Cell-Free Systems: Ideal Platforms for Accelerating the Discovery and Production of Peptide-Based Antibiotics.

Peptide-based antibiotics (PBAs), including antimicrobial peptides (AMPs) and their synthetic mimics, have received significant interest due to their diverse and unique bioactivities. The integration of high-throughput sequencing and bioinformatics tools has dramatically enhanced the discovery of enzymes, allowing researchers to identify specific genes and metabolic pathways responsible for producing novel PBAs more precisely. Cell-free systems (CFSs) that allow precise control over transcription and translation in vitro are being adapted, which accelerate the identification, characterization, selection, and production of novel PBAs. Furthermore, these platforms offer an ideal solution for overcoming the limitations of small-molecule antibiotics, which often lack efficacy against a broad spectrum of pathogens and contribute to the development of antibiotic resistance. In this review, we highlight recent examples of how CFSs streamline these processes while expanding our ability to access new antimicrobial agents that are effective against antibiotic-resistant infections.

Bioactive compounds from food-grade Bacillus.

Bacillus species have attracted significant attention in recent years due to their potential for producing various bioactive compounds with diverse functional properties. This review highlights bioactive substances from food-grade Bacillus strains and their applications in functional foods and nutraceuticals. The metabolic capacities of Bacillus species have allowed them to generate a wide range of bioactive substances, including vitamins, enzymes, anti-microbial peptides, and other non-ribosomal peptides. These substances have a variety of positive effects, including potential cholesterol-lowering and immune-modulatory qualities in addition to anti-oxidant and anti-bacterial actions. The uses and mechanisms of action of these bioactive chemicals can be used to improve the functional qualities and nutritional profile of food products. Examples include the use of anti-microbial peptides to increase safety and shelf life, as well as the use of Bacillus-derived enzymes in food processing to improve digestibility and sensory qualities. The exploitation of bioactive compounds from food-grade Bacillus strains presents a promising frontier in the development of functional foods and nutraceuticals with enhanced health benefits. Due to their wide range of activity and applications, they are considered as important resources for the development of novel medications, agricultural biocontrol agents, and industrial enzymes. Ongoing research into the biosynthetic pathways, functional properties, and applications of these compounds is essential to fully realize their potential in the food industry. This review underscores the significance of various bioactive compounds generated from Bacillus in tackling global issues like environmental sustainability, sustainable agriculture, and antibiotic resistance. Future developments in microbiology and biotechnology will enable us to fully utilize the potential of these amazing microbes, resulting in novel approaches to industry, agriculture, and health. © 2024 Society of Chemical Industry.

Advances in the adenylation domain: discovery of diverse non-ribosomal peptides.

Non-ribosomal peptide synthetases are mega-enzyme assembly lines that synthesize many clinically useful compounds. As a gatekeeper, they have an adenylation (A)-domain that controls substrate specificity and plays an important role in product structural diversity. This review summarizes the natural distribution, catalytic mechanism, substrate prediction methods, and in vitro biochemical analysis of the A-domain. Taking genome mining of polyamino acid synthetases as an example, we introduce research on mining non-ribosomal peptides based on A-domains. We discuss how non-ribosomal peptide synthetases can be engineered based on the A-domain to obtain novel non-ribosomal peptides. This work provides guidance for screening non-ribosomal peptide-producing strains, offers a method to discover and identify A-domain functions, and will accelerate the engineering and genome mining of non-ribosomal peptide synthetases. KEY POINTS: • Introducing adenylation domain structure, substrate prediction, and biochemical analysis methods • Advances in mining homo polyamino acids based on adenylation domain analysis • Creating new non-ribosomal peptides by engineering adenylation domains.

Compilation of the Antimicrobial Compounds Produced by Burkholderia Sensu Stricto.

