Nonribosomal Peptide Research Articles

A Comprehensive Genome Mining Analysis of Biosynthetic Gene Clusters in Pseudomonas sp. SXM-1

Very resistant pathogenic microorganisms have been reported to current antibiotics in the last decade. Therefore, there is a great need to understand not only resistance metabolism but also secondary metabolites of pathogenic microorganisms. Genome mining tools have so far been improved to understand secondary metabolites from biosynthetic gene clusters. Microorganisms predicted for their genomes and secondary metabolites using these tools are widely employed in pharmaceutical and industrial studies. Pseudomonas spp. are widely used in recombinant DNA technology to produce commercial products. Bioinformatics-based in silico tools significantly contribute to the discovery of new bioactive compounds for pharmacy and medicine. This study aims to conduct a comprehensive gene cluster analysis of the Pseudomonas sp. SXM-1 strain isolated from the coastal seawater of Xiamen Bay using antiSMASH (7.0.1). The accession number of Pseudomonas sp. SXM-1 strain was retrieved from NCBI. 14 regions were found, including non-ribosomal peptides metallophores (NRP-metallophore), nonribosomal peptide-synthetase (NRPS), NRPS-like, ribosomally synthesized and post-translationally modified peptide-like (RiPP-like), betalactone, nonribosomal peptide-synthetase (NRPS), ectoine and N-acetylglutaminylglutamine amide (NAGGN). Analysis of all 14 regions revealed secondary metabolites with potential applications in diverse fields. Microbiologists are strongly advised to conduct wet-lab experiments to validate the secondary metabolites discussed in this study.

Biosynthetic Incorporation of Non-native Aryl Acid Building Blocks into Peptide Products Using Engineered Adenylation Domains.

Nonribosomal peptides (NRPs), one of the most widespread secondary metabolites in nature, with therapeutically significant activities, are biosynthesized by modular nonribosomal peptide synthetases (NRPSs). Aryl acids contribute to the structural diversity of NRPs as well as nonproteinogenic amino acids and keto acids. We previously confirmed that a single Asn-to-Gly substitution in the 2,3-dihydroxybenzoic acid-activating adenylation (A) domain EntE involved in enterobactin biosynthesis accepts monosubstituted benzoic acid derivatives with nitro, cyano, bromo, and iodo functionalities at the 2 or 3 positions. Here, we showed that the mutant EntE (N235G) accommodates various disubstituted benzoic acid derivatives with halogen, methyl, methoxy, nitro, and cyano functionalities at the 2 and 3 positions and monosubstituted benzoic acid with an alkyne at the 3 position. Structural analysis of the mutant EntE (N235G) with nonhydrolyzable aryl-AMP analogues using 3-chloro-2-methylbenzoic acid and 3-prop-2-ynoxybenzoic acid revealed how bulky 3-chloro-2-methylbenzoic acid and clickable 3-prop-2-ynoxybenzoic acid are recognized by enlarging the substrate-binding pocket of the enzyme. When engineered EntE mutants were coupled with enterobactin and vibriobactin biosynthetic enzymes, 3-hydroxybenzoic acid-, salicylic acid-, and 3-bromo-2-fluorobenzoic acid-containing peptides were produced as early stage intermediates, highlighting the potential of NRP biosynthetic pathway engineering for constructing diverse aryl acid-containing metabolites.

Genomic Characterization of Bacillus sp. THPS1: A Hot Spring-Derived Species with Functional Features and Biotechnological Potential

Bacillus sp. THPS1 is a novel strain isolated from a high-temperature hot spring in Thailand, exhibiting distinctive genomic features that enable adaptation to an extreme environment. This study aimed to characterize the genomic and functional attributes of Bacillus sp. THPS1 to understand its adaptation strategies and evaluate its potential for biotechnological applications. The draft genome is 5.38 Mbp with a GC content of 35.67%, encoding 5606 genes, including those linked to stress response and sporulation, which are essential for survival in high-temperature conditions. Phylogenetic analysis and average nucleotide identity (ANI) values confirmed its classification as a distinct species within the Bacillus genus. Pangenome analysis involving 19 others closely related thermophilic Bacillus species identified 1888 singleton genes associated with heat resistance, sporulation, and specialized metabolism, suggesting adaptation to nutrient-deficient, high-temperature environments. Genomic analysis revealed 12 biosynthetic gene clusters (BGCs), including those for polyketides and non-ribosomal peptides, highlighting its potential for synthesizing secondary metabolites that may facilitate its adaptation. Additionally, the presence of three Siphoviridae phage regions and 96 mobile genetic elements (MGEs) suggests significant genomic plasticity, whereas the existence of five CRISPR arrays implies an advanced defense mechanism against phage infections, contributing to genomic stability. The distinctive genomic features and functional capacities of Bacillus sp. THPS1 make it a promising candidate for biotechnological applications, particularly in the production of heat-stable enzymes and the development of resilient bioformulations.

