A systematic comparison of natural product potential, with an emphasis on RiPPs, by mining of bacteria of three large ecosystems.

The implementation of several global microbiome studies has yielded extensive insights into the biosynthetic potential of natural microbial communities. However, studies on the distribution of several classes of ribosomally synthesized and post-translationally modified peptides (RiPPs), non-ribosomal peptides (NRPs) and polyketides (PKs) in different large microbial ecosystems have been very limited. Here, we collected a large set of metagenome-assembled bacterial genomes from marine, freshwater and terrestrial ecosystems to investigate the biosynthetic potential of these bacteria. We demonstrate the utility of public dataset collections for revealing the different secondary metabolite biosynthetic potentials among these different living environments. We show that there is a higher occurrence of RiPPs in terrestrial systems, while in marine systems, we found relatively more terpene-, NRP-, and PK encoding gene clusters. Among the many new biosynthetic gene clusters (BGCs) identified, we analyzed various Nif-11-like and nitrile hydratase leader peptide (NHLP) containing gene clusters that would merit further study, including promising products, such as mersacidin-, LAP- and proteusin analogs. This research highlights the significance of public datasets in elucidating the biosynthetic potential of microbes in different living environments and underscores the wide bioengineering opportunities within the RiPP family.

Uncovering the diversity and distribution of biosynthetic gene clusters of prochlorosins and other putative RiPPs in marine Synechococcus strains.

IMPORTANCE: Genome mining studies have revealed the remarkable combinatorial diversity of ribosomally synthesized and post-translationally modified peptides (RiPPs) in marine bacteria, including prochlorosins. However, mining strategies also prove valuable in investigating the genomic landscape of associated genes within biosynthetic gene cluster (BGC) specific to targeted RiPPs of interest. Our study contributes to the enrichment of knowledge regarding prochlorosin diversity. It offers insights into potential mechanisms involved in their biosynthesis and modification, such as hyper-modification, which may give rise to active lantibiotics. Additionally, our study uncovers putative novel promiscuous post-translational enzymes, thereby expanding the chemical space explored within the Synechococcus genus. Moreover, this research extends the applications of mining techniques beyond the discovery of new RiPP-like clusters, allowing for a deeper understanding of genomics and diversity. Furthermore, it holds the potential to reveal previously unknown functions within the intriguing RiPP families, particularly in the case of prochlorosins.

Bioinformatic mining for RiPP biosynthetic gene clusters in Bacteroidales reveals possible new subfamily architectures and novel natural products.

The Bacteroidales order, widely distributed among diverse human populations, constitutes a key component of the human microbiota. Members of this Gram-negative order have been shown to modulate the host immune system, play a fundamental role in the gut's microbial food webs, or be involved in pathogenesis. Bacteria inhabiting such a complex environment as the human microbiome are expected to display social behaviors and, hence, possess factors that mediate cooperative and competitive interactions. Different types of molecules can mediate interference competition, including non-ribosomal peptides (NRPs), polyketides, and bacteriocins. The present study investigates the potential of Bacteroidales bacteria to biosynthesize class I bacteriocins, which are ribosomally synthesized and post-translationally modified peptides (RiPPs). For this purpose, 1,136 genome-sequenced strains from this order were mined using BAGEL4. A total of 1,340 areas of interest (AOIs) were detected. The most commonly identified enzymes involved in RiPP biosynthesis were radical S-adenosylmethionine (rSAM), either alone or in combination with other biosynthetic enzymes such as YcaO. A more comprehensive analysis of a subset of 9 biosynthetic gene clusters (BGCs) revealed a consistent association in Bacteroidales BGCs between peptidase-containing ATP-binding transporters (PCATs) and precursor peptides with GG-motifs. This finding suggests a possibly shared mechanism for leader peptide cleavage and transport of mature products. Notably, human metagenomic studies showed a high prevalence and abundance of the RiPP BGCs from Phocaeicola vulgatus and Porphyromonas gulae. The mature product of P. gulae BGC is hypothesized to display γ-thioether linkages and a C-terminal backbone amidine, a potential new combination of post-translational modifications (PTM). All these findings highlight the RiPP biosynthetic potential of Bacteroidales bacteria, as a rich source of novel peptide structures of possible relevance in the human microbiome context.

