Double-reporter-guided targeted activation of the oxytetracycline silent gene cluster in Streptomyces rimosus M527.

In Streptomyces rimosus M527, the oxytetracycline (OTC) biosynthetic gene cluster is not expressed under laboratory conditions. In this study a reported-guided mutant selection (RGMS) procedure was used to activate the cluster. The double-reporter plasmid pAGT was constructed in which gusA encoding a ß-glucuronidase and tsr encoding a thiostrepton resistance methyltransferase were placed under the control of the native promoter of oxyA gene (PoxyA ). Plasmid pAGT was introduced and integrated into the chromosome of S. rimosus M527 by conjugation, yielding initial strain M527-pAGT. Subsequently, mutants of M527-pAGT were generated by using ribosome engineering technology. The mutants harboring activated OTC gene cluster were selected based on visual observation of GUS activity and thiostrepton resistance. Finally, mutant M527-pAGT-R7 was selected producing OTC in a concentration of 235.2 mg/L. In this mutant transcriptional levels of oxysr genes especial oxyAsr gene were increased compared to wild-type strain S. rimosus M527. The mutant M527-pAGT-R7 showed antagonistic activities against Gram-negative and Gram-positive strains. All data indicate that the OTC gene cluster was successfully activated using the RGMS method.

Transposon-based identification of genes involved in the rimocidin biosynthesis in Streptomyces rimosus M527.

The transposon mutagenesis strategy has been employed to generate random insertion mutants and analyze the correlation between genes and secondary metabolites in the genus Streptomyces. In this study, our primary objective was to identify an unknown gene involved in rimocidin biosynthesis and elucidate its role in rimocidin production in Streptomyces rimosus M527. To achieve this, we established a random mutant library of S. rimosus M527 using a Tn5 transposon-mediated random mutagenesis strategy. Among the 137 isolated mutants, M527-G10 and M527-W5 exhibited the most significant variations in antagonistic activity against the plant pathogenic fungus Fusarium oxysporum f. sp. cucumerinum. Specifically, M527-G10 displayed a 72.93% reduction, while M527-W5 showed a 49.8% increase in rimocidin production compared to the wild-type (WT) strain S. rimosus M527. Subsequently, we employed a plasmid rescue strategy to identify the insertion loci of the transposon in the genomes of mutants M527-G10 and M527-W5, revealing a response regulator transcription factor (rrt) and a hypothetical protein (hyp), respectively. The roles of rrt and hyp in rimocidin biosynthesis were determined through gene deletion, overexpression in the WT strain, and complemented expression in the transposon mutants. Notably, the gene-deletion mutants M527-ΔRRT and M527-ΔHYP exhibited similar behavior in rimocidin production compared to the corresponding transposon mutants M527-G10 and M527-W5, suggesting that transposon insertions in genes rrt and hyp led to alterations in rimocidin production. Furthermore, both gene deletion and overexpression of rrt and hyp had no discernible effects on cell growth. These results reveal that genes rrt and hyp have positive and negative impacts on rimocidin production in S. rimosus M527, respectively.

Development and optimization of an intergeneric conjugation system and analysis of promoter activity in Streptomyces rimosus M527.

An efficient genetic transformation system and suitable promoters are essential prerequisites for gene expression studies and genetic engineering in streptomycetes. In this study, firstly, a genetic transformation system based on intergeneric conjugation was developed in Streptomyces rimosus M527, a bacterial strain which exhibits strong antagonistic activity against a broad range of plant-pathogenic fungi. Some experimental parameters involved in this procedure were optimized, including the conjugative media, ratio of donor to recipient, heat shock temperature, and incubation time of mixed culture. Under the optimal conditions, a maximal conjugation frequency of 3.05×10-5 per recipient was obtained. Subsequently, based on the above developed and optimized transformation system, the synthetic promoters SPL-21 and SPL-57, a native promoter potrB, and a constitutive promoter permE* commonly used for gene expression in streptomycetes were selected and their activity was analyzed using gusA as a reporter gene in S. rimosus M527. Among the four tested promoters, SPL-21 exhibited the strongest expression activity and gave rise to a 2.2-fold increase in ß-glucuronidase (GUS) activity compared with the control promoter permE*. Promoter SPL-57 showed activity comparable to that of permE*. Promoter potrB, which showed the lowest activity, showed a 50% decrease in GUS activity compared with the control permE*. The transformation system developed in this study and the tested promotors provide a basis for the further modification of S. rimosus M527.