Due to the increase in multidrug-resistant microorganisms, the investigation of novel or more efficient antimicrobial compounds is essential. The World Health Organization issued a list of priority multidrug-resistant bacteria whose eradication will require new antibiotics. Among them, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae are in the "critical" (most urgent) category. As a result, major investigations are ongoing worldwide to discover new antimicrobial compounds. Burkholderia, specifically Burkholderia sensu stricto, is recognized as an antimicrobial-producing group of species. Highly dissimilar compounds are among the molecules produced by this genus, such as those that are unique to a particular strain (like compound CF66I produced by Burkholderia cepacia CF-66) or antimicrobials found in a number of species, e.g., phenazines or ornibactins. The compounds produced by Burkholderia include N-containing heterocycles, volatile organic compounds, polyenes, polyynes, siderophores, macrolides, bacteriocins, quinolones, and other not classified antimicrobials. Some of them might be candidates not only for antimicrobials for both bacteria and fungi, but also as anticancer or antitumor agents. Therefore, in this review, the wide range of antimicrobial compounds produced by Burkholderia is explored, focusing especially on those compounds that were tested in vitro for antimicrobial activity. In addition, information was gathered regarding novel compounds discovered by genome-guided approaches.

Classification and Multifaceted Potential of Secondary Metabolites Produced by Bacillus subtilis Group: A Comprehensive Review.

Despite their remarkable biosynthetic potential, Bacillus subtilis have been widely overlooked. However, their capability to withstand harsh conditions (extreme temperature, Ultraviolet (UV) and γ-radiation, and dehydration) and the promiscuous metabolites they synthesize have created increased commercial interest in them as a therapeutic agent, a food preservative, and a plant-pathogen control agent. Nevertheless, the commercial-scale availability of these metabolites is constrained due to challenges in their accessibility via synthesis and low fermentation yields. In the context of this rising in interest, we comprehensively visualized the antimicrobial peptides produced by B. subtilis and highlighted their prospective applications in various industries. Moreover, we proposed and classified these metabolites produced by the B. subtilis group based on their biosynthetic pathways and chemical structures. The biosynthetic pathway, bioactivity, and chemical structure are discussed in detail for each class. We believe that this review will spark a renewed interest in the often disregarded B. subtilis and its remarkable biosynthetic capabilities.

Newest perspectives of glycopeptide antibiotics: biosynthetic cascades, novel derivatives, and new appealing antimicrobial applications.

Glycopeptide antibiotics (GPAs) are a family of non-ribosomal peptide natural products with polypeptide skeleton characteristics, which are considered the last resort for treating severe infections caused by multidrug-resistant Gram-positive pathogens. Over the past few years, an increasing prevalence of Gram-positive resistant strain "superbugs" has emerged. Therefore, more efforts are needed to study and modify the GPAs to overcome the challenge of superbugs. In this mini-review, we provide an overview of the complex biosynthetic gene clusters (BGCs), the ingenious crosslinking and tailoring modifications, the new GPA derivatives, the discoveries of new natural GPAs, and the new applications of GPAs in antivirus and anti-Gram-negative bacteria. With the development and interdisciplinary integration of synthetic biology, next-generation sequencing (NGS), and artificial intelligence (AI), more GPAs with new chemical structures and action mechanisms will constantly be emerging.

[Latest Findings on Polyketides/Non-ribosomal Peptides That Are Secondary Metabolites of Streptococcus mutans].

Dental caries is a chronic infectious disease that occurs in the hard tissue of teeth under the influence of multiple factors, among which bacteria being a key factor. Streptococcus mutans ( S. mutans) is considered a major pathogen that causes caries. Secondary metabolites, including bacteriocins and polyketides/non-ribosomal peptides, are a class of small-molecule compounds synthesized by S. mutans. To date, polyketides/non-ribosomal peptides identified in S. mutans include mutanobactin, mutanocyclin, and mutanofactin, which are synthesized by the mub, muc, and muf biosynthetic gene clusters, respectively. These polyketides/non-ribosomal peptides play important roles in bacterial inter-species competition, oxidative stress, and biofilm formation. In this review, we provided an overview of the synthesis, function and regulation of three polyketides/non-ribosomal peptides of S. mutans, including mutanobactin, mutanocyclin, and mutanofactin, aiming to provide new insights into the cariogenic mechanism of S. mutans and to promote the better management of dental caries.

Momomycin, an Antiproliferative Cryptic Metabolite from the Oxytetracycline Producer Streptomyces rimosus.

