This review provides a comprehensive update of the advances in discovery, biosynthesis, and engineering of ribosomally-synthesized and post-translationally modified peptides (RiPPs).

Lactic acid bacteria (LAB) constitute a heterogeneous group of microorganisms that produce lactic acid as the major product during the fermentation process. LAB are Gram-positive bacteria with great biotechnological potential in the food industry. They can produce bacteriocins, which are proteinaceous antimicrobial molecules with a diverse genetic origin, posttranslationally modified or not, that can help the producer organism to outcompete other bacterial species. In this review, we focus on the various types of bacteriocins that can be found in LAB and the organization and regulation of the gene clusters responsible for their production and biosynthesis, and consider the food applications of the prototype bacteriocins from LAB. Furthermore, we propose a revised classification of bacteriocins that can accommodate the increasing number of classes reported over the last years.

Identifying genes encoding bacteriocins and ribosomally synthesized and posttranslationally modified peptides (RiPPs) can be a challenging task. Especially those peptides that do not have strong homology to previously identified peptides can easily be overlooked. Extensive use of BAGEL2 and user feedback has led us to develop BAGEL3. BAGEL3 features genome mining of prokaryotes, which is largely independent of open reading frame (ORF) predictions and has been extended to cover more (novel) classes of posttranslationally modified peptides. BAGEL3 uses an identification approach that combines direct mining for the gene and indirect mining via context genes. Especially for heavily modified peptides like lanthipeptides, sactipeptides, glycocins and others, this genetic context harbors valuable information that is used for mining purposes. The bacteriocin and context protein databases have been updated and it is now easy for users to submit novel bacteriocins or RiPPs. The output has been simplified to allow user-friendly analysis of the results, in particular for large (meta-genomic) datasets. The genetic context of identified candidate genes is fully annotated. As input, BAGEL3 uses FASTA DNA sequences or folders containing multiple FASTA formatted files. BAGEL3 is freely accessible at http://bagel.molgenrug.nl.

Lantibiotics are ribosomally synthesized and posttranslationally modified antimicrobial compounds containing lanthionine and methyl-lanthionine residues. Nisin, one of the most extensively studied and used lantibiotics, has been shown to display very potent activity against Gram-positive bacteria, and stable resistance is rarely observed. By binding to lipid II and forming pores in the membrane, nisin can cause the efflux of cellular constituents and inhibit cell wall biosynthesis. However, the activity of nisin against Gram-negative bacteria is much lower than that against Gram-positive bacteria, mainly because lipid II is located at the inner membrane, and the rather impermeable outer membrane in Gram-negative bacteria prevents nisin from reaching lipid II. Thus, if the outer membrane-traversing efficiency of nisin could be increased, the activity against Gram-negative bacteria could, in principle, be enhanced. In this work, several relatively short peptides with activity against Gram-negative bacteria were selected from literature data to be fused as tails to the C terminus of either full or truncated nisin species. Among these, we found that one of three tails (tail 2 [T2; DKYLPRPRPV], T6 [NGVQPKY], and T8 [KIAKVALKAL]) attached to a part of nisin displayed improved activity against Gram-negative microorganisms. Next, we rationally designed and reengineered the most promising fusion peptides. Several mutants whose activity significantly outperformed that of nisin against Gram-negative pathogens were obtained. The activity of the tail 16 mutant 2 (T16m2) construct against several important Gram-negative pathogens (i.e., Escherichia coli , Klebsiella pneumoniae , Acinetobacter baumannii , Pseudomonas aeruginosa , Enterobacter aerogenes ) was increased 4- to 12-fold compared to that of nisin. This study indicates that the rational design of nisin can selectively and significantly improve its outer membrane-permeating capacity as well as its activity against Gram-negative pathogens. IMPORTANCE Lantibiotics are antimicrobial peptides that are highly active against Gram-positive bacteria but that have relatively poor activity against most Gram-negative bacteria. Here, we modified the model lantibiotic nisin by fusing parts of it to antimicrobial peptides with known activity against Gram-negative bacteria. The appropriate selection of peptidic moieties that could be attached to (parts of) nisin could lead to a significant increase in its inhibitory activity against Gram-negative bacteria. Using this strategy, hybrids that outperformed nisin by displaying 4- to 12-fold higher levels of activity against relevant Gram-negative bacterial species were produced. This study shows the power of modified peptide engineering to alter target specificity in a desired direction.

This review highlights the main genetic features of circular bacteriocins, which require the co-ordinated expression of several genetic determinants