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Applied and Environmental Microbiology, February 2009, p. 931-936, Vol. 75, No. 4
0099-2240/09/$08.00+0     doi:10.1128/AEM.02186-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Bioproduction of p-Hydroxystyrene from Glucose by the Solvent-Tolerant Bacterium Pseudomonas putida S12 in a Two-Phase Water-Decanol Fermentation {triangledown}

Suzanne Verhoef,1,2,3,{dagger}* Nick Wierckx,1,2,{dagger} R. G. Maaike Westerhof,1,{ddagger} Johannes H. de Winde,2,3 and Harald J. Ruijssenaars1,2

TNO Quality of Life, Business Unit Food and Biotechnology Innovations, Julianalaan 67, 2628 BC Delft, The Netherlands,1 B-Basic, Julianalaan 67, 2628 BC Delft, The Netherlands,2 Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands3

Received 22 September 2008/ Accepted 26 November 2008

Two solvent-tolerant Pseudomonas putida S12 strains, originally designed for phenol and p-coumarate production, were engineered for efficient production of p-hydroxystyrene from glucose. This was established by introduction of the genes pal and pdc encoding L-phenylalanine/L-tyrosine ammonia lyase and p-coumaric acid decarboxylase, respectively. These enzymes allow the conversion of the central metabolite L-tyrosine into p-hydroxystyrene, via p-coumarate. Degradation of the p-coumarate intermediate was prevented by inactivating the fcs gene encoding feruloyl-coenzyme A synthetase. The best-performing strain was selected and cultivated in the fed-batch mode, resulting in the formation of 4.5 mM p-hydroxystyrene at a yield of 6.7% (C-mol of p-hydroxystyrene per C-mol of glucose) and a maximum volumetric productivity of 0.4 mM h–1. At this concentration, growth and production were completely halted due to the toxicity of p-hydroxystyrene. Product toxicity was overcome by the application of a second phase of 1-decanol to extract p-hydroxystyrene during fed-batch cultivation. This resulted in a twofold increase of the maximum volumetric productivity (0.75 mM h–1) and a final total p-hydroxystyrene concentration of 21 mM, which is a fourfold improvement compared to the single-phase fed-batch cultivation. The final concentration of p-hydroxystyrene in the water phase was 1.2 mM, while a concentration of 147 mM (17.6 g liter–1) was obtained in the 1-decanol phase. Thus, a P. putida S12 strain producing the low-value compound phenol was successfully altered for the production of the toxic value-added compound p-hydroxystyrene.

* Corresponding author. Mailing address: TNO Quality of Life, Julianalaan 67, 2628 BC Delft, The Netherlands. Phone: 31 15 2785019. Fax: 31 15 2782355. E-mail: suzanne.verhoef{at}tno.nl

{triangledown} Published ahead of print on 5 December 2008.

{dagger} S.V. and N.W. contributed equally to this work.

{ddagger} Present address: Dyadic Nederland BV, Nieuwe Kanaal 7, 6709 PA Wageningen, The Netherlands.

Applied and Environmental Microbiology, February 2009, p. 931-936, Vol. 75, No. 4
0099-2240/09/$08.00+0     doi:10.1128/AEM.02186-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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