Natural products provide an important source of pharmaceuticals and chemical tools. Traditionally, assessment of unexplored microbial phyla has led to new natural products. However, with every new microbe, the number of orphan biosynthetic gene clusters (BGC) grows. As such, the more difficult proposition is finding new molecules from well-studied strains. Herein, we targeted Streptomyces rimosus, the widely-used oxytetracycline producer, for the discovery of new natural products. Using MALDI-MS-guided high-throughput elicitor screening (HiTES), we mapped the global secondary metabolome of S. rimosus and structurally characterized products of three cryptic BGCs, including momomycin, an unusual cyclic peptide natural product with backbone modifications and several non-canonical amino acids. We elucidated important aspects of its biosynthesis and evaluated its bioactivity. Our studies showcase HiTES as an effective approach for unearthing new chemical matter from "drained" strains.

Culture and genome-based analysis of four soil Clostridium isolates reveal their potential for antimicrobial production.

BACKGROUND: Soil bacteria are a major source of specialized metabolites including antimicrobial compounds. Yet, one of the most diverse genera of bacteria ubiquitously present in soil, Clostridium, has been largely overlooked in bioactive compound discovery. As Clostridium spp. thrive in extreme environments with their metabolic mechanisms adapted to the harsh conditions, they are likely to synthesize molecules with unknown structures, properties, and functions. Therefore, their potential to synthesize small molecules with biological activities should be of great interest in the search for novel antimicrobial compounds. The current study focused on investigating the antimicrobial potential of four soil Clostridium isolates, FS01, FS2.2 FS03, and FS04, using a genome-led approach, validated by culture-based methods. RESULTS: Conditioned/spent media from all four Clostridium isolates showed varying levels of antimicrobial activity against indicator microorganism; all four isolates significantly inhibited the growth of Pseudomonas aeruginosa. FS01, FS2.2, and FS04 were active against Bacillus mycoides and FS03 reduced the growth of Bacillus cereus. Phylogenetic analysis together with DNA-DNA hybridization (dDDH), average nucleotide identity (ANI), and functional genome distribution (FGD) analyses confirmed that FS01, FS2.2, and FS04 belong to the species Paraclostridium bifermentans, Clostridium cadaveris, and Clostridium senegalense respectively, while FS03 may represent a novel species of the genus Clostridium. Bioinformatics analysis using antiSMASH 5.0 predicted the presence of eight biosynthetic gene clusters (BGCs) encoding for the synthesis of ribosomally synthesized post-translationally modified peptides (RiPPs) and non-ribosomal peptides (NRPs) in four genomes. All predicted BGCs showed no similarity with any known BGCs suggesting novelty of the molecules from those predicted gene clusters. In addition, the analysis of genomes for putative virulence factors revealed the presence of four putative Clostridium toxin related genes in FS01 and FS2.2 genomes. No genes associated with the main Clostridium toxins were identified in the FS03 and FS04 genomes. CONCLUSIONS: The presence of BGCs encoding for uncharacterized RiPPs and NRPSs in the genomes of antagonistic Clostridium spp. isolated from farm soil indicated their potential to produce novel secondary metabolites. This study serves as a basis for the identification and characterization of potent antimicrobials from these soil Clostridium spp. and expands the current knowledge base, encouraging future research into bioactive compound production in members of the genus Clostridium.

Natural products in the predatory defence of the filamentous fungal pathogen Aspergillus fumigatus.

The kingdom of fungi comprises a large and highly diverse group of organisms that thrive in diverse natural environments. One factor to successfully confront challenges in their natural habitats is the capability to synthesize defensive secondary metabolites. The genetic potential for the production of secondary metabolites in fungi is high and numerous potential secondary metabolite gene clusters have been identified in sequenced fungal genomes. Their production may well be regulated by specific ecological conditions, such as the presence of microbial competitors, symbionts or predators. Here we exemplarily summarize our current knowledge on identified secondary metabolites of the pathogenic fungus Aspergillus fumigatus and their defensive function against (microbial) predators.

