Circumventing the effect of product toxicity: development of a novel two-stage production process for the lantibiotic gallidermin

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

^* To whom correspondence should be addressed. Email: panke{at}ipe.mavt.ethz.ch.

	Abstract

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 type A-lantibiotics. A Staphylococcus gallinarum Tü3928 mutant was constructed whose gene for the extracellular pregallidermin protease GdmP had been substituted by a kanamycin resistance gene. Mass spectrometry (MS) analysis indicated that this mutant produced fully post-translationally 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/L (corresponding to approximately 2.35 mM), while gallidermin produced clear inhibitory zones already at concentrations as low as 0.25 g/L (0.12 mM). We could show that the lack in toxicity is entirely due to the presence of the truncated leader, since MS as well as bioassay analysis showed that the peptide resulting from tryptic cleavage of pregallidermins and gallidermin produced by S. gallinarum Tü3928 had identical mass and approximately the same specific activity. This demonstrates that even a shortened leader sequence is sufficient to prevent the toxicity of mature gallidermin. In non-optimized 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.