Investigating the Specificity of the Dehydration and Cyclization Reactions in Engineered Lanthipeptides by Synechococcal SyncM.

ProcM-like enzymes are class II promiscuous lanthipeptide synthetases that are an attractive tool in synthetic biology for producing lanthipeptides with biotechnological or clinically desired properties. SyncM is a recently described modification enzyme from this family used to develop a versatile expression platform for engineering lanthipeptides. Most remarkably, SyncM can modify up to 79 SyncA substrates in a single strain. Six SyncAs were previously characterized from this pool of substrates. They showed particular characteristics, such as the presence of one or two lanthionine rings, different flanking residues influencing ring formation, and different ring directions, demonstrating the relaxed specificity of SyncM toward its precursor peptides. To gain a deeper understanding of the potential of SyncM as a biosynthetic tool, we further explored the enzyme's capabilities and limits in dehydration and ring formation. We used different SyncA scaffolds for peptide engineering, including changes in the ring's directionality (relative position of Ser/Thr to Cys in the peptide) and size. We further aimed to rationally design mimetics of cyclic antimicrobials and introduce macrocycles in prochlorosin-related and nonrelated substrates. This study highlights the largest lanthionine ring with 15 amino acids (ring-forming residues included) described to date. Taking advantage of the amino acid substrate tolerance of SyncM, we designed the first single-SyncA-based antimicrobial. The insights gained from this work will aid future bioengineering studies. Additionally, it broadens SyncM's application scope for introducing macrocycles in other bioactive molecules.

Whole Genome Sequencing of Chinese White Dolphin (Sousa chinensis) for High-Throughput Screening of Antihypertensive Peptides.

Chinese white dolphin (Sousa chinensis), also known as the Indo-Pacific humpback dolphin, has been classified as "Vulnerable" on the IUCN Red List of Threatened Species. It is a special cetacean species that lives in tropical and subtropical nearshore waters, with significant differences from other cetaceans. Here, we sequenced and assembled a draft genome of the Chinese white dolphin with a total length of 2.3 Gb and annotation of 18,387 protein-coding genes. Genes from certain expanded families are potentially involved in DNA replication and repairing, suggesting that they may be related to adaptation of this marine mammal to nearshore environments. We also discovered that its historical population had undergone a remarkable bottleneck incident before the Mindel glaciation. In addition, a comparative genomic survey on antihypertensive peptides (AHTPs) among five representative mammals with various residential habitats (such as remarkable differences in exogenous ion concentrations and sea depth) revealed that these small bioactive peptides were highly conserved among these examined mammals, and they had the most abundant hits in collagen subunit proteins, especially for two putative AHTP peptides Gly-Leu-Pro (GLP) and Leu-Gly-Pro (LGP). Our genome assembly will be a valuable resource for further genetic researches on adaptive ecology and conservation biology of cetaceans, and for in-depth investigations into bioactive peptides in aquatic and terrestrial mammals for development of peptide-based drugs to treat various human cardiovascular diseases.

High-Throughput Identification of Putative Antimicrobial Peptides from Multi-Omics Data of the Lined Seahorse (Hippocampus erectus).

Lined seahorse (Hippocampus erectus), the most widely cultivated seahorse in China, has been in short supply because of its important medicinal value; meanwhile, unnatural deaths caused by various diseases (especially enteritis) have limited their practical large-scale aquaculture. Antimicrobial peptides (AMPs), as the best alternative to antibiotics, have been extensively applied in agricultural practices. In this study, we identified 290 putative AMP sequences from our previously published genome and transcriptome data of the lined seahorse. Among them, 267 are novel, and 118 were validated by our proteome data generated in the present study. It seems that there is a tissue preference in the distribution of AMP/AMP precursor transcripts, such as lectins in the male pouch. In addition, their transcription levels usually varied during development. Interestingly, the representative lectins kept extremely high levels at the pre-pregnancy stage while at relatively lower levels at other stages. Especially Lectin25, with the highest transcription levels and significant developmental changes, has been reported to be involved in seahorse and human pregnancy. The comparison of transcriptome data between one-day and three-month juveniles indicated that Hemoglobin2 (Hemo2) was significantly upregulated in the body, haslet, and brain. Our proteome data of female and male individuals revealed three putative AMP precursors with sexual specificity, including two male-biased cyclin-dependent kinases (CDK-like16 and CDK-like23) and one female-biased bovine pancreatic trypsin inhibitor 2 (BPTI2). In conclusion, our present high-throughput identification of putative AMP sequences from multi-omics (including genomics, transcriptomics, and proteomics) data provides an overview of AMPs in the popular lined seahorse, which lays a solid foundation for further development of AMP-based fish food additives and human drugs.

