Monday, July 27, 2009 - 10:00 AM
S4

Use of Halogenases in Combinatorial Biosynthesis

Lutz Heide1, Lucia Westrich1, Christine Anderle1, Manuel Wolpert1, Bertolt Gust1, Bernd Kammerer1, and Jörn Piel2. (1) Pharmaceutical Institute, University of Tübingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany, (2) Kekulé-Institute for Organic Chemistry and Biochemistry, University of Bonn, Gerhard-Domagk Str. 1, Bonn, 53121, Germany

The aminocoumarin antibiotic coumermycin A1 is a potent inhibitor of gyrase. We assembled the entire coumermycin A1 biosynthetic gene cluster (38.6 kb) onto a single cosmid clone by using the RED/ET recombination technology. Heterologous expression of the reconstituted gene cluster in Streptomyces coelicolor M512 resulted in the heterologous production of coumermycin A1. Inactivation of the methyltransferase gene couO which is responsible for the C-methylation at position 8 of the aminocoumarin moieties in coumermycin A1 resulted in an accumulation of a C-8 unsubstituted coumermycin A1 derivative. Subsequent expression of the halogenase gene clo-hal from the clorobiocin gene cluster in the heterologous producer strain led to the formation of two new hybrid antibiotics, containing either one or two 3-amino-4,7-dihydroxy-8-chlorocoumarin moieties.

The depsipeptide antibiotic hormaomycin, produced by Streptomyces griseoflavus W-384, contains a 5-chloro-pyrrole moiety. In the producer strain we identified the gene hrmQ which shows sequence similarity to FADH2-dependent halogenases. This gene was cloned and heterologously expressed in Streptomyces roseochromogenes var. oscitans DS12.976, the producer of the aminocoumarin antibiotic clorobiocin which genuinely contains a 5-methyl-pyrrole moiety. We used a mutant of this strain in which the respective pyrrole-5-methyltransferase had been inactivated.

Expression of the halogenase hrmQ in this mutant strain led to the formation of two new clorobiocin derivatives which carried a 5-chloro-pyrrole moiety. The substrate of HrmQ is likely to be a pyrrole-2-carboxyl-S-[acyl carrier protein] thioester. This is the first time that a halogenase acting on an acyl carrier protein-bound substrate is successfully used in combinatorial biosynthesis.


See more of Biosynthetic enzyme mechanisms and combinatorial biosynthesis
See more of Invited Oral Papers

See more of The Annual Meeting and Exhibition 2009 (July 26 - 30, 2009)