Curated Optogenetic Publication Database

Abstract: 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.

Abstract: The LitR/CarH family comprises adenosyl B12-based photosensory transcriptional regulators that control light-inducible carotenoid production in nonphototrophic bacteria. In this study, we established a blue-green light-inducible hyperexpression system using LitR and its partner ECF-type sigma factor LitS in streptomycin-producing Streptomyces griseus NBRC 13350. The constructed multiple-copy number plasmid, pLit19, carried five genetic elements: pIJ101rep, the thiostrepton resistance gene, litR, litS, and σLitS-recognized light-inducible crtE promoter. Streptomyces griseus transformants harboring pLit19 exhibited a light-dependent hyper-production of intracellular reporter enzymes including catechol-2,3-dioxygenase and β-glucuronidase, extracellular secreted enzymes including laccase and transglutaminase, and secondary metabolites including melanin, flaviolin, and indigoidine. Cephamycin-producing Streptomyces sp. NBRC 13304, carrying an entire actinorhodin gene cluster, exhibited light-dependent actinorhodin production after the introduction of the pLit19 shuttle-type plasmid with the pathway-specific activator actII-ORF4. Insertion of sti fragment derived from Streptomyces phaeochromogenes pJV1 plasmid into pLit19 increased its light sensitivity, allowing gene expression under weak light irradiation. The two constructed Escherichia coli-Streptomyces shuttle-type pLit19 plasmids were found to have abilities similar to those of pLit19. We successfully established an optogenetically controlled hyperproduction system for S. griseus NBRC 13350 and Streptomyces sp. NBRC 13304.