2026/01/21 by Victor Gigante Pereira, Paloma Rezende Corrêa, Rodrigo Martins Barros +4 · 1 voice
Biochemistry, Genetics and Molecular Biology · Medicine · #Bacterial Genetics and Biotechnology #RNA and protein synthesis mechanisms #Tuberculosis Research and Epidemiology
paper · pdf · doi:10.1021/acssynbio.5c00966
openalex publication_date 2026/01/21 · openalex created_date 2026/01/22 · openalex updated_date 2026/07/02
High Resolution Image Download MS PowerPoint Slide Recombinant protein expression in mycobacteria faces two major challenges: limited regulatory tools for inducible expression and inefficient secretion of heterologous products. In this study, we developed plasmid-based systems that enable translationally gated secretion in Mycobacterium smegmatis, coupling riboswitch-mediated translational control with efficient extracellular export. The platform integrates the M. tuberculosis antigen 85A promoter and signal peptide for constitutive secretion combined with synthetic riboswitches for inducible translational regulation. We tested two theophylline-responsive riboswitches (riboE and riboE+) and a temperature-sensitive variant (riboU9) by using mCherry as a reporter. Fluorescence assays, RT-PCR, and Western blotting confirmed efficient secretion and strict translational control. The theophylline-inducible systems exhibited a dose-dependent response with maximal expression at 2 mM inducer, while the riboU9 construct showed a clean ON/OFF phenotype triggered by temperature shift. In all cases, transcripts were detected irrespective of induction, confirming regulation at the translational rather than transcriptional level. Secretion was highly efficient, with 10–20 fold higher protein levels in extracellular versus intracellular fractions. Induction during early- and mid-log phases yielded maximal protein, whereas late-log induction reduced output by ∼50%. Together, these results define translationally gated secretion as a new control layer in mycobacterial protein production. This modular platform expands the genetic toolkit available for Mycobacterium research, providing new opportunities for the study of antigens and virulence factors from slow-growing pathogens and offering potential applications in structural biology, vaccine development, and drug target validation.