Lon protease reprograms cellular physiology of Streptomyces coelicolor resulting in enhance antibiotic production

The genus Streptomyces is widely recognized as a rich source of natural compounds, including antibiotics, immunosuppressants, and herbicides. Synthesis of secondary metabolites is initiated by cellular differentiation and is a complex process regulated by intracellular and extracellular signals, as well as numerous regulatory proteins. ATP-dependent Lon protease plays a key role in cellular proteostasis and stress adaptation. Overexpression of this protease has been shown to increase the production of actinorhodin (ACT) and undecylprodigiosin (RED) in Streptomyces coelicolor . However, the systems-level mechanisms underlying this phenotype remain unclear. In this study, we employed a multifaceted approach encompassing whole-genome sequencing (WGS), transcriptomics, and high-resolution imaging to analyze how Lon reprograms the cellular physiology of the hyper-antibiotic-producer recombinant strain, Sco-pRA lon . WGS revealed that the pRA lon recombinant vector, which has a ΦC31 int/attP site, integrates not only at the canonical attB site within SCO3798 of Streptomyces species, but also at a previously uncharacterized attB -like locus in SCO3793. Transcriptome profiling at the 24 th and 72 nd hours of fermentation revealed extensive Lon-dependent remodeling, particularly in key functional categories such as secondary metabolism, stress response, primary metabolism, and morphological differentiation. Confocal microscopy confirmed that lon overexpression shifts programmed cell death dynamics, triggers earlier MII (antibiotic-producing mycelia) formation, and supports sustained viability and homogeneous pellet morphology, even in the later stages of fermentation. These structural features coincide with elevated antibiotic titers. Together, these results position Lon as a systems-level regulator, which couples proteostasis to metabolic flux, nutrient signaling, developmental progression, and secondary metabolism. By mapping Lon-dependent regulatory networks, linking transcriptional signatures to quantitative morphological phenotypes, and identifying a new chromosomal attB -like integration site, this work provides a valuable framework for protease-guided strain engineering and highlights Lon as a promising lever for rational improvement of antibiotic production in Streptomyces . To the best of our knowledge, this is the first comprehensive study investigating the systems-level effects of overexpression of Lon protease gene in Streptomyces .