The non-haem chloroperoxidase from Pseudomonas fluorescens and its relationship to pyrrolnitrin biosynthesis

Institut für Mikrobiologie, Universität Hohenheim, Garbenstrasse 30, D-70593 Stuttgart, Germany Department of Molecular Genetics, CIBA Agricultural Biotechnology, Research Triangle Park, North Carolina, USA Institut für Biochemie, Technische Universität Dresden, D-01062 Dresden, Germany 4 Author for correspondence: Karl-Heinz van Pée. Tel: +49 351 463 4494. Fax: +49 351 463 5566. e-mail: chr@ctch02.chm.tu-dresden.de ABSTRACT The non-haem chloroperoxidase gene (cpoF ) from the pyrrolnitrin producer Pseudomonas fluorescens BL914 was cloned using an oligonucleotide derived from part of the N-terminal amino acid sequence of chloroperoxidase (CPO-P) from Pseudomonas pyrrocina as a probe. Based on the overexpression of cpoF in Escherichia coli and the stabilty of CPO-F against higher temperatures and proteases, the enzyme was purified to homogeneity. Partial characterization of the enzyme showed that it belongs to the class of bacterial non-haem CPOs. To investigate the role of CPO-F in pyrrolnitrin biosynthesis, the cpoF gene was inactivated by insertion of a kanamycin cassette. Exchange of the chromosomal cpoF gene against the disrupted copy had no influence on pyrrolnitrin production demonstrating that CPO-F was not involved in pyrrolnitrin biosynthesis. Keywords: Pseudomonas fluorescens , pyrrolnitrin, chloroperoxidase, gene disruption