Adaptive Laboratory Evolution Restores Solvent Tolerance in Plasmid-Cured Pseudomonas putida S12: a Molecular Analysis.

S12 is inherently solvent tolerant and constitutes a promising platform for biobased production of aromatic compounds and biopolymers. The megaplasmid pTTS12 of S12 carries several gene clusters involved in solvent tolerance, and the removal of this megaplasmid caused a significant reduction in solvent tolerance. In this study, we succeeded in restoring solvent tolerance in plasmid-cured S12 using adaptive laboratory evolution (ALE), underscoring the innate solvent tolerance of this strain. Whole-genome sequencing identified several single nucleotide polymorphisms (SNPs) and a mobile element insertion enabling ALE-derived strains to survive and sustain growth in the presence of a high toluene concentration (10% [vol/vol]). We identified mutations in an RND efflux pump regulator, , that resulted in constitutive upregulation of the multifunctional efflux pump ArpABC. SNPs were also found in the intergenic region and subunits of ATP synthase, RNA polymerase subunit β', a global two-component regulatory system (GacA/GacS), and a putative AraC family transcriptional regulator, Afr. Transcriptomic analysis further revealed a constitutive downregulation of energy-consuming activities in ALE-derived strains, such as flagellar assembly, FF ATP synthase, and membrane transport proteins. In summary, constitutive expression of a solvent extrusion pump in combination with high metabolic flexibility enabled the restoration of the solvent tolerance trait in S12 lacking its megaplasmid. Sustainable production of high-value chemicals can be achieved by bacterial biocatalysis. However, bioproduction of biopolymers and aromatic compounds may exert stress on the microbial production host and limit the resulting yield. Having a solvent tolerance trait is highly advantageous for microbial hosts used in the biobased production of aromatics. The presence of a megaplasmid has been linked to the solvent tolerance trait of ; however, the extent of innate, intrinsic solvent tolerance in this bacterium remained unclear. Using adaptive laboratory evolution, we successfully adapted the plasmid-cured S12 strain to regain its solvent tolerance. Through these adapted strains, we began to clarify the causes, origins, limitations, and trade-offs of the intrinsic solvent tolerance in This work sheds light on the possible genetic engineering targets to enhance solvent tolerance in as well as other bacteria.