Adaptive laboratory evolution restores solvent tolerance in the plasmid-cured Pseudomonas putida S12; a global transcriptional analysis

Pseudomonas putida S12 is an inherently solvent-tolerant strain and constitutes a promising platform for biotechnology applications in whole-cell biocatalysis of aromatic compounds. The genome of P. putida S12 consists of a 5.8 Mbp chromosome and a 580 kbp megaplasmid pTTS12. pTTS12 encodes several genes which enable the tolerance to various stress conditions, including the main solvent efflux pump SrpABC. Removal (curing) of megaplasmid pTTS12 and subsequent loss of solvent efflux pump SrpABC caused a significant reduction in solvent tolerance of the resulting strain. In this study, we succeeded in restoring solvent tolerance in the megaplasmid-cured P. putida S12 using adaptive laboratory evolution (ALE) and molecular analysis to investigate the intrinsic solvent tolerance of P. putida S12. RNA-seq was performed to study the global transcriptomic response of the solvent-adapted plasmid-cured P. putida S12 in the presence of toluene. This analysis revealed the downregulation of ATP synthase, flagella and other RND efflux pumps, which indicates the importance of maintaining proton motive force during solvent stress. RNA-seq analysis and comparison of the wildtype, plasmid-cured, and ALE derived plasmid-cured Pseudomonas putida S12 grown in liquid Lysogeny broth in the absence and presence of toluene (0.1 %)