Synthetic gene circuits for programmable Pseudomonas catalytic biofilms

Nowadays, industrial fermentations rely almost entirely on the use of planktonic cells. However, biofilms (the most common form of bacterial growth in nature), offer several advantages to be exploited in modern fermentation processes. Bacteria in biofilms are more tolerant to several stresses than free cells, including toxic chemicals and shear stress. Furthermore, the adhesion of cells to surfaces can be exploited to operate a continuous fermentation process without excessive loss of biomass, thereby facilitating downstream processing. A programmable switch between planktonic and biofilm lifestyle is desirable to harness the advantages of both lifestyles. On this premise, we constructed a genetic gene circuit for biofilm formation in the platform strains Pseudomonas putida and Pseudomonas taiwanensis. Both P. putida and P. taiwanensis are robust, non-pathogenic soil bacteria and promising chassis for biotechnology as they can thrive under harsh operating conditions, displaying high tolerance towards several chemicals and can metabolize a broad range of substrates. These characteristics make them ideal for the production of a wide spectrum of chemicals. The synthetic circuit initiates biofilm formation upon detection of substrate or intermediate metabolites of the desired biotransformation, thus no additional inducer is needed. The circuit also allows for the propagation of cells in planktonic state prior employment in the bioreactor, which facilitates handling and speed up expansion of the culture. The design proposed herein employs a feedback-resistant diguanylate cyclase (DGC) from Caulobacter crescentus, which increases the concentration of DGC and therefore triggers biofilm formation. The resulting engineered strains were utilized for the biotransformation and degradation of chemicals (cyclohexanol) in continuous cultivation systems. This approach led to a ~300-fold increase in biofilm formation in microtiter plates, and was successfully used in diverse fermentation systems displaying increased catalytic efficiency.