A novel toxin-antitoxin module SlvT–SlvA governs megaplasmid stability and incites solvent tolerance inPseudomonas putidaS12

AbstractPseudomonas putidaS12 is highly tolerant towards organic solvents in saturating concentrations, rendering this microorganism suitable for the industrial production of various aromatic compounds. Previous studies reveal thatP. putidaS12 contains a single-copy 583 kbp megaplasmid pTTS12. This pTTS12 encodes several important operons and gene clusters facilitatingP. putidaS12 to survive and grow in the presence of toxic compounds or other environmental stresses. We wished to revisit and further scrutinize the role of pTTS12 in conferring solvent tolerance. To this end, we cured the megaplasmid fromP. putidaS12 and conclusively confirmed that the SrpABC efflux pump is the major contributor of solvent tolerance on the megaplasmid pTTS12. Importantly, we identified a novel toxin-antitoxin module (proposed gene namesslvTandslvArespectively) encoded on pTTS12 which contributes to the solvent tolerant phenotype and is essential in conferring genetic stability to the megaplasmid. Chromosomal introduction of thesrpoperon in combination withslvAT gene pair created a solvent tolerance phenotype in non-solvent tolerant strains such asP. putidaKT2440,E. coliTG1, andE. coliBL21(DE3).ImportanceSustainable alternatives for high-value chemicals can be achieved by using renewable feedstocks in bacterial biocatalysis. However, during bioproduction of such chemicals and biopolymers, aromatic compounds that function as products, substrates or intermediates in the production process may exert toxicity to microbial host cells and limit the production yield. Therefore, solvent-tolerance is a highly preferable trait for microbial hosts in the biobased production of aromatic chemicals and biopolymers. In this study, we revisit the essential role of megaplasmid pTTS12 from solvent-tolerantP. putidaS12 for molecular adaptation to organic solvent. In addition to the RND efflux pump (SrpABC), we identified a novel toxin-antitoxin module (SlvAT) which contributes to tolerance in low solvent concentration as well as to genetic stability of pTTS12. These two gene clusters were successfully transferred to non-solvent tolerant strains ofP. putidaand toE. colistrains to confer and enhance solvent tolerance.