PARAGEN is Syngulon’s bacteriocin collection, bringing together natural and synthetic bacteriocins selected for their diverse antimicrobial spectra and potential applications. Bacteriocins are genetically encoded antimicrobial peptides capable of targeting specific microbial strains or species, making them valuable tools for industrial fermentation, contamination control, animal and human health, and other applications in which microbial populations need to be selectively controlled.
Current bacteriocin databases classify bacteriocins based on their structural features such as extensive post-translational modification or bacteriocins that are active as two-component systems.
An Application-Oriented Bacteriocin Collection
To take advantage of the wide range of potential applications, Syngulon has developed and continues to expand PARAGEN, an in-house collection based on a bacteriocin DNA library. In addition to “natural” bacteriocins, we will use synthetic biology to tune and create de novo peptides based on the needs of our industrial partners. These can be used in combination (“cocktails”) to finely adjust microbial community composition. This bacteriocin collection is being classified according to characteristics relevant to industrial applications, including target spectrum, operating conditions and the performance of bacteriocin combinations. Indeed, our experimental data show the potential for bacteriocins to work in combination.
Natural, Engineered and Combined Bacteriocins
Bacteriocin qualities can be changed through simple modifications, from minor changes to the amino acid sequence to the removal or addition of secondary modifications. These changes can affect the level of bacteriocin activity as well as species or strain target specificity.
For example, the bacteriocin Enterocin AS-48 is a circular peptide with a C-terminal tryptophan joined to an N-terminal methionine in the mature peptide. Circularization occurs through the activity of several accessory genes in the bacteriocin genetic locus. However, activity of this bacteriocin can still be observed when it is produced as a linear peptide; this can be done by mutating the C-terminal tryptophan to an alanine residue unable to bind to the N-terminal methionine, or through expression of the bacteriocin gene (Sanchez-Hidalgo et al. 2011).
Additionally, bacteriocin hybrids have been demonstrated to enhance the target spectrum. Swapping the N-termini of Pediocin PA-1 and Enterocin 50-52 demonstrated that a hybrid bacteriocin capable of killing both Gram-positive and Gram-negative targets even when one of the wild-type bacteriocins has no activity against Gram-negative bacteria (Tiwari et al. 2015).
We currently use bacteriocins as selective agents in microbial fermentation. We envision expanding the use of bacteriocins to reshape the microbial community as needed. To this end, bacteriocins can be used not only individually, but in “cocktails” containing multiple bacteriocins, each one targeting a specific microbial strain or species. The desired result can be either the elimination of unwanted strains, or the maintenance of a certain proportion of strains.
To facilitate this “microbial community engineering”, we are building a collection of bacteriocins that we can deploy against any possible contaminant, or for shaping a microbial community as needed.
The current academic classification system separates bacteriocins into lantibiotics and non-lantibiotics, and further subdivides this second group based on similarity to pediocins (a certain type of bacteriocin), two-component systems, circular bacteriocins, and larger, protein-like bacteriocins. This system is updated and restructured as novel bacteriocins and bacteriocins with differing characteristics are discovered.
A Functional Classification for Industrial Applications
We wish to build a classification system for our PARAGEN collection based on the needs of our industrial partners. This could include bacteriocins specific for certain species, or bacteriocins that work well at certain temperatures or pH levels. It would also include the functionality of certain bacteriocin combinations, as some may complement each other better than others. This will be determined by data collected at bench and industrial scales, as well as through systems modelling of bacteriocin and microbe interaction.
PARAGEN supports Syngulon’s antibiotic-free selection, contamination control and therapeutic applications by providing a diverse source of bacteriocins for screening, engineering and rational combination.
Finally, we will continue to expand the PARAGEN collection with bacteriocins created or adapted through synthetic biology, providing a versatile toolbox to shape microbial communities and develop targeted bacteriocin solutions for industrial and therapeutic applications.