Comparative Characterization of σ³²-Dependent Promoters for the Heat-Inducible Expression of FAST-PETase in...

Efficient regulation of recombinant enzyme expression is an important consideration for the development of microbial biocatalysts. Heat-inducible promoters regulated by the alternative sigma factor σ³² provide an inducer-free strategy for controlling gene expression in Escherichia coli. In this study, four σ³²-dependent promoters (PdnaK, PgrpE, PibpA, and PclpB) were comparatively characterized using the PET-degrading enzyme FAST-PETase fused to superfolder green fluorescent protein as a model recombinant protein. Promoter performance was evaluated based on basal leakage, induction kinetics, and expression strength following heat induction. Among the promoters examined, PdnaK exhibited the strongest heat-inducible expression and was dissected to examine the autonomous and combinatorial behavior of its promoter-derived elements. Molecular docking analysis further supported the experimental observations by showing qualitative agreement between predicted σ³²-DNA interactions and promoter performance. Together, these findings provide a comparative characterization of σ³²-dependent promoters and identify promoter architectures that may facilitate the development of heat-inducible recombinant enzyme expression systems in E. coli.