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Applied and Environmental Microbiology, December 2006, p. 7510-7517, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.01541-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Identification of Novel Benzoylformate Decarboxylases by Growth Selection^,

Helge Henning,^1, Christian Leggewie,^1, Martina Pohl,¹ Michael Müller,² Thorsten Eggert,¹ and Karl-Erich Jaeger^1*

Institute of Molecular Enzyme Technology, Heinrich Heine University Duesseldorf, Research Centre Juelich, D-52426 Juelich, Germany,¹ Institute of Pharmaceutical Sciences, Albert Ludwigs University, D-79104 Freiburg, Germany²

Received 4 July 2006/ Accepted 23 September 2006

A growth selection system was established using Pseudomonas putida, which can grow on benzaldehyde as the sole carbon source. These bacteria presumably metabolize benzaldehyde via the ß-ketoadipate pathway and were unable to grow in benzoylformate-containing selective medium, but the growth deficiency could be restored by expression in trans of genes encoding benzoylformate decarboxylases. The selection system was used to identify three novel benzoylformate decarboxylases, two of them originating from a chromosomal library of P. putida ATCC 12633 and the third from an environmental-DNA library. The novel P. putida enzymes BfdB and BfdC exhibited 83% homology to the benzoylformate decarboxylase from P. aeruginosa and 63% to the enzyme MdlC from P. putida ATCC 12633, whereas the metagenomic BfdM exhibited 72% homology to a putative benzoylformate decarboxylase from Polaromonas naphthalenivorans. BfdC was overexpressed in Escherichia coli, and the enzymatic activity was determined to be 22 U/ml using benzoylformate as the substrate. Our results clearly demonstrate that P. putida KT2440 is an appropriate selection host strain suitable to identify novel benzoylformate decarboxylase-encoding genes. In principle, this system is also applicable to identify a broad range of different industrially important enzymes, such as benzaldehyde lyases, benzoylformate decarboxylases, and hydroxynitrile lyases, which all catalyze the formation of benzaldehyde.

Published ahead of print on 29 September 2006.

This paper is dedicated to Maria-Regina Kula on the occasion of her 70th birthday.

H.H. and C.L. contributed equally to this work.