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Transcriptome Analysis of a Phenol-Producing Pseudomonas putida S12 Construct: Genetic and Physiological Basis for Improved Production ^,

Nick J. P. Wierckx,^1,2* Hendrik Ballerstedt,^1,2 Jan A. M. de Bont,^2, Johannes H. de Winde,^2,3 Harald J. Ruijssenaars,^1,2 and Jan Wery^2,

TNO Quality of Life, Department of Bioconversion, Julianalaan 67, 2628 BC, Delft, The Netherlands,¹ Kluyver Centre for Genomics of Industrial Fermentation, P.O. Box 5057, 2600 GB, Delft, The Netherlands,² Delft University of Technology, Department of Biotechnology, Julianalaan 67, 2628 BC, Delft, The Netherlands³

Received 24 August 2007/ Accepted 29 October 2007

The unknown genetic basis for improved phenol production by a recombinant Pseudomonas putida S12 derivative bearing the tpl (tyrosine-phenol lyase) gene was investigated via comparative transcriptomics, nucleotide sequence analysis, and targeted gene disruption. We show upregulation of tyrosine biosynthetic genes and possibly decreased biosynthesis of tryptophan caused by a mutation in the trpE gene as the genetic basis for the enhanced phenol production. In addition, several genes in degradation routes connected to the tyrosine biosynthetic pathway were upregulated. This either may be a side effect that negatively affects phenol production or may point to intracellular accumulation of tyrosine or its intermediates. A number of genes identified by the transcriptome analysis were selected for targeted disruption in P. putida S12TPL3. Physiological and biochemical examination of P. putida S12TPL3 and these mutants led to the conclusion that the metabolic flux toward tyrosine in P. putida S12TPL3 was improved to such an extent that the heterologous tyrosine-phenol lyase enzyme had become the rate-limiting step in phenol biosynthesis.

Published ahead of print on 9 November 2007.

Supplemental material for this article may be found at http://jb.asm.org/.

Present address: Royal Nedalco, Van Konijnenburgweg 100, Bergen op Zoom, 4612 PL, The Netherlands.

Present address: Dyadic Nederland BV, Nieuwe Kanaal 7^s, 6709 PA Wageningen, The Netherlands.