Automated workflow for characterization of bacteriocin production in natural producers Lactococcus lactis and Latilactobacillus sakei.

BACKGROUND: Lactic acid bacteria are commonly used as protective starter cultures in food products. Among their beneficial effects is the production of ribosomally synthesized peptides termed bacteriocins that kill or inhibit food-spoiling bacteria and pathogens, e.g., members of the Listeria species. As new bacteriocins and producer strains are being discovered rapidly, modern automated methods for strain evaluation and bioprocess development are required to accelerate screening and development processes. RESULTS: In this study, we developed an automated workflow for screening and bioprocess optimization for bacteriocin producing lactic acid bacteria, consisting of microcultivation, sample processing and automated antimicrobial activity assay. We implemented sample processing workflows to minimize bacteriocin adsorption to producer cells via addition of Tween 80 and divalent cations to the cultivation media as well as acidification of culture broth prior to cell separation. Moreover, we demonstrated the applicability of the automated workflow to analyze influence of media components such as MES buffer or yeast extract for bacteriocin producers Lactococcus lactis B1629 and Latilactobacillus sakei A1608. CONCLUSIONS: Our automated workflow provides advanced possibilities to accelerate screening and bioprocess optimization for natural bacteriocin producers. Based on its modular concept, adaptations for other strains, bacteriocin products and applications are easily carried out and a unique tool to support bacteriocin research and bioprocess development is provided.

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.

Microfluidic single-cell scale-down bioreactors: A proof-of-concept for the growth of Corynebacterium glutamicum at oscillating pH values.

In large-scale bioreactors, gradients in cultivation parameters such as oxygen, substrate, and pH result in fluctuating cell environments. pH fluctuations were identified as a critical parameter for bioprocess performance. Traditionally, scale-down systems at the laboratory scale are used to analyze the effects of fluctuating pH values on strains and thus process performance. Here, we demonstrate the application of dynamic microfluidic single-cell cultivation (dMSCC) as a novel scale-down system for the characterization of Corynebacterium glutamicum growth using oscillating pH conditions as a model stress factor. A detailed comparison between two-compartment reactor (two-CR) scale-down experiments and dMSCC was performed for one specific pH oscillation between reference pH 7 (~8 min) and disturbed pH 6 (~2 min). Similar reductions in growth rates were observed in both systems (dMSCC 21% and two-CR 27%) compared to undisturbed cultivation at pH 7. Afterward, systematic experiments at symmetric and asymmetric pH oscillations, between pH ranges of 4-6 and 8-11 and different intervals from 1 to 20 min, were performed to demonstrate the unique application range and throughput of the dMSCC system. Finally, the strength of the dMSCC application was demonstrated by mimicking fluctuating environmental conditions of a putative large-scale bioprocess, which is difficult to conduct using two-CRs.