Inducible promoters such as P_lac are of limited usability for industrial protein production with Pseudomonas putida. We therefore utilized cell density-dependent auto-inducible promoters for recombinant gene expression in P. putida KT2440 based on the RoxS/RoxR Quorum Sensing (QS) system of the bacterium. To this end, genetic regions upstream of the RoxS/RoxR-regulated genes ddcA (P_R _ox132 ) and PP_3332 (P_R _ox306 ) were inserted into plasmids that mediated the expression of superfolder green fluorescent protein (sfGFP) and surface displayed mCherry, confirming their promoter functionalities. Mutation of the Pribnow box of P_R _ox306 to the σ⁷⁰ consensus sequence (P_R _ox3061 ) resulted in a more than threefold increase of sfGFP production. All three promoters caused cell density-dependent expression, starting transcription at optical densities (OD₅₇₈ ) of approximately 1.0 (P_R _ox132 , P_R _ox306 ) or 0.7 (P_R _ox3061 ) as determined by RT-qPCR. The QS dependency of P_R _ox306 was further shown by cultivating P. putida in media that had already been used for cultivation and thus contained bacterial signal molecules. The longer P. putida had grown in these media before, the earlier protein expression in freshly inoculated P. putida appeared with P_R _ox306 . This confirmed previous findings that a bacterial compound accumulates within the culture and induces protein expression.

Katrin Gesing

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