Analysis Of The Main Secondary Metabolites, And Cloning And Functional Study Of Their Biosynthetic Gene Cluster From Streptomyces Vietnamensis

Streptomyces vietnamensis GIMV4.0001 is a newly streptomycete species isolated from tropical forest soil designated by our laboratory. Previous study revealed that this strain posseses the ability to produce a diffusible violet-blue pigment. In this study, an optimization for pigment production was carried out. The Gauze's synthetic No.1 medium was found to be the suitable medium for pigment production. This medium is also suitable for industrial production since its materials are relatively cheep. The optimized fermentation conditions are as follows: inoculation quantity 5%, culture temperature 30°C, rotation speed 180 r/min and culture time 7days. A further study was carried out on its secondary metabolites. The ethyl acetate extracts of the fermentation broth exhibited broad antibacterial activity against Gram-positive bacteria as well as some strains of Gram-negative bacteria to different extents. The crude extracts also showed excellent anticancer activity. Two blue pigments (B1, B2) in the extract content are the main active compounds as revealed by TLC-bioautography. By a combination of physical and chemical characterization, nuclear magnetic resonance and mass spectrometry, these two blue pigments were identified as granticin and granaticin B, respectively. Five more related compounds were also isolated and identified as quinizarin Zg, Zgg, dihydro-Zg, MM44785 and L-rhodinoside-deglycosylated MM44785, among which dihydro-Zg and L-rhodinoside-deglycosylated possess novel structures.The diversity of secondary metabolite pathways of S. vietnamensis was investigated. Except polyketide synthase (PKS) for granaticin, there are still at least three more PKS pathways possibly responsible for macrolides, angucyclines or spore pigments in S. vietnamensis. This provided guidance for deeper mining the bioactive secondary metabolites potentially produced by this strain.By a sequential cloning strategy, a 37480-long fragment containing the whole granaticin biosynthetic gene cluster (gra) was sequenced. All granaticin biosynthetic genes (orf9~orf34) were found as expected, and the gene arrangement was identical with that of S. violaceoruber Tü22 (AJ011500). When aligning the two gra cluster, about one hundred short insertions or deletions (hereinafter referred as indels) were observed throughout the whole cluster. The full gra cluster from S. vietnamensis was 1260 bp shorter than that of S. violaceoruber. This result suggested that insertion and deletion of tiny fragment might be one of the basic evolution mechanisms for streptomycete genomes.High overall homology of the two gra clusters clearly shows that they share a very recent ancestor, whereas phylogenetic analysis of the genus Streptomyces based on 16S rDNA, revealed a distant evolutionary relationship between S. vietnamensis and S. violaceoruber, and a scattered distribution of the granaticin producers within the genus. This provided compelling evidence that antibiotic biosynthetic gene cluster can be acquired horizontally. The remnants of the disabled flanking gene orf35 found in the gra cluster from S. vietnamensis not only indicated the full orf35 gene once resided in this host genome, making the current case a more convincing example of horizontal gene transfer, but also present a scenario how the antibiotic gene clusters evolved after horizontal gene transfer. Then the contemporary gra cluster held by S. vietnamensis can be interpreted as a combination of horizontal gene transfer and variable vertical transmission.The assay of resisitance level of S. vietnamensis to common antibiotics showed that apramycin, kanamycin, streptomycin, spectromycin and thiostrepton can be used as selection markers in genetic manipulation experiments.An efficient genetic manipulation system was established by intergeneric conjugation between E. coli and S. vietnamensis. Plasmids derived from the TA cloning vector pCR2.1, substituted for genomic library cosmid, were applied to S. vietnamensi for gene disruption. A granaticin-deficient mutant was constructed by replacement of the biosynthetic genes orf1, orf2, orf3 with the aac(3)-IV cassette, demonstrating the modified PCR targeting system was successfully applied to S. vietnamensis. To our knowledge, this is the first report of efficient genetic manipulation on the granaticin-producing strain and can pave a way to study the mechanisms of regulations and biosynthesis of granaticin in vivo.Functional studies were carried out on the biosynthetic gene orf20 by in vivo disruption and in vitro expression. E. coli carrying the recombinant plasmid pET28b-orf20 received an elevated resistance to paraquat, whereas S. vietnamensis orf20 disrupant showed no visible changes of resistance to paraquat. Unexpectedly, the granaticin production of the disrupant was improved for more than three folds. These data suggested that the orf20 gene can complement the soxR gene in E. coli, but is not involved in the regulation of anti-oxidative stress response in S. vietnamensis. Instead, it imposes a negative effect on granaticin production.Transcription of the granaticin-export gene orf15 was analysed by real time PCR. The transcription level of orf15 was abruptly elevated just before the granaticin production and then dropped down with speed in S. vietnamensis wild type, whereas the transcription level of orf15 was much more elevated at that stage and longer duration time of high transcription level was observed in the orf20 disrupant. This accounted for the promotion of granaticin production in the orf20 disrupant.