More P450s Are Involved in Secondary Metabolite Biosynthesis in Streptomyces Compared to Bacillus, Cyanobacteria, and Mycobacterium.

Unraveling the role of cytochrome P450 monooxygenases (CYPs/P450s), heme-thiolate proteins present in living and non-living entities, in secondary metabolite synthesis is gaining momentum. In this direction, in this study, we analyzed the genomes of 203 Streptomyces species for P450s and unraveled their association with secondary metabolism. Our analyses revealed the presence of 5460 P450s, grouped into 253 families and 698 subfamilies. The CYP107 family was found to be conserved and highly populated in Streptomyces and Bacillus species, indicating its key role in the synthesis of secondary metabolites. Streptomyces species had a higher number of P450s than Bacillus and cyanobacterial species. The average number of secondary metabolite biosynthetic gene clusters (BGCs) and the number of P450s located in BGCs were higher in Streptomyces species than in Bacillus, mycobacterial, and cyanobacterial species, corroborating the superior capacity of Streptomyces species for generating diverse secondary metabolites. Functional analysis via data mining confirmed that many Streptomyces P450s are involved in the biosynthesis of secondary metabolites. This study was the first of its kind to conduct a comparative analysis of P450s in such a large number (203) of Streptomyces species, revealing the P450s' association with secondary metabolite synthesis in Streptomyces species. Future studies should include the selection of Streptomyces species with a higher number of P450s and BGCs and explore the biotechnological value of secondary metabolites they produce.

Heterologous expression of intact biosynthetic gene clusters in Fusarium graminearum.

Filamentous fungi such as species from the genus Fusarium are capable of producing a wide palette of interesting metabolites relevant to health, agriculture and biotechnology. Secondary metabolites are formed from large synthase/synthetase enzymes often encoded in gene clusters containing additional enzymes cooperating in the metabolite's biosynthesis. The true potential of fungal metabolomes remain untapped as the majority of secondary metabolite gene clusters are silent under standard laboratory growth conditions. One way to achieve expression of biosynthetic pathways is to clone the responsible genes and express them in a well-suited heterologous host, which poses a challenge since Fusarium polyketide synthase and non-ribosomal peptide synthetase gene clusters can be large (e.g. as large as 80 kb) and comprise several genes necessary for product formation. The major challenge associated with heterologous expression of fungal biosynthesis pathways is thus handling and cloning large DNA sequences. In this paper we present the successful workflow for cloning, reconstruction and heterologous production of two previously characterized Fusarium pseudograminearum natural product pathways in Fusarium graminearum. In vivo yeast recombination enabled rapid assembly of the W493 (NRPS32-PKS40) and the Fusarium Cytokinin gene clusters. F. graminearum transformants were obtained through protoplast-mediated and Agrobacterium tumefaciens-mediated transformation. Whole genome sequencing revealed isolation of transformants carrying intact copies the gene clusters was possible. Known Fusarium cytokinin metabolites; fusatin, 8-oxo-fusatin, 8-oxo-isopentenyladenine, fusatinic acid together with cis- and trans-zeatin were detected by liquid chromatography and mass spectrometry, which confirmed gene functionality in F. graminearum. In addition the non-ribosomal lipopeptide products W493 A and B was heterologously produced in similar amounts to that observed in the F. pseudograminearum doner. The Fusarium pan-genome comprises more than 60 uncharacterized putative secondary metabolite gene clusters. We nominate the well-characterized F. graminearum as a heterologous expression platform for Fusarium secondary metabolite gene clusters, and present our experience cloning and introducing gene clusters into this species. We expect the presented methods will inspire future endevours in heterologous production of Fusarium metabolites and potentially aid the production and characterization of novel natural products.

Nature's Combinatorial Biosynthesis Produces Vatiamides A-F.

Hybrid type I PKS/NRPS biosynthetic pathways typically proceed in a collinear manner wherein one molecular building block is enzymatically incorporated in a sequence that corresponds to gene arrangement. In this work, genome mining combined with the use of a fluorogenic azide-based click probe led to the discovery and characterization of vatiamides A-F, three structurally diverse alkynylated lipopeptides, and their brominated analogues, from the cyanobacterium Moorea producens ASI16Jul14-2. These derive from a unique combinatorial non-collinear PKS/NRPS system encoded by a 90 kb gene cluster in which an upstream PKS cassette interacts with three separate cognate NRPS partners. This is facilitated by a series of promiscuous intermodule PKS-NRPS docking motifs possessing identical amino acid sequences. This interaction confers a new type of combinatorial capacity for creating molecular diversity in microbial systems.

