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

Pseudomonas putida S12 is inherently solvent tolerant and constitutes a promising platform for biobased production of aromatic compounds and biopolymers. The megaplasmid pTTS12 of P. putida 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 P. putida 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, arpR, 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, F(o)F(1) 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 P. putida S12 lacking its megaplasmid. IMPORTANCE 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 Pseudomonas putida; however, the extent of innate, intrinsic solvent tolerance in this bacterium remained unclear. Using adaptive laboratory evolution, we successfully adapted the plasmid-cured P. putida 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 P. putida. This work sheds light on the possible genetic engineering targets to enhance solvent tolerance in Pseudomonas putida as well as other bacteria.