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Applied and Environmental Microbiology, December 2005, p. 8221-8227, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8221-8227.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Engineering of Solvent-Tolerant Pseudomonas putida S12 for Bioproduction of Phenol from Glucose

Nick J. P. Wierckx,* Hendrik Ballerstedt, Jan A. M. de Bont, and Jan Wery

TNO Quality of Life, Business Unit Bioconversion and Food Processes, P.O. Box 342, 7300 AH Apeldoorn, The Netherlands

Received 13 June 2005/ Accepted 24 August 2005

Efficient bioconversion of glucose to phenol via the central metabolite tyrosine was achieved in the solvent-tolerant strain Pseudomonas putida S12. The tpl gene from Pantoea agglomerans, encoding tyrosine phenol lyase, was introduced into P. putida S12 to enable phenol production. Tyrosine availability was a bottleneck for efficient production. The production host was optimized by overexpressing the aroF-1 gene, which codes for the first enzyme in the tyrosine biosynthetic pathway, and by random mutagenesis procedures involving selection with the toxic antimetabolites m-fluoro-DL-phenylalanine and m-fluoro-L-tyrosine. High-throughput screening of analogue-resistant mutants obtained in this way yielded a P. putida S12 derivative capable of producing 1.5 mM phenol in a shake flask culture with a yield of 6.7% (mol/mol). In a fed-batch process, the productivity was limited by accumulation of 5 mM phenol in the medium. This toxicity was overcome by use of octanol as an extractant for phenol in a biphasic medium-octanol system. This approach resulted in accumulation of 58 mM phenol in the octanol phase, and there was a twofold increase in the overall production compared to a single-phase fed batch.

* Corresponding author. Mailing address: TNO Quality of Life, P.O. Box 342, 7300 AH Apeldoorn, The Netherlands. Phone: (31) 55-5493512. Fax: (31) 55-5493410. E-mail: nick.wierckx{at}tno.nl.

Applied and Environmental Microbiology, December 2005, p. 8221-8227, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8221-8227.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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