An immunoinformatics approach to define T cell epitopes from polyketide and non-ribosomal peptide synthesis proteins of Mycobacterium tuberculosis as potential vaccine candidates.

The role of polyketide and non-ribosomal proteins from the class of small molecule metabolism of Mycobacterium tuberculosis is well documented in envelope organization, virulence, and pathogenesis. Consequently, the identification of T cell epitopes from these proteins could serve to define potential antigens for the development of vaccines. Fourty-one proteins from polyketide and non-ribosomal peptide synthesis of small molecule metabolism proteins of M tuberculosis H37Rv were analyzed computationally for the presence of HLA class I binding nanomeric peptides. All possible overlapping nanomeric peptide sequences from 41 small molecule metabolic proteins were generated through in silico and analyzed for their ability to bind to 33 alleles belonging to A, B, and C loci of HLA class I molecule. Polyketide and non-ribosomal protein analyses revealed that 20% of generated peptides were predicted to bind HLA with halftime of dissociation T1/2  ≥ 100 minutes, and 77% of them were mono-allelic in their binding. The structural bases for recognition of nanomers by different HLA molecules were studied by structural modeling of HLA class I-peptide complexes. Pathogen peptides that could mimic as self-peptides or partially self-peptides in the host were excluded using a comparative study with the human proteome; thus, subunit or DNA vaccines will have more chance of success.

Mining of Egypt's Red Sea invertebrates for potential bioactive producers.

OBJECTIVE: The objective of this work was to isolate bacteria from Red Sea invertebrates, determine their antimicrobial activity, and screen for the biosynthetic gene clusters [polyketides (PKs) and nonribosomal peptides (NRPs)] which could be involved in the production of bioactive secondary metabolites. RESULT: Eleven different samples of marine invertebrates' were collected from Egypt's Red Sea (El-Tor-Sharm El-Sheikh and Hurghada) by scuba diving, and a total 80 isolates of the associated microorganisms were obtained from the cultivation on six different cultural medium. Seven isolates of them showed an antimicrobial activity against five pathogenic reference strains, while the most active antimicrobial agent was isolate number HFF-8 which was 99% identical to Bacillus amyloliquefaciens. HFF-8's extract showed positive results against Gram negative bacteria, Gram positive bacteria and yeast. Moreover, the isolates gave positive bands when screened for the presence of PK synthase (PKS) I and II and NRP synthetase (NRPS) I and II biosynthetic genes, those biosynthetic fragments when cloned and sequenced were primitively predicted as biosynthetic fragments for kirromycin and leinamycin production by NaPDoS program with 56 and 55%, respectively. CONCLUSION: The Red Sea can provide a sustainable solution to combat bacterial resistance. The contribution of this work is that B. amyloliquefaciens was isolated from Heteroxenia fuscescens, Red Sea, Egypt. Moreover, the bacterial extract showed a broad spectrum with a potent antimicrobial activity.

Genome-Based Identification of a Plant-Associated Marine Bacterium as a Rich Natural Product Source.

The large number of sequenced bacterial genomes provides the opportunity to bioinformatically identify rich natural product sources among previously neglected microbial groups. Testing this discovery strategy, unusually high biosynthetic potential was suggested for the Oceanospirillales member Gynuella sunshinyii, a Gram-negative marine bacterium from the rhizosphere of the halophilic plant Carex scabrifolia. Its genome contains numerous unusual biosynthetic gene clusters for diverse types of metabolites. Genome-guided isolation yielded representatives of four different natural product classes, of which only alteramide A was known. Cytotoxic lacunalides were identified as products of a giant trans-acyltransferase polyketide synthase gene cluster, one of six present in this strain. Cytological profiling against HeLa cells suggested that lacunalide A disrupts CDK signaling in the cell cycle. In addition, chemical studies on model compounds were conducted, suggesting the structurally unusual ergoynes as products of a conjugated diyne-thiourea cyclization reaction.

