title: Solvent resistance pumps of Pseudomonas putida S12: Applications in 1-naphthol production and biocatalyst engineering.
abstract: The solvent resistance capacity of Pseudomonas putida S12 was applied by using the organism as a host for biocatalysis and through cloning and expressing solvent resistant pump genes into Escherichia coli. P. putida S12 expressing toluene ortho mononooxygenase (TOM-Green) was used for 1-naphthol production in a water-organic solvent biphasic system. Application of P. putida S12 improved 1-naphthol production per gram cell dry weight by approximately 42% compared to E. coli. Moreover, P. putida S12 enabled the use of a less expensive solvent, decanol, for 1-naphthol production. The solvent resistant pump (srpABC) genes of P. putida S12 were cloned into a solvent sensitive E. coli strain to transfer solvent tolerance. Recombinant strains bearing srpABC genes in either a low-copy number or a high-copy number plasmid grew in the presence of saturated concentration of toluene. Both of the recombinant strains were more tolerant to 1% v/v of toxic solvents, decanol and hexane, reaching similar cell density as the no-solvent control. Reverse-transcriptase analysis revealed that the srpABC genes were transcribed in engineered strains. The results demonstrate successful transfer of the proton-dependent solvent resistance mechanism and suggest that the engineered strain could serve as more robust biocatalysts in media with organic solvents.
authors.affiliation: The Department of Chemical and Biochemical Engineering, University of Iowa, Iowa City, Iowa 52242, United States.
authors.forename: S V B
authors.initials: SV
authors.lastname: Janardhan Garikipati
authors.affiliation: The Department of Chemical and Biochemical Engineering, University of Iowa, Iowa City, Iowa 52242, United States. Electronic address: tonya-peeples@uiowa.edu.
authors.forename: Tonya L
authors.initials: TL
authors.lastname: Peeples
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D055162:Biocatalysis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development*; D015964:Gene Expression Regulation, Bacterial; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D009284:Naphthols / Q000378:metabolism; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D012997:Solvents / Q000378:metabolism*; D014050:Toluene / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D009284:Naphthols; D012997:Solvents; C029350:1-naphthol; D014050:Toluene; D006899:Mixed Function Oxygenases; C093480:toluene ortho-monooxygenase
keywords: P. putida S12; Solvent resistant pump (srp); Solvent tolerance; srpABC