Jeella Z. Acedo

A bioinformatics-driven course-based undergraduate research experience (CURE) project was developed and implemented in an upper-level biochemistry course to address the need for an accessible, lecture-based framework that integrates contemporary computational protein analysis into undergraduate biochemistry education. Students predicted the physicochemical and structural properties of uncharacterized, putative antimicrobial peptides using freely accessible databases, software, and web-based servers, including GenBank and UniProt databases, ExPASy ProtParam, SWISS-MODEL, AlphaFold 3, and PyMOL. Students were asked to identify key molecular features, formulate hypotheses to inform subsequent purification and functional investigations, and present their outputs in abstract, poster, and video formats. Student perceptions of learning and skill development were collected using a Likert scale survey and open-ended feedback. Strong perceived learning gains were highlighted by the Likert scale data, while positive learning experiences and skill development were prominent themes captured in the qualitative feedback. Group dynamics emerged as an important factor influencing the student experience, with both positive and negative perspectives reported. Altogether, the overall feedback was positive, showcasing that the CURE enhanced students’ understanding of protein biochemistry while developing computational, analytical, and scientific communication skills. Lastly, the CURE project described here provides a framework that can be adapted to various protein families beyond antimicrobial proteins.

Bacteriocins are ribosomally synthesized antimicrobial peptides that are emerging as natural preservatives and alternatives to conventional antibiotics in response to the growing threat of antimicrobial resistance. Here, we provide a comprehensive experimental characterization of corynacin K411, previously reported as K411/AucA. The corynacin K411 biosynthetic gene cluster encodes a set of proteins that support a coordinated system for peptide production, secretion, and self-immunity. Corynacin K411 exhibits potent activity against various bacteria, including the foodborne pathogen Listeria monocytogenes . It demonstrates stability across acidic and alkaline pH (pH 2 to 10) and high temperatures (37 °C and 60 °C). It exerts bactericidal activity by disrupting membrane integrity, causing leakage of nucleic acids, proteins, potassium and magnesium ions. Importantly, corynacin K411 is non-hemolytic and non-cytotoxic to human keratinocytes. In addition, it inhibits L. monocytogenes biofilm formation, reduces the biomass of pre-formed biofilms, and lowers bacterial cell counts in a milk preservation model system. Structural analysis of the AlphaFold-predicted three-dimensional structure indicates a saposin-like fold, consistent with other characterized saposin-like leaderless bacteriocins.

The emergence of antimicrobial resistance has prompted the search for antibiotic alternatives, and bacteriocins have emerged as promising alternatives to traditional antibiotics. Bacteriocins refer to a large family of antimicrobial peptides that are ribosomally synthesized by bacteria. The class of bacteriocins called leaderless bacteriocins is a unique group distinguished by the absence of a leader peptide and post-translational modifications, and hence their linear structures. Members of this class have been discovered by a traditional approach of screening bacterial isolates for antimicrobial activity and subsequently identifying the active molecule. This study presents the first extensive genome mining approach for leaderless bacteriocin discovery. In detail, we performed a precursor peptide-based genome mining to search for novel leaderless bacteriocins. Over 400 producer organisms with putative leaderless bacteriocins encoded in their genomes were identified, revealing the widespread occurrence of leaderless bacteriocins. Among the identified putative bacteriocins, 122 are unique peptide sequences. To validate our genome mining results, a novel bacteriocin we termed miticin, encoded in the genome of Streptococcus mitis, was obtained through chemical synthesis. Miticin was indeed found to be active, specifically against a wide range of Gram-positive bacteria. With this study, we provide the foundation for exploring a repertoire of potential novel antimicrobials identified through an innovative bioinformatics-guided approach, representing a significant leap compared to traditional screening and isolation methods for the discovery of bioactive molecules, specifically leaderless bacteriocins.

The rise of drug-resistant bacteria is a major threat to public health, highlighting the urgent need for new antimicrobial compounds and treatments. Bacteriocins, which are ribosomally synthesized antimicrobial peptides produced by bacteria, hold promise as alternatives to conventional antibiotics. In this study, we identified and characterized a novel leaderless bacteriocin, bawcin, the first bacteriocin to be characterized from a Bacillus wiedmannii species. Chemically synthesized and purified bawcin was shown to be active against a broad range of Gram-positive bacteria, including foodborne pathogens Staphylococcus aureus, Bacillus cereus, and Listeria monocytogenes. Stability screening revealed that bawcin is stable over a wide range of pH (2.0–10.0), temperature conditions (25–100 °C), and against the proteases, papain and pepsin. Lastly, three-dimensional structure homology modeling suggests that bawcin contains a saposin-fold with amphipathic helices and a highly cationic surface that may be critical for membrane interaction and the subsequent cell death of its targets. This study provides the foundational understanding of the activity and properties of bawcin, offering valuable insights into its applications across different antimicrobial uses, including as a natural preservative in food and livestock industries.

