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

Abstract

Lantibiotics such as gallidermin are lanthionine-containing polypeptide antibiotics produced by grampositive bacteria that might become relevant for the treatment of various infectious diseases. So far, selftoxicity 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.Antibiotic resistance has spread dramatically among pathogens throughout the last two decades. Of particular concern is the increasing emergence of multiresistant pathogens in health care settings. Nowadays, about 70% of nosocomial infections in the United States are resistant to at least one antibiotic (35), and in 2002, the first clinical isolate of Staphylococcus aureus with high-level resistance to vancomycin, a drug of last resort, was isolated (8). As the number of resistant pathogens continues to grow, the number of new antibiotics to fight them is declining steeply (37). Therefore, the development of new antimicrobial agents should be encouraged.Type A lantibiotics are cationic, amphiphilic peptides that are produced by a wide range of gram-positive bacteria and show bactericidal activity against other gram-positive bacteria. They show a dual mode of action at nanomolar concentrations which involves pore formation and inhibition of peptidoglycan biosynth...

Highly Efficient Staphylococcus carnosus Mutant Selection System Based on Suicidal Bacteriocin Activation

“…4B). This finding is in accordance with the recently described productivity of a gdmP mutant at nonoptimized culture conditions (49).…”

“…2A and C). The running behavior of the new peak corresponded to the previously described peak of pregallidermin (49), and it exhibited the characteristic absorption at 270 nm resulting from posttranslational oxidative decarboxylation of the pregallidermin C terminus (32). Preparative amounts of the putative pregallidermin were isolated from S. gallinarum Tü3928 ⌬gdmP::erm mutant cultures grown in YE4 medium, which had proven useful for high-level gallidermin production (30).…”

“…Since the published sequence of the gallidermin gene cluster ends close to the start codon of gdmP (23,49) (GenBank accession no. DQ367437), it was necessary to determine its upstream sequence, which was achieved by direct chromosomal sequencing with the LI-COR 4200 system (with the first five primers listed in Table 2).…”

SmbFT, a Putative ABC Transporter Complex, Confers Protection against the Lantibiotic Smb in Streptococci

“…Since the strain was constructed by insertion of an ermB gene (erythromycin resistance), it might have interfered with the transcription of the downstream genes in the smb operon and so, as a result, less smbAB was produced. Often lantibiotic production is subject to feedback inhibition and is positively influenced by the immunity proteins (64,65). Therefore, it is also possible that SmbFT positively influences smb production.…”

Development of a high cell-density protocol for the production of pregallidermin, a non-toxic precursor of the lantibiotic gallidermin

“…The most important parameter to consider is the low production titer typically obtained in lantibiotic fermentation as opposed to the high product concentrations that are considered a prerequisite for an economic production process (Nielsen et al, 2002). Presumably, final concentrations remain low because of product inhibition (Ungermann et al, 1991;Valsesia et al, 2007), which is thought to be the major limitation for the achievement of high product titers. With nisin, the most comprehensively studied lantibiotic (class I), final concentrations of up to 170 mg L −1 have been reported in batch and fed-batch protocols (de Vuyst, 1994).…”