Investigating and Engineering an 1,2-Propanediol-Responsive Transcription Factor-Based Biosensor

Area of Science:

• Metabolic Engineering
• Synthetic Biology
• Molecular Biology

Background:

• Transcription factor (TF)-based biosensors are crucial for metabolic engineering but limited in variety.
• A need exists for novel biosensors to meet diverse bioproduction targets.

Purpose of the Study:

• To engineer the 1,2-propanediol (1,2-PD)-responsive transcription activator PocR from *Salmonella typhimurium* to expand the biosensor repertoire.
• To enhance the dynamic and operational ranges of PocR for improved performance in *E. coli*.

Main Methods:

• Heterologous characterization and promiscuity testing of PocR in *E. coli*.
• Protein structure prediction (AlphaFold) and mutagenesis to explore PocR's mechanism.
• Engineering of PocR variants (PocR-F46R/G105D, PocR-ARE) and promoter engineering to create a bifunctional regulator.

Main Results:

• Initial PocR characterization showed limited operational and dynamic ranges due to promoter binding.
• Engineered variants demonstrated altered inducer specificity (glycerol) and significantly improved dynamic (6.7-fold) and operational (0-20 mM 1,2-PD) ranges.
• Successful conversion of PocR to a repressor and development of a 1,2-PD-induced bifunctional regulatory system.

Conclusions:

• The study successfully engineered a 1,2-PD-responsive transcriptional activator, PocR, significantly enhancing its performance.
• Developed variants offer a glycerol-responsive activator and a versatile 1,2-PD-induced bifunctional regulator, expanding the biosensor toolbox.
• This work highlights the potential of exploring and exploiting underexplored transcriptional activators for biotechnological applications.