Lanthipeptides are polycyclic peptides characterized by the presence of lanthionine (Lan) and/or methyllanthionine (MeLan). They are members of the ribosomally synthesized and post-translationally modified peptides (RiPPs). The stereochemical configuration of (Me)Lan cross-links is important for the bioactivity of lanthipeptides. To date, MeLan residues in characterized lanthipeptides have either the 2S,3S or 2R,3R stereochemistry. Herein, we reconstituted in Escherichia coli the biosynthetic pathway toward SapT, a class I lanthipeptide that exhibits morphogenetic activity. Through the synthesis of standards, the heterologously produced peptide was shown to possess three MeLan residues with the 2S,3R stereochemistry (d-allo-l-MeLan), the first time such stereochemistry has been observed in a lanthipeptide. Bioinformatic analysis of the biosynthetic enzymes suggests this stereochemistry may also be present in other lanthipeptides. Analysis of another gene cluster in Streptomyces coelicolor that is widespread in actinobacteria confirmed another example of d-allo-l-MeLan and verified the bioinformatic prediction. We propose a mechanism for the origin of the unexpected stereochemistry and provide support using site-directed mutagenesis.

Head-to-tail cyclized bacteriocins are ribosomally synthesized antimicrobial peptides that are defined by peptide backbone cyclization involving the N- and C- terminal amino acids. Their cyclic nature and overall three-dimensional fold confer superior stability against extreme pH and temperature conditions, and protease degradation. Most of the characterized head-to-tail cyclized bacteriocins were discovered through a traditional approach that involved the screening of bacterial isolates for antimicrobial activity and subsequent isolation and characterization of the active molecule. In this study, we performed genome mining using transporter protein sequences associated with experimentally validated head-to-tail cyclized bacteriocins as driver sequences to search for novel bacteriocins. Biosynthetic gene cluster analysis was then performed to select the high probability functional gene clusters. A total of 387 producer strains that encode putative head-to-tail cyclized bacteriocins were identified. Sequence and phylogenetic analyses revealed that this class of bacteriocins is more diverse than previously thought. Furthermore, our genome mining strategy captured hits that were not identified in precursor-based bioprospecting, showcasing the utility of this approach to expanding the repertoire of head-to-tail cyclized bacteriocins. This work sets the stage for future isolation of novel head-to-tail cyclized bacteriocins to serve as possible alternatives to traditional antibiotics and potentially help address the increasing threat posed by resistant pathogens.

Correction for ‘Structural determinants of macrocyclization in substrate-controlled lanthipeptide biosynthetic pathways’ by Silvia C. Bobeica et al., Chem. Sci., 2020, DOI: 10.1039/d0sc01651a.

Lanthipeptides are characterized by thioether crosslinks formed by post-translational modifications. The cyclization process that favors a single ring pattern over many other possible ring patterns has been the topic of much speculation. Recent studies suggest that for some systems the cyclization pattern and stereochemistry is determined not by the enzyme, but by the sequence of the precursor peptide. However, the factors that govern the outcome of the cyclization process are not understood. This study presents the three-dimensional structures of seven lanthipeptides determined by nuclear magnetic resonance spectroscopy, including five prochlorosins and the two peptides that make up cytolysin, a virulence factor produced by Enterococcus faecalis that is directly linked to human disease. These peptides were chosen because their substrate sequence determines either the ring pattern (prochlorosins) or the stereochemistry of cyclization (cytolysins). We present the structures of prochlorosins 1.1, 2.1, 2.8, 2.10 and 2.11, the first three-dimensional structures of prochlorosins. Our findings provide insights into the molecular determinants of cyclization as well as why some prochlorosins may be better starting points for library generation than others. The structures of the large and small subunits of the enterococcal cytolysin show that these peptides have long helical stretches, a rare observation for lanthipeptides characterized to date. These helices may explain their pore forming activity and suggest that the small subunit may recognize a molecular target followed by recruitment of the large subunit to span the membrane.

Class II lanthipeptides belong to a diverse group of natural products known as ribosomally synthesized and post-translationally modified peptides (RiPPs). Most RiPP precursor peptides contain an N-terminal recognition sequence known as the leader peptide, which is typically recognized by biosynthetic enzymes that catalyze modifications on a C-terminal core peptide. For class II lanthipeptides these are carried out by a bifunctional lanthipeptide synthetase (LanM) that catalyzes dehydration and cyclization reactions on peptidic substrates to generate thioether containing, macrocyclic molecules. Some lanthipeptide synthetases are extraordinarily substrate tolerant, making them promising candidates for biotechnological applications such as combinatorial biosynthesis and cyclic peptide library construction. In this study, we characterized the mode of substrate recognition of HalM2, the lanthipeptide synthetase responsible for the production of the antimicrobial peptide haloduracin β. Using NMR spectroscopic techniques, in vitro binding assays, and enzyme activity assays, we identified substrate residues that are important for binding to HalM2 and for post-translational modification of the peptide substrates. Additionally, we provide evidence of the binding site on the enzyme using binding assays with enzyme truncants, hydrogen-deuterium exchange mass spectrometry, and photoaffinity labeling. Understanding the mechanism by which lanthipeptide synthetases recognize their substrate will facilitate their use in biotechnology, as well as further our general understanding of how RiPP enzymes recognize their substrates.