Study Of The Phenazine Biosynthetic Gene Cluster In Streptomyces Diastaticus W2

Streptomyces diastaticus W2 and W4 are endophytes isolated from the herb Artemisia annus,which produce two phenazine compounds,izumiphenazine C and diastaphenazine.Diastaphenazine is a new dimeric phenazine with cytotoxic and anti-Staphylococcus aureus activities.So far,the biosynthetic mechanism of dimeric phenazines remains a mystery.In this study,morphological observation,antibiotic sensitivity and antimicrobial spectrum of the diastaphenazine producers S.diastaticus W2 and W4 were conducted.Further homologous analysis of genome sequence of two strains suggested the close relationship between them.The sequence of an 8,074,405 bp chromosome and three large linear plasmids were acquired after the whole-genome sequencing of S.diastaticus W2 on the PacBio RSII platform.Bioinformatics analyses discovered 7993 genes in the chromosome,102 of which were supposed to be related to the biosynthesis of terpene and polyketide and 65 of which were considered to be responsible for other secondary metabolism.There are 24 putative gene clusters for secondary metabolite biosynthesis in the genome of S.diastaticus W2.The cluster9 for bacteriocin biosynthesis,the cluster12 for siderophore biosynthesis and the cluster16 for terpene biosynthesis showing no similarity with known gene clusters in the database,are likely to produce new metabolites.Only one gene cluster cluster17 were found to be related to the phenazine biosynthesis.A cosmid genomic library of S.diastaticus W2 was constructed by using the vector pMSB152 B.Based on the sequences of 15 secondary metabolite biosynthetic gene clusters less than 40 kb,the specific primers were designed for PCR-based screening of the S.diastaticus W2 genomic library and 67 positive clones were selected out.The positive clones carrying the cluster9 for bacteriocin biosynthesis were introduced into Streptomyces albus for heterologous expression by conjugation.The resulting heterologous expression strains S.albus::21C7 and S.albus::22A11 showed antibacterial activity against Bacillus subtilis,and the absorption peak of the putative correspondingmetabolite was tracked by HPLC analysis of the fermentation products of heterologous expression strains.The positive clones carrying the cluster12 for siderophore biosynthesis were introduced into S.albus and the absorption peaks of the corresponding metabolite were found as well.5 positive clones containing the phenazine biosynthetic gene cluster were transferred into S.albus for heterologous expression.TLC analysis disclosed that two heterologous expression strains S.albus::23C5 and S.albus::20G10could produce extra metabolites with specific ultraviolet absorption.Further HPLC and LC-MS analyses verified that these two strains could produce phenazine-1-carboxylic acid(PCA),izumiphenazine C and diastaphenazine,which indicated cluster 17 were responsible for the biosynthesis of the dimeric phenazine diastaphenazine.