Analisi dell’attivazione di un sistema RiPPs ibrido in Streptomyces griseus S4-7 mediante la sovra espressione dei regolatori pathway-specifici

Streptomyces griseus S4-7 is the representative strain producing antifungal metabolites in Fusarium-suppressive soils. Among the biosynthetic gene clusters (BGCs) proven to be related to the ecological role of S4-7, one cluster encoding a hybrid ribosomally synthesized and post-translationally modified peptides (RiPPs) system was selected for further investigation. However, despite several cultivation and heterologous expression experiments, the peptides connected to this cluster were never detected, leaving the question of their structure and biosynthesis unresolved. In this study, we aimed to activate this pathway by overexpressing the cluster-situated regulators. For this purpose, we used the replicative plasmid pGM1202 to clone the predicted regulator genes, both individually and in combination, under the control of the inducible promoter tipA. Obtained recombinant plasmids were then transferred to S. griseus S4-7 via intergeneric conjugation. The engineered strains were cultivated using different media, and crude extracts were prepared and subjected to agar diffusion test. Samples with an increased activity against Bacillus megaterium (in comparison to the strain harboring the empty vector) were then selected for HPLC-MS/MS analysis. Overexpression of a SARP and a LacI transcriptional regulator was accomplished. However, although data provided by HPLC-MS/MS highlighted the presence of promising masses, the ions resulted in poor fragmentation, allowing the analysis of only a few MS2 spectra. These results did not allow further investigations that would confirm or deny the production of the cryptic RiPPs. Interestingly, it was observed that overexpression of these two regulators affected the secondary metabolism of the strain beyond its specific pathway. Concerning regulation within the cluster, results suggested that the LacI serves as a repressor, while the SARP regulator is likely to activate the biosynthetic machinery. Furthermore, our results support a new hypothesis where the SARP may be involved in a more complex interposition in the regulatory network, and perhaps a crucial mutual action with the neighboring LuxR-type regulator. Finally, co-expression of both SARP and LuxR-type regulators would lead to a better understanding of the regulation of the hybrid RiPPs system from S. griseus S4-7. ​