Computation-driven redesign of an NRPS-like carboxylic acid reductase improves activity and selectivity.

Engineering nonribosomal peptide synthetases (NRPSs) has been a "holy grail" in synthetic biology due to their modular nature and limited understanding of catalytic mechanisms. Here, we reported a computational redesign of the "gate-keeper" adenylation domain of the model NRPS-like enzyme carboxylic acid reductases (CARs) by using approximate mechanism-based geometric criteria and the Rosetta energy score. Notably, MabCAR3 mutants ACA-1 and ACA-4 displayed a remarkable improvement in catalytic efficiency (kcat/KM) for 6-aminocaproic acid, up to 101-fold. Furthermore, G418K exhibited an 86-fold enhancement in substrate specificity for adipic acid compared to 6-aminocaproic acid. Our work provides not only promising biocatalysts for nylon monomer biosynthesis but also a strategy for efficient NRPSs engineering.

Genomic Features of Taiwanofungus gaoligongensis and the Transcriptional Regulation of Secondary Metabolite Biosynthesis

Fungal secondary metabolites (SMs) have broad applications in biomedicine, biocontrol, and the food industry. In this study, whole-genome sequencing and annotation of Taiwanofungus gaoligongensis were conducted, followed by comparative genomic analysis with 11 other species of Polyporales to examine genomic variations and secondary metabolite biosynthesis pathways. Additionally, transcriptome data were used to analyze the differential expression of polyketide synthase (PKS), terpene synthase (TPS) genes, and transcription factors (TFs) under different culture conditions. The results show that T. gaoligongensis differs from other fungal species in genome size (34.58 Mb) and GC content (50.72%). The antibiotics and Secondary Metabolites Analysis Shell (AntiSMASH) analysis reveals significant variation in the number of SM biosynthetic gene clusters (SMBGCs) across the 12 species (12–29), with T. gaoligongensis containing 25 SMBGCs: 4 PKS, 6 non-ribosomal peptide synthetase (NRPS), and 15 TPS clusters. The TgPKS1 gene is hypothesized to be involved in the biosynthesis of orsellinic acid or its derivatives, while TgPKS2 might catalyze the synthesis of 6-methylsalicylic acid (6MSA) and its derivatives. The TgTRI5 genes are suggested to synthesize tetracyclic sesquiterpene type B trichothecene compounds, while TgPentS may be involved in the synthesis of δ-cadinol, β-copaene, and α-murolene analogs or derivatives. Comparative genomic analysis shows that the genome size of T. gaoligongensis is similar to that of T. camphoratus, with comparable SMs. Both species share four types of PKS domains and five distinct types of TPS. Additionally, T. gaoligongensis exhibits a high degree of similarity to Laetiporus sulphureus, despite belonging to a different genus within the same family. Transcriptome analysis reveals significant variation in the expression levels of PKS and TPS genes across different cultivation conditions. The TgPKS1 and TgPKS4 genes, along with nine TgTFs, are significantly upregulated under three solid culture conditions. In contrast, under three different liquid culture conditions, the TgPKS3, TgTRI5-1, and TgTRI5-2 genes, along with twelve TgTFs, exhibit higher activity. Co-expression network analysis and TgTFs binding site prediction in the promoter regions of TgPKS and TgTPS genes suggest that TgMYB9 and TgFTD4 regulate TgPKS4 expression. TgHOX1, TgHSF2, TgHSF3, and TgZnF4 likely modulate TgPKS3 transcriptional activity. TgTRI5-1 and TgTRI5-5 expression is likely regulated by TgbZIP2 and TgZnF15, respectively. This study provides new insights into the regulatory mechanisms of SMs in T. gaoligongensis and offers potential strategies for enhancing the biosynthesis of target compounds through artificial intervention.

Cyclic natural product oligomers: diversity and (bio)synthesis of macrocycles.

