title: Novel and tightly regulated resorcinol and cumate-inducible expression systems for Streptomyces and other actinobacteria.
abstract: Inducible expression is a versatile genetic tool for controlling gene transcription, determining gene functions and other uses. Herein, we describe our attempts to create several inducible systems based on a cumate or a resorcinol switch, a hammerhead ribozyme, the LacI repressor, and isopropyl β-d-thiogalactopyranoside (IPTG). We successfully developed a new cumate (p-isopropylbenzoic acid)-inducible gene switch in actinobacteria that is based on the CymR regulator, the operator sequence (cmt) from the Pseudomonas putida cumate degradation operon and P21 synthetic promoter. Resorcinol-inducible expression system is also functional and is composed of the RolR regulator and the PA3 promoter fused with the operator (rolO) from the Corynebacterium glutamicum resorcinol catabolic operon. Using the gusA (β-glucuronidase) gene as a reporter, we showed that the newly generated expression systems are tightly regulated and hyper-inducible. The activity of the uninduced promoters is negligible in both cases. Whereas the induction factor reaches 45 for Streptomyces albus in the case of cumate switch and 33 in the case of resorcinol toggle. The systems are also dose-dependent, which allows the modulation of gene expression even from a single promoter. In addition, the cumate system is versatile, given that it is functional in different actinomycetes. Finally, these systems are nontoxic and inexpensive, as these are characteristics of cumate and resorcinol, and they are easy to use because inducers are water-soluble and easily penetrate cells. Therefore, the P21-cmt-CymR and PA3-rolO-RolR systems are powerful tools for engineering actinobacteria.
authors.affiliation: Helmholtz Institute for Pharmaceutical Research Saarland, Saarland University Campus, Building C2 3, 66123, Saarbrücken, Germany.
authors.forename: Liliya
authors.initials: L
authors.lastname: Horbal
authors.forename: Victor
authors.initials: V
authors.lastname: Fedorenko
authors.forename: Andriy
authors.initials: A
authors.lastname: Luzhetskyy
mesh_terms: D039903:Actinobacteria / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D001565:Benzoates / Q000378:metabolism*; D048230:Corynebacterium glutamicum / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods; D005966:Glucuronidase / Q000032:analysis / Q000235:genetics; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D056968:Lac Repressors / Q000235:genetics / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D011401:Promoter Regions, Genetic; D016958:Pseudomonas putida / Q000235:genetics; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism; D012118:Resorcinols / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000235:genetics; D015533:Transcriptional Activation / Q000187:drug effects*
chemical_list: D001565:Benzoates; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D016337:RNA, Catalytic; D012118:Resorcinols; D014157:Transcription Factors; C427204:hammerhead ribozyme; D007544:Isopropyl Thiogalactoside; C055607:cumic acid; D005966:Glucuronidase; C031389:resorcinol