Systematic promoter design for plasmid-encoded S-adenosylmethionine sensing systems

Area of Science:

• Biochemistry
• Synthetic Biology
• Molecular Biology

Background:

• S-adenosylmethionine (SAM) is a crucial biomolecule acting as a primary methyl donor.
• SAM's availability is a rate-limiting factor in the biosynthesis of essential methylated compounds.
• A high-throughput sensing system is needed to monitor and reconfigure SAM homeostasis.

Purpose of the Study:

• To construct a minimal plasmid-based biosensor for detecting changes in intracellular SAM availability.
• To identify genetic or environmental factors influencing SAM levels.
• To optimize SAM biosensing through synthetic promoter engineering.

Main Methods:

• Engineered a plasmid with a fluorescent protein reporter under the control of the SAM-dependent transcription factor MetJ.
• Systematically reconstructed and tested 10 synthetic MetJ-regulated promoters with varying MetJ binding site configurations.
• Validated the system's responsiveness to factors altering intracellular SAM concentrations.

Main Results:

• Identified an optimal MetJ binding site position between the -35 and -10 promoter boxes for effective repression.
• Demonstrated the biosensor's capability to detect factors that deplete or restore intracellular SAM availability.
• Characterized the response patterns of synthetic MetJ-regulated promoters.

Conclusions:

• The developed MetJ-based biosensor provides a sensitive tool for monitoring SAM availability.
• Optimized synthetic promoters enhance the potential for developing advanced SAM biosensing systems.
• This work facilitates the study and manipulation of SAM homeostasis in biological systems.