Cyclic compounds are generally preferred over linear compounds for functional studies due to their enhanced bioavailability, stability towards metabolic degradation, and selective receptor binding. This has led to a need for effective cyclization strategies for compound synthesis and hence increased interest in macrocyclization mediated by thioesterase (TE) domains, which naturally boost the chemical diversity and bioactivities of cyclic natural products. Many non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) derived natural products are assembled to form cyclodimeric compounds, with these molecules possessing diverse structures and biological activities. There is significant interest in identifying the biosynthetic pathways that produce such molecules given the challenge that cyclodimerization represents from a biosynthetic perspective. In the last decade, many groups have pursued the characterization of TE domains and have provided new insights into this biocatalytic machinery: however, the enzymes involved in formation of cyclodimeric compounds have proven far more elusive. In this review we focus on natural products that involve macrocyclization in their biosynthesis and chemical synthesis, with an emphasis on the function and biosynthetic investigation on the special family of TE domains responsible for forming cyclodimeric natural products. We also introduce additional macrocyclization catalysts, including butelase and the CT-mediated cyclization of peptides, alongside the formation of cyclodipeptides mediated by cyclodipeptide synthases (CDPS) and single-module NRPSs. Due to the interdisciplinary nature of biosynthetic research, we anticipate that this review will prove valuable to synthetic chemists, drug discovery groups, enzymologists, and the biosynthetic community in general, and inspire further efforts to identify and exploit these biocatalysts for the formation of novel bioactive molecules.

Exploring the biosynthesis potential of permafrost microbiomes

BackgroundPermafrost microbiomes are of paramount importance for the biogeochemistry of high latitude soils and while endemic biosynthetic domain sequences involved in secondary metabolism have been found in polar surface soils, the biosynthetic potential of permafrost microbiomes remains unexplored. Moreover, the nature of these ecosystems facilitates the unique opportunity to study the distribution and diversity of biosynthetic genes in relic DNA from ancient microbiomes. To explore the biosynthesis potential in permafrost, we used adenylation (AD) domain sequencing to evaluate non-ribosomal peptide (NRP) production in permafrost cores housing microbiomes separated at kilometer and kiloyear scales.ResultsPermafrost microbiomes represented NRP repertoires significantly different from that of temperate soil microbiomes, but as for temperate soils, the estimated domain richness and diversity was strongly correlated to the bacterial taxonomic diversity across locations. Furthermore, we found significant differences in both community composition and AD domain composition across geographical and temporal distances. Overall, the vast majority of biosynthetic domains showed below 90% amino acid similarity to characterized BGCs, confirming the high degree of novelty of NRPs inherent to permafrost microbiomes. Using available metagenomic sequences, we further identified a high biosynthetic diversity beyond NRPs throughout arctic surface soils down to deep and ancient (megayear old) permafrost microbiomes.ConclusionWe have shown that arctic permafrost microbiomes harbor a unique biosynthetic repertoire rich in hitherto undescribed NRPs. This diversity is driven by geographic separation across kilometer scales and by the bacterial taxonomic diversity between microbiomes confined in separate permafrost layers. Hence the permafrost biome represents a unique resource for studying secondary metabolism, and potentially for the discovery of novel drug leads.

Directed Evolution of an Adenylation Domain Alters Substrate Specificity and Generates a New Catechol Siderophore in Escherichia coli.

Nonribosomal peptide synthetases (NRPS) biosynthesize numerous natural products with therapeutic, agricultural, and industrial significance. Reliably altering substrate selection in these enzymes has been a longstanding goal, as this would enable the production of tailor-made peptides with desired activities. In this study, the NRPS EntF and the associated biosynthesis of the siderophore enterobactin (ENT) were used as a model system to interrogate substrate selection by an adenylation (A) domain. We employed a directed evolution pipeline that harnesses an in vivo genetic selection for siderophore production to alter A domain substrate selection. Surprisingly, this led to the formation of a new, physiologically active catechol siderophore in Escherichia coli. We characterized the enzyme variants in vitro and demonstrated transferability of our findings to the well-studied TycC and GrsB NRPSs. This work identifies critical binding pocket residues that allow for altered substrate selection in our model system and expands upon our understanding of iron acquisition in E. coli.

Animal-encoded nonribosomal pathway to bursatellin analogs.

