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Cascading recombinase memory switch for programmable and stable gene expression in Pseudomonas putida.

Javier M Hernández-Sancho¹, Daniel C Volke¹, Pablo I Nikel¹

¹BRIGHT, Technical University of Denmark, 2800 Kongens Lyngby, Denmark.

Nucleic Acids Research

|June 10, 2026

Summary

We developed a genetic toggle switch for reliable gene control in Pseudomonas putida. This system enables stable microbial phenotypes and programmable bioprocesses using genetic memory.

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Area of Science:

Synthetic Biology
Microbial Engineering
Biotechnology

Background:

Genetic circuits often fail in non-model bacteria, hindering industrial applications.
Developing robust gene regulation systems for industrial microbes like Pseudomonas putida is crucial.

Purpose of the Study:

Engineer a stable, programmable genetic toggle switch for Pseudomonas putida.
Create a genetic memory element to control bioprocess-relevant phenotypes.

Main Methods:

Designed an integrase-based cascading toggle switch using Bxb1 serine integrase and a T7 RNA polymerase system.
Optimized gene expression for tight OFF states and strong induction.
Integrated the switch into a 2-fluoro-cis,cis-muconic acid production strain to control biofilm formation.

Main Results:

Achieved consistent and programmable gene expression with stable phenotypes after inducer removal.
Demonstrated efficient switching and genetic memory.
Successfully engineered switch-mediated catalytic biofilms for continuous chemical production.

Conclusions:

The developed toggle switch expands the synthetic biology toolbox for Pseudomonas.
This system links heritable genetic memory to stable microbial phenotypes for bioprocesses.
Enables robust control of microbial functions in industrially relevant organisms.