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Applied and Environmental Microbiology, June 2004, p. 3637-3643, Vol. 70, No. 6
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.6.3637-3643.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Biotransformation in Double-Phase Systems: Physiological Responses of Pseudomonas putida DOT-T1E to a Double Phase Made of Aliphatic Alcohols and Biosynthesis of Substituted Catechols

Antonia Rojas,¹ Estrella Duque,¹ Andreas Schmid,² Ana Hurtado,¹ Juan-Luis Ramos,^1* and Ana Segura¹

Department of Biochemistry and Molecular and Cellular Biology of Plants, CSIC-Estación Experimental del Zaidín, E-18008 Granada, Spain,¹ Institute of Biotechnology, Swiss Federal Institute of Technology (ETH), CH-8093 Zurich, Switzerland²

Received 17 December 2003/ Accepted 27 February 2004

Pseudomonas putida strain DOT-T1E is highly tolerant to organic solvents, with a logP_ow (the logarithm of the partition coefficient of a solvent in a two-phase water-octanol system of 2.5. Solvent tolerant microorganisms can be exploited to develop double-phase (organic solvent and water) biotransformation systems in which toxic substrates or products are kept in the organic phase. We tested P. putida DOT-T1E tolerance to different aliphatic alcohols with a logP_ow value between 2 and 4, such as decanol, nonanol, and octanol, which are potentially useful in biotransformations in double-phase systems in which compounds with a logP_ow around 1.5 are produced. P. putida DOT-T1E responds to aliphatic alcohols as the second phase through cis-to-trans isomerization of unsaturated cis fatty acids and through efflux of these aliphatic alcohols via a series of pumps that also extrude aromatic hydrocarbons. These defense mechanisms allow P. putida DOT-T1E to survive well in the presence of high concentrations of the aliphatic alcohols, and growth with nonanol or decanol occurred at a high rate, whereas in the presence of an octanol double-phase growth was compromised. Our results support that the logP_ow of aliphatic alcohols correlates with their toxic effects, as octanol (logP_ow = 2.9) has more negative effects in P. putida cells than 1-nonanol (logP_ow = 3.4) or 1-decanol (logP_ow = 4). A P. putida DOT-T1E derivative bearing plasmid pWW0-xylE::Km transforms m-xylene (logP_ow = 3.2) into 3-methylcatechol (logP_ow = 1.8). The amount of 3-methylcatechol produced in an aliphatic alcohol/water bioreactor was 10- to 20-fold higher than in an aqueous medium, demonstrating the usefulness of double-phase systems for this particular biotransformation.

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* Corresponding author. Mailing address: CSIC-Estacion Experimental del Zaidin, Profesor Albareda 1, E-18008 Granada, Spain. Phone: 34-958-181608. Fax: 34-958-135740. E-mail: jlramos{at}eez.csic.es.

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Applied and Environmental Microbiology, June 2004, p. 3637-3643, Vol. 70, No. 6
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.6.3637-3643.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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