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Bacteriocins are ribosomally synthesized antimicrobial peptides, which are produced by different bacteria and show high antimicrobial activity against a broad range of Gram-positive bacteria. A very well characterized bacteriocin is the lantibiotic nisin, which is naturally synthesized by Lactococcus lactis strains and undergoes post translationally modification including the introduction of characteristic (methyl )lanthionine rings. By targeting the universal peptidoglycan precursor lipid II, nisin shows strong antimicrobial activity against various Gram-positive bacteria including the food pathogen Listeria monocytogenes. A heterologous production using well established organisms like C. glutamicum, which are easy to cultivate on defined fermentation media, would be highly desirable. In order to examine the potential of C. glutamicum as a nisin producer, this organism was first characterized regarding sensitivity and resistance against nisin. Different experimental approaches clearly indicated that Corynebacterium glutamicum is rather sensitive towards nisin, facing a major challenge to overcome for a nisin production with this organism. Strategies to increase the nisin resistance included the introduction of immunity genes nisI and nisFEG from a natural L. lactis nisin producer, test of different ABC-transporter systems and attempts to reduce the attraction for the positively charged nisin by altering the negatively charged cell envelope of C. glutamicum. A screening for homologues to known resistance proteins from the phylum Firmicutes in 114 different organisms of the genus Corynebacterium revealed a broad distribution of resistance cluster within this genus, which could indeed confer some sort of resistance when expressed in the C. glutamicum reference strain. Furthermore, involvement of mycolic membrane porins in nisin translocation through the cell envelope was demonstrated. A combinatorial approach of different resistance mechanisms resulted in four-fold increase of nisin tolerance. Considering a heterologous production of nisin with C. glutamicum, the resistance was still too low for a successful biotechnological production. As an alternative, a two-step production approach was developed and established. The strategy was based on the synthesis of the inactive precursor peptide prenisin and a downstream cleavage step of the leader sequence, resulting in active nisin in cell culture supernatants. For the heterologous production of prenisin, the synthesis and modification genes nisZ, nisB, nisC and nisT from L. lactis were codon-optimized for expression in C. glutamicum and expressed under the control of the inducible Ptac promotor. For detection of active nisin, a highly specific and sensitive fluorescence sensor was constructed. This sensor is based on the autoregulation system of L. lactis nisin producers. Using the strain L. lactis NZ9000, which harbors a copy of the two component system genes nisR and nisK integrated in the genome, the presence of ≥ 0.25 ng/ml of nisin was sufficient to activate expression of the fluorescence reporter gene mCherryLlac, which was codon-optimized for expression in L. lactis subsp. cremoris and fused behind the nisin inducible promotor PnisZ. With this mCherry sensor strain, optimal cultivation and induction conditions were developed, enabling detection of active nisin directly from culture supernatants of C. glutamicum producer cells. Moreover, fully modified prenisin and active nisin Z was verified by mass spectrometric analyses with the help of cooperation partners. To improve the activation of prenisin, a soluble variant of the natural nisin protease NisP (sNisP) was constructed and recombinantly produced with Escherichia coli BL21 cells. After purification of both prenisin and sNisP, a four-fold higher amount of active nisin was obtained in vitro compared to activation by digestion with trypsin. With contributions to a further study a heterologous production of the unmodified bacteriocin pediocin PA-1 with C. glutamicum, which lacks a mannose-phosphotransferase system (Man-PTS) as target for pediocin, was achieved. Hence, C. glutamicum turned out as suitable organism for recombinant bacteriocin production. An important prerequisite for the establishment of bacteriocin production processes are appropriate assays to evaluate antimicrobial activity of produced bacteriocins. One example is the use of the pH-dependent reporter protein pHluorin, which has been cloned on an expression plasmid and introduced into L. monocytogenes. This sensor strain was used as a tool to analyze disruption of the cell membrane caused by the action of the tested bacteriocin. This sensor strategy can thus also be applied for semi-quantitative determination of produced bacteriocin levels. The herein presented results show the efforts been made to establish a heterologous synthesis of bacteriocins with C. glutamicum, expanding the production portfolio of this organism by this further product class. The results of this thesis could provide a basis for the establishment of further heterologous production processes for different bacteriocins.

Additional file 1: Figure S1. Generation and properties of a C. glutamicum biosensor for detection of membrane damage. Figure S2. Generation and properties of L. lactis NZ9000/pNZ-Pnis-mcherryLl biosensor for specific detection of nisin. Figure S3. Genetic organization of natural nisin Z operon of L. lactis B1629 and structure of prenisin Z. Table S1. Bacterial strains and plasmids used in this study. Table S2. Oligonucleotide primers and synthetic gene sequences used in this study.

Bacteriocins are antimicrobial peptides produced by bacteria to inhibit competitors in their natural environ- ments. Some of these peptides have emerged as commercial food preservatives and, due to the rapid increase in antibiotic resistant bacteria, are also discussed as interesting alternatives to antibiotics for therapeutic purposes. Currently, commercial bacteriocins are produced exclusively with natural producer organisms on complex substrates and are sold as semi-purified preparations or crude fermentates. To allow clinical application, efficacy of production and purity of the product need to be improved. This can be achieved by shifting production to recombinant microorganisms. Here, we identify Corynebacterium glutamicum as a suitable production host for the bacteriocin pediocin PA-1. C. glutamicum CR099 shows resistance to high concentrations of pediocin PA-1 and the bacteriocin was not inactivated when spiked into growing cultures of this bacterium. Recombinant C. glutamicum expressing a syn- thetic pedACDCgl operon releases a compound that has potent antimicrobial activity against Listeria monocytogenes and Listeria innocua and matches size and mass:charge ratio of commercial pediocin PA-1. Fermentations in shake flasks and bioreactors suggest that low levels of dissolved oxygen are favorable for production of pediocin. Under these conditions, however, reduced activity of the TCA cycle resulted in decreased availability of the important pediocin precursor L-asparagine suggesting options for further improvement. Overall, we demonstrate that C. glutamicum is a suitable host for recombinant production of bacteriocins of the pediocin family., publishedVersion