Deciphering bioactive peptides and their action mechanisms through proteomics.

INTRODUCTION: Bioactive peptides such as antimicrobial peptides (AMPs), ribosomally synthesized and post translationally modified peptides (RiPPs) and the non-ribosomal peptides (NRPs) have emerged with promising applications in medicine, agriculture and industry. However, their development has been limited by several difficulties making it necessary to search for novel discovery methods. In this context, proteomics has been considered a reliable tool. Areas covered: This review highlights recent developments in proteomic tools that facilitate the discovery of AMPs, RiPPs and NRPs as well as the elucidation of action mechanisms of AMPs and resistance mechanisms of pathogens to them. Expert commentary: Proteomic approaches have emerged as useful tools for the study of bioactive peptides, especially mass spectrometry-based peptidomics profiling, a promising strategy for AMP discovery. Furthermore, the rapidly expanding fields of genome mining and genome sequencing techniques, as well as mass spectrometry, have revolutionized the discovery of novel RiPPs and NRPs from complex biological samples.

Chemical and Genetic Diversity of Nodularia spumigena from the Baltic Sea.

Nodularia spumigena is a toxic, filamentous cyanobacterium occurring in brackish waters worldwide, yet forms extensive recurrent blooms in the Baltic Sea. N. spumigena produces several classes of non-ribosomal peptides (NRPs) that are active against several key metabolic enzymes. Previously, strains from geographically distant regions showed distinct NRP metabolic profiles. In this work, conspecific diversity in N. spumigena was studied using chemical and genetic approaches. NRP profiles were determined in 25 N. spumigena strains isolated in different years and from different locations in the Baltic Sea using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Genetic diversity was assessed by targeting the phycocyanin intergenic spacer and flanking regions (cpcBA-IGS). Overall, 14 spumigins, 5 aeruginosins, 2 pseudaeruginosins, 2 nodularins, 36 anabaenopeptins, and one new cyanopeptolin-like peptide were identified among the strains. Seven anabaenopeptins were new structures; one cyanopeptolin-like peptide was discovered in N. spumigena for the first time. Based on NRP profiles and cpcBA-IGS sequences, the strains were grouped into two main clusters without apparent influence of year and location, indicating persistent presence of these two subpopulations in the Baltic Sea. This study is a major step in using chemical profiling to explore conspecific diversity with a higher resolution than with a sole genetic approach.

Plasticity of the malleobactin pathway and its impact on siderophore action in human pathogenic bacteria.

The human pathogenic bacteria Burkholderia mallei, Burkholderia pseudomallei, and Burkholderia thailandensis harbor a highly conserved gene cluster coding for the biosynthesis of the long sought-after malleobactins. Four new, unexpected congeners of the malleobactin family that were isolated and fully characterized in this study feature unusual deviations from the parent, ornibactin-like architecture. Thus, the malleobactin non-ribosomal peptide synthetase (NRPS) has a rare flexibility that yields diverse peptide backbones, of which one candidate confers pronounced siderophore activity (EC50: 8.4 µM, CAS assay). These findings not only unveil a highly diverse assembly line but also are an important addition to the knowledgebase of the pathogens' metabolomes.

Isolation and Synthesis of Laxaphycin B-Type Peptides: A Case Study and Clues to Their Biosynthesis.

The laxaphyci's B family constitutes a group of five related cyclic lipopeptides isolated from diverse cyanobacteria from all around the world. This group shares a typical structure of 12 amino acids from the l and d series, some of them hydroxylated at the beta position, and all containing a rare beta-amino decanoic acid. Nevertheless, they can be differentiated due to slight variations in the composition of their amino acids, but the configuration of their alpha carbon remains conserved. Here, we provide the synthesis and characterization of new laxaphycin B-type peptides. In doing so we discuss how the synthesis of laxaphycin B and analogues was developed. We also isolate minor acyclic laxaphycins B, which are considered clues to their biosynthesis.