High Throughput Identification of Antihypertensive Peptides from Fish Proteome Datasets.

Antihypertensive peptides (AHTPs) are a group of small peptides with the main role to block key enzymes or receptors in the angiotensin genesis pathway. A great number of AHTPs have been isolated or digested from natural food resources; however, comprehensive studies on comparisons of AHTPs in various species from the perspective of big data are rare. Here, we established a simplified local AHTP database, and performed in situ mapping for high throughput identification of AHTPs with high antihypertensive activity from high-quality whole proteome datasets of 18 fish species. In the 35 identified AHTPs with reported high activity, we observed that Gly-Leu-Pro, Leu-Pro-Gly, and Val-Ser-Val are the major components of fish proteins, and AHTP hit numbers in various species demonstrated a similar distributing pattern. Interestingly, Atlantic salmon (Salmo salar) is in possession of far more abundant AHTPs compared with other fish species. In addition, collagen subunit protein is the largest group with more matching AHTPs. Further exploration of two collagen subunits (col4a5 and col8a1) in more fish species suggested that the hit pattern of these conserved proteins among teleost is almost the same, and their phylogeny is consistent with the evolution of these fish species. In summary, our present study provides basic information for the relationship of AHTPs with fish proteins, which sheds light on rapid discovery of marine drugs or food additives from fish protein hydrolysates to alleviate hypertension.

High-Throughput Identification of Antimicrobial Peptides from Amphibious Mudskippers.

Widespread existence of antimicrobial peptides (AMPs) has been reported in various animals with comprehensive biological activities, which is consistent with the important roles of AMPs as the first line of host defense system. However, no big-data-based analysis on AMPs from any fish species is available. In this study, we identified 507 AMP transcripts on the basis of our previously reported genomes and transcriptomes of two representative amphibious mudskippers, Boleophthalmus pectinirostris (BP) and Periophthalmus magnuspinnatus (PM). The former is predominantly aquatic with less time out of water, while the latter is primarily terrestrial with extended periods of time on land. Within these identified AMPs, 449 sequences are novel; 15 were reported in BP previously; 48 are identically overlapped between BP and PM; 94 were validated by mass spectrometry. Moreover, most AMPs presented differential tissue transcription patterns in the two mudskippers. Interestingly, we discovered two AMPs, hemoglobin ß1 and amylin, with high inhibitions on Micrococcus luteus. In conclusion, our high-throughput screening strategy based on genomic and transcriptomic data opens an efficient pathway to discover new antimicrobial peptides for ongoing development of marine drugs.

A Transcriptomic Survey of Ion Channel-Based Conotoxins in the Chinese Tubular Cone Snail (Conus betulinus).

Conotoxins in the venom of cone snails (Conus spp.) are a mixture of active peptides that work as blockers, agonists, antagonists, or inactivators of various ion channels. Recently we reported a high-throughput method to identify 215 conotoxin transcripts from the Chinese tubular cone snail, C. betulinus. Here, based on the previous datasets of four transcriptomes from three venom ducts and one venom bulb, we explored ion channel-based conotoxins and predicted their related ion channel receptors. Homologous analysis was also performed for the most abundant ion channel protein, voltage-gated potassium (Kv; with Kv1.1 as the representative), and the most studied ion channel receptor, nicotinic acetylcholine receptor (nAChR; with α2-nAChR as the representative), in different animals. Our transcriptomic survey demonstrated that ion channel-based conotoxins and related ion channel proteins/receptors transcribe differentially between the venom duct and the venom bulb. In addition, we observed that putative κ-conotoxins were the most common conotoxins with the highest transcription levels in the examined C. betulinus. Furthermore, Kv1.1 and α2-nAChR were conserved in their functional domains of deduced protein sequences, suggesting similar effects of conotoxins via the ion channels in various species, including human beings. In a word, our present work suggests a high-throughput way to develop conotoxins as potential drugs for treatment of ion channel-associated human diseases.