Single copy optogenetic system for Streptomyces

LitR is a blue-green light-sensing transcriptional regulator that uses coenzyme B₁₂ as a chromophore. In this study, we developed a genome-integrative light-inducible expression (iLiEX) system in Streptomyces griseus NBRC 13350, a Gram-positive bacterium that produces streptomycin. The system incorporates LitR, transcriptional amplification module T7 RNA polymerase, and a serine integrase. Using iLiEX, we achieved light-dependent overproduction of catechol-2,3-dioxygenase and β-glucuronidase (GUS) at levels comparable to those from a high-copy plasmid. Notably, GUS activity was 39-fold higher than with the constitutively strong ermE* promoter. The iLiEX system was also functional in S. coelicolor, S. lividans, S. albus J1074, and S. avermitilis. We improved iLiEX in two key ways: by optimizing the ribosome-binding site of T7 RNA polymerase to increase expression, and by introducing the T7 lysozyme gene to reduce leaky transcription. The system's versatility was improved by shortening the T7 promoter from 89 to 44 bp. For simple visualization on agar plates, light-dependent overexpression of fluorescent proteins, a chromogenic protein, and a brown pigment synthesis enzyme was demonstrated. High-level production of secreted enzymes, including laccase and transglutaminase, was also confirmed. Overall, we developed a single-copy light-inducible overexpression system with broad functionality across multiple Streptomyces species.