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Journal of Bacteriology, September 2004, p. 6093-6100, Vol. 186, No. 18
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.18.6093-6100.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Biosynthesis of Chloro-ß-Hydroxytyrosine, a Nonproteinogenic Amino Acid of the Peptidic Backbone of Glycopeptide Antibiotics

Oliver Puk,^1, Daniel Bischoff,^2, Claudia Kittel,¹ Stefan Pelzer,^1, Stefan Weist,² Efthimia Stegmann,¹ Roderich D. Süssmuth,² and Wolfgang Wohlleben^1*

Mikrobiologie/Biotechnologie, Mikrobiologisches Institut, Fakultät für Biologie,¹ Institut für Organische Chemie, Universität Tübingen, Tübingen, Germany²

Received 20 April 2004/ Accepted 17 June 2004

The role of the putative P450 monooxygenase OxyD and the chlorination time point in the biosynthesis of the glycopeptide antibiotic balhimycin produced by Amycolatopsis balhimycina were analyzed. The oxyD gene is located directly downstream of the bhp (perhydrolase) and bpsD (nonribosomal peptide synthetase D) genes, which are involved in the synthesis of the balhimycin building block ß-hydroxytyrosine (ß-HT). Reverse transcriptase experiments revealed that bhp, bpsD, and oxyD form an operon. oxyD was inactivated by an in-frame deletion, and the resulting mutant was unable to produce an active compound. Balhimycin production could be restored (i) by complementation with an oxyD gene, (ii) in cross-feeding studies using A. balhimycina JR1 (a null mutant with a block in the biosynthesis pathway of the building blocks hydroxy- and dihydroxyphenylglycine) as an excretor of the missing precursor, and (iii) by supplementation of ß-HT in the growth medium. These data demonstrated an essential role of OxyD in the formation pathway of this amino acid. Liquid chromatography-electrospray ionization-mass spectrometry analysis indicated the biosynthesis of completely chlorinated balhimycin by the oxyD mutant when culture filtrates were supplemented with nonchlorinated ß-HT. In contrast, supplementation with 3-chloro-ß-HT did not restore balhimycin production. These results indicated that the chlorination time point was later than the stage of free ß-HT, most likely during heptapeptide synthesis.

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* Corresponding author. Mailing address: Mikrobiologie/Biotechnologie, Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany. Phone: 49 7071 2976944. Fax: 49 7071 295979. E-mail: wolfgang.wohlleben{at}biotech.uni-tuebingen.de.

Present address: GSF-Forschungszentrum für Umwelt und Gesundheit, Institut für Entwicklungsgenetik, D-85764 Neuherberg, Germany.

Present address: Boehringer Ingelheim Pharma GmbH & Co. KG, 88397 Biberach, Germany.

Present address: Combinature Biopharm AG, D-13125 Berlin, Germany.

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Journal of Bacteriology, September 2004, p. 6093-6100, Vol. 186, No. 18
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.18.6093-6100.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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