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Applied and Environmental Microbiology, March 2007, p. 1635-1645, Vol. 73, No. 5
0099-2240/07/$08.00+0     doi:10.1128/AEM.01969-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Circumventing the Effect of Product Toxicity: Development of a Novel Two-Stage Production Process for the Lantibiotic Gallidermin

G. Valsesia,^1, G. Medaglia,^1, M. Held,¹ W. Minas,² and S. Panke^1*

Bioprocess Laboratory, Institute for Process Engineering, ETH Zurich, Zurich, Switzerland,¹ Biotech Concepts GmbH, Zurich, Switzerland²

Received 18 August 2006/ Accepted 16 December 2006

Lantibiotics such as gallidermin are lanthionine-containing polypeptide antibiotics produced by gram-positive bacteria that might become relevant for the treatment of various infectious diseases. So far, self-toxicity has prevented the isolation of efficient overproducing strains, thus hampering their thorough investigation and preventing their exploitation in fields other than the food area. We wanted to investigate the effect of lantibiotic precursor peptides on the producing strains in order to evaluate novel strategies for the overproduction of these promising peptides. In this study, gallidermin was chosen as a representative example of the type A lantibiotics. A Staphylococcus gallinarum Tü3928 mutant, whose gene for the extracellular pregallidermin protease GdmP was replaced by a kanamycin-resistance gene, was constructed. Mass spectrometry (MS) analysis indicated that this mutant produced fully posttranslationally modified gallidermin precursors with truncated versions of the leader peptide, but not the entire leader as predicted from the gdmA sequence. In filter-on-plate assays, these truncated pregallidermins showed no toxicity against Staphylococcus gallinarum Tü3928 up to a concentration of 8 g/liter (corresponding to approximately 2.35 mM), while gallidermin produced clear inhibitory zones at concentrations as low as 0.25 g/liter (0.12 mM). We showed that the lack of toxicity is due entirely to the presence of the truncated leader, since MS as well as bioassay analysis showed that the peptides resulting from tryptic cleavage of pregallidermins and gallidermin produced by S. gallinarum Tü3928 had identical masses and approximately the same specific activity. This demonstrates that even a shortened leader sequence is sufficient to prevent the toxicity of mature gallidermin. In nonoptimized fermentations, the gdmP mutant produced pregallidermin to a 50%-higher molar titer, suggesting that the absence of self-toxicity has a beneficial effect on gallidermin production and giving a first confirmation of the suitability of the overproduction strategy.

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* Corresponding author. Mailing address: ETH Zentrum CNB E122.1, Universitätstr. 6, 8092 Zürich, Switzerland. Phone: (41) 44 6320413. Fax: (41) 44 6321325. E-mail: panke{at}ipe.mavt.ethz.ch.

Published ahead of print on 28 December 2006.

G.V. and G.M. contributed equally to this work.

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Applied and Environmental Microbiology, March 2007, p. 1635-1645, Vol. 73, No. 5
0099-2240/07/$08.00+0     doi:10.1128/AEM.01969-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.