Cell density-dependent auto-inducible promoters for expression of recombinant proteins in Pseudomonas putida

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.