The bursatellin-oxazinin family is a series of tyrosine-derived, nitrile-containing marine natural products from gastro-pod and bivalve molluscs. Although the first analogs were identified and associated with toxicity forty years ago, their biosynthetic origins were unknown. During an investigation of published mollusc genomes and transcriptomes, we serendipitously identified a putative bursatellin biosynthetic gene cluster (referred hereafter as the bur-ox pathway). Through biochemical characterization of some bur-ox genes, we provide evidence suggesting that bursatellin-type metabolites are produced by molluscs themselves rather than by their microbial symbionts. We show that the reductive domain from a monomodular nonribosomal peptide synthetase (NRPS) protein FmtATR performs a four-electron reduction to produce tyrosinols from tyrosine derivatives. Moreover, an aminocarboxypro-pyltransferase enzyme, ACT, uses S -adenosylmethionine (SAM) to transform tyrosinols into their phenolic homoserine ethers, which in bursatellin is further modified to the nitrile. Widespread occurrence of bur-ox in molluscs suggests a common biosynthetic origin for bursatellins and oxazinins as well as an important but currently unidentified physiological role for this metabolite family in molluscs inhabiting diverse ecological niches. Further, the presence of bur-ox pathway homologs in many culinary bivalves such as mussels and geoducks suggests that possible impacts on human consumers should be investigated. As one of the few NRPS pathways of animal origin to be characterized, bur-ox sheds light on underappreciated chemical and biochemical diversity in animals.

In Vivo Quantification of Surfactin Nonribosomal Peptide Synthetase Complexes in Bacillus subtilis.

Surfactin, a potent biosurfactant produced by Bacillus subtilis, is synthesized using a non-ribosomal peptide synthetase (NRPS) encoded by the srfAA-AD operon. Despite its association with quorum sensing via the ComX pheromone, the dynamic behavior and in vivo quantification of the NRPS complex remain underexplored. This study established an in vivo quantification system using fluorescence labeling to monitor the availability of surfactin-forming NRPS subunits (SrfAA, SrfAB, SrfAC, and SrfAD) during bioprocesses. Four Bacillus subtilis sensor strains were constructed by fusing these subunits with the megfp gene, resulting in strains BMV25, BMV26, BMV27, and BMV28. These strains displayed growth and surfactin productivity similar to those of the parental strain, BMV9. Fluorescence signals indicated varying NRPS availability, with BMV27 showing the highest and BMV25 showing the lowest relative fluorescence units (RFUs). RFUs were converted to the relative number of NRPS molecules using open-source FPCountR package. During bioprocesses, NRPS availability peaked at the end of the exponential growth phase and declined in the stationary phase, suggesting reduced NRPS productivity under nutrient-limited conditions and potential post-translational regulation. This study provides a quantitative framework for monitoring NRPS dynamics in vivo, offering insights into optimizing surfactin production. The established sensor strains and quantification system enable the real-time monitoring of NRPS availability, aiding bioprocess optimization for industrial applications of surfactin and potentially other non-ribosomal peptides.

Chemical structure metagenomics of microbial natural products: surveying nonribosomal peptides and beyond

Bioactivity-guided fractionation (BGF) has historically been a fruitful natural product discovery workflow. However, it is plagued by increasing rediscovery rates in recent years and new methods capable of exploring the natural product chemical space more broadly and more efficiently is in urgent need. Chemical structure metagenomics as one such method is the theme of this Perspective. It emphasizes a chemical-structure-centered viewpoint toward natural product research. Key to chemical structure metagenomics is the ability to predict the structure of a natural product based on its biosynthetic gene sequences, which facilitated the discovery of numerous new bioactive molecules and helped uncover oversampled/underexplored niches of decades of BGF based discovery. While microbial nonribosomal peptides have been the focus of chemical structure metagenomics efforts thus far, it is in principle applicable to other natural product families. The future outlook of this new approach will also be discussed.

Environmental Stability Determines the Cytotoxicity of Metal-Organic Frameworks to a Nitrogen-Fixing Bacterium Azotobacter vinelandii.

During widespread applications of metal-organic frameworks (MOFs), the environmental hazards and risks of MOFs have aroused great concerns. In this study, we aimed to reveal the importance of the environmental stability of MOFs on their toxicity. Two Zn-MOFs, namely, ZIF-8 with high aqueous stability and Zn-BDC with low aqueous stability, were compared directly in the toxicological evaluations of a nitrogen-fixing bacterium Azotobacter vinelandii. Zn-BDC showed strong cytotoxicity at 100 mg/L and higher, inducing growth inhibition, cell apoptosis, structural changes, oxidative damage, and, consequently, loss of nitrogen fixation ability. In contrast, ZIF-8 was nearly nontoxic to A. vinelandii. The transcriptome analysis showed that Zn-BDC directly disturbed the ribosome pathway and lowered the expression level of nitrogen-fixing nif cluster genes. On the other hand, ZIF-8 stress could regulate the flagellar assembly, siderophore group nonribosomal peptide biosynthesis, bacterial chemotaxis, and amino sugar and nucleotide sugar metabolism pathways to promote the cell growth of A. vinelandii. Beyond that, the toxicity of Zn-MOFs to A. vinelandii was associated with the release of Zn2+, but Zn-MOFs were less toxic than the mixtures of their starting materials. Overall, our results suggested that the environmental stability of Zn-MOFs determined their environmental toxicity through different molecular pathways. Designing stable MOFs is preferred due to environment-friendly considerations.

Streptomyces hygroscopicus and rapamycinicus Evaluated from a U.S. Marine Sanctuary: Biosynthetic Gene Clusters Encode Antibiotic and Chemotherapeutic Secondary Metabolites

Cancer remains a leading cause of death worldwide. Also threatening the public is the emergence of antibiotic resistance to existing medicines. Despite the challenge to produce viable natural products to market, there continues to be a need within public health to provide new chemotherapeutic drugs such as those exhibiting cytotoxicity and tumor cell growth-inhibitory properties. As marine genomic research advances, it is apparent that marine-derived sediment harbors uniquely potent bioactive compounds compared to their terrestrial counterparts. The Streptomyces genus in particular produces more than 30% of all secondary metabolites currently approved for human health, thus harboring unexplored reservoirs of chemotherapeutic and antibiotic agents to combat emerging disease. The present study identifies the presence of Streptomyces hygroscopicus and rapamycinicus in environmental sediment at locations within the U.S. Stellwagen Bank National Marine Sanctuary (SBNMS) from 2017 to 2022. Sequencing and bioinformatics methods catalogued biosynthetic gene clusters (BGCs) that drive cytotoxic and antibiotic biochemical processes in samples collected from sites permittable and protected to fishing activity. Poisson regression models confirmed that Sites 1 and 3 had significantly higher occurrences of rapamycinicus than other sites (p < 0.01). Poisson regression models confirmed that Sites 1, 2 and 3 had significantly higher occurrence for Streptomyces hygroscopicus across sites (p < 0.05). Interestingly, permitted fishing sites showed a greater prevalence of both species. Statistical analyses showed a significant difference in aligned hits with polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) by site and between species with hygroscopicus showing a greater quantity than rapamycinicus among Streptomyces spp. (p < 0.05; F = 4.7 > F crit).

HiFiBGC: an ensemble approach for improved biosynthetic gene cluster detection in PacBio HiFi-read metagenomes

BackgroundMicrobes produce diverse bioactive natural products with applications in fields such as medicine and agriculture. In their genomes, these natural products are encoded by physically clustered genes known as biosynthetic gene clusters (BGCs). Genome and metagenome sequencing advances have enabled high-throughput identification of BGCs as a promising avenue for natural product discovery. BGC mining from (meta)genomes using in silico tools has allowed access to a vast diversity of potentially novel natural products. However, a fundamental limitation has been the ability to assemble complete BGCs, especially from complex metagenomes. With their fragmented assemblies, short-read technologies struggle to recover complete BGCs, such as the long and repetitive nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS). Recent advances in long-read sequencing, such as the High Fidelity (HiFi) technology from PacBio, have reduced this limitation and can help retrieve both accurate and complete BGCs from metagenomes, warranting improvement in the existing BGC identification approach for better utilization of HiFi data.ResultsHere, we present HiFiBGC, a command-line-based workflow to identify BGCs in PacBio HiFi metagenomes. HiFiBGC leverages an ensemble of assemblies from three HiFi-tailored metagenome assemblers and the reads not represented in these assemblies. Based on our analyses of four HiFi metagenomic datasets from four different environments, we show that HiFiBGC identifies, on average, 78% more BGCs than the top-performing single-assembler-based method. This increase is due to HiFiBGC’s ensemble assembly approach, which improves recovery by 25%, as well as from the inclusion of mostly fragmented BGCs identified in the unmapped reads.ConclusionsHiFiBGC is a computational workflow for identifying BGCs in long-read HiFi metagenomes, implemented majorly using Python programming language and workflow manager Snakemake. HiFiBGC is available on GitHub at https://github.com/ay-amityadav/HiFiBGC