1.18 times, and the productivity of griseoluteic acid increased 11.64 times, from 3.97±0.39mg/L to 49±4.50mg/L after 96 hour fermentation.The discovery of phenazine derivatives from Streptomyces is of significant importance for the phenazine compounds research and its pathway engineering. This research will provide fundamental knowleges for the further study of the phenazine compounds and its pathway engineering in Streptomyces P510.