title: Engineering of Pseudomonas taiwanensis VLB120 for constitutive solvent tolerance and increased specific styrene epoxidation activity.
abstract: The application of whole cells as biocatalysts is often limited by the toxicity of organic solvents, which constitute interesting substrates/products or can be used as a second phase for in situ product removal and as tools to control multistep biocatalysis. Solvent-tolerant bacteria, especially Pseudomonas strains, are proposed as promising hosts to overcome such limitations due to their inherent solvent tolerance mechanisms. However, potential industrial applications suffer from tedious, unproductive adaptation processes, phenotypic variability, and instable solvent-tolerant phenotypes. In this study, genes described to be involved in solvent tolerance were identified in Pseudomonas taiwanensis VLB120, and adaptive solvent tolerance was proven by cultivation in the presence of 1% (vol/vol) toluene. Deletion of ttgV, coding for the specific transcriptional repressor of solvent efflux pump TtgGHI gene expression, led to constitutively solvent-tolerant mutants of P. taiwanensis VLB120 and VLB120ΔC. Interestingly, the increased amount of solvent efflux pumps enhanced not only growth in the presence of toluene and styrene but also the biocatalytic performance in terms of stereospecific styrene epoxidation, although proton-driven solvent efflux is expected to compete with the styrene monooxygenase for metabolic energy. Compared to that of the P. taiwanensis VLB120ΔC parent strain, the maximum specific epoxidation activity of P. taiwanensis VLB120ΔCΔttgV doubled to 67 U/g of cells (dry weight). This study shows that solvent tolerance mechanisms, e.g., the solvent efflux pump TtgGHI, not only allow for growth in the presence of organic compounds but can also be used as tools to improve redox biocatalysis involving organic solvents.
authors.affiliation: Laboratory of Chemical Biotechnology, Department of Biochemical and Chemical Engineering, TU Dortmund University, Dortmund, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Volmer
authors.affiliation: Laboratory of Chemical Biotechnology, Department of Biochemical and Chemical Engineering, TU Dortmund University, Dortmund, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Neumann
authors.affiliation: Laboratory of Chemical Biotechnology, Department of Biochemical and Chemical Engineering, TU Dortmund University, Dortmund, Germany bruno.buehler@bci.tu-dortmund.de.
authors.forename: Bruno
authors.initials: B
authors.lastname: Bühler
authors.affiliation: Laboratory of Chemical Biotechnology, Department of Biochemical and Chemical Engineering, TU Dortmund University, Dortmund, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Schmid
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D024881:Drug Resistance, Bacterial; D004852:Epoxy Compounds / Q000378:metabolism / Q000494:pharmacology; D017353:Gene Deletion; D005818:Genetic Engineering / Q000379:methods*; D008969:Molecular Sequence Data; D011549:Pseudomonas / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D012997:Solvents / Q000494:pharmacology; D020058:Styrene / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D004852:Epoxy Compounds; D012997:Solvents; D020058:Styrene; C013690:styrene oxide