RT Journal Article SR Electronic T1 Adaptive laboratory evolution restores solvent tolerance in plasmid-cured Pseudomonas putida S12; a molecular analysis JF bioRxiv FD Cold Spring Harbor Laboratory SP 2020.08.01.232264 DO 10.1101/2020.08.01.232264 A1 Kusumawardhani, Hadiastri A1 Furtwängler, Benjamin A1 Blommestijn, Matthijs A1 Kaltenytė, Adelė A1 van der Poel, Jaap A1 Kolk, Jevan A1 Hosseini, Rohola A1 de Winde, Johannes H. YR 2020 UL http://biorxiv.org/content/early/2020/08/01/2020.08.01.232264.abstract AB Pseudomonas putida S12 is intrinsically solvent-tolerant and constitutes a promising platform for biobased production of aromatic compounds and biopolymers. The genome of P. putida S12 consists of a 5.8 Mbp chromosome, and a 580 kbp megaplasmid pTTS12 that carries several gene clusters involved in solvent tolerance. Removal of pTTS12 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 revealed 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% v/v). Mutations were identified in an RND efflux pump regulator arpR, resulting 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’, global two-component regulatory system (GacA/GacS) and a putative AraC-family transcriptional regulator Afr. RNA-seq analysis further revealed a constitutive down-regulation of energy consuming activities in ALE-derived strains, including flagellar assembly, F0F1 ATP synthase, and membrane transport proteins. Out results indicate that constitutive expression of an alternative solvent extrusion pump in combination with high metabolic flexibility ensures restoration of solvent-tolerance in P. putida S12 lacking its megaplasmid.Competing Interest StatementThe authors have declared no competing interest.