Garvicin Q: characterization of biosynthesis and mode of action.

Bacteriocins are ribosomally synthesized antimicrobial peptides, that either kill target bacteria or inhibit their growth. Bacteriocins are used in food preservation and are of increasing interest as potential alternatives to conventional antibiotics. In the present study, we show that Lactococcus petauri B1726, a strain isolated from fermented balsam pear, produces a heat-stable and protease-sensitive compound. Following genome sequencing, a gene cluster for production of a class IId bacteriocin was identified consisting of garQ (encoding for the bacteriocin garvicin Q), garI (for a putative immunity protein), garC, and garD (putative transporter proteins). Growth conditions were optimized for increased bacteriocin activity in supernatants of L. petauri B1726 and purification and mass spectrometry identified the compound as garvicin Q. Further experiments suggest that garvicin Q adsorbs to biomass of various susceptible and insusceptible bacteria and support the hypothesis that garvicin Q requires a mannose-family phosphotransferase system (PTSMan) as receptor to kill target bacteria by disruption of membrane integrity. Heterologous expression of a synthetic garQICD operon was established in Corynebacterium glutamicum demonstrating that genes garQICD are responsible for biosynthesis and secretion of garvicin Q. Moreover, production of garvicin Q by the recombinant C. glutamicum strain was improved by using a defined medium yet product levels were still considerably lower than with the natural L. petauri B1726 producer strain.Collectively, our data identifies the genetic basis for production of the bacteriocin garvicin Q by L. petauri B1726 and provides insights into the receptor and mode of action of garvicin Q. Moreover, we successfully performed first attempts towards biotechnological production of this interesting bacteriocin using natural and heterologous hosts.

Optimized recombinant production of the bacteriocin garvicin Q by Corynebacterium glutamicum.

Bacteriocins are antimicrobial peptides applied in food preservation and are interesting candidates as alternatives to conventional antibiotics or as microbiome modulators. Recently, we established Corynebacterium glutamicum as a suitable production host for various bacteriocins including garvicin Q (GarQ). Here, we establish secretion of GarQ by C. glutamicum via the Sec translocon achieving GarQ titers of about 7 mg L-1 in initial fermentations. At neutral pH, the cationic peptide is efficiently adsorbed to the negatively charged envelope of producer bacteria limiting availability of the bacteriocin in culture supernatants. A combination of CaCl2 and Tween 80 efficiently reduces GarQ adsorption to C. glutamicum. Moreover, cultivation in minimal medium supplemented with CaCl2 and Tween 80 improves GarQ production by C. glutamicum to about 15 mg L-1 but Tween 80 resulted in reduced GarQ activity at later timepoints. Using a reporter strain and proteomic analyses, we identified HtrA, a protease associated with secretion stress, as another potential factor limiting GarQ production. Transferring production to HtrA-deficient C. glutamicum K9 improves GarQ titers to close to 40 mg L-1. Applying conditions of low aeration prevented loss in activity at later timepoints and improved GarQ titers to about 100 mg L-1. This is about 50-fold higher than previously shown with a C. glutamicum strain employing the native GarQ transporter GarCD for secretion and in the range of levels observed with the native producer Lactococcus petauri B1726. Additionally, we tested several synthetic variants of GarQ and were able to show that exchange of the methionine in position 5 to a phenylalanine (GarQM5F) results in markedly increased activity against Lactococcus lactis and Listeria monocytogenes. In summary, our findings shed light on several aspects of recombinant GarQ production that may also be of relevance for production with natural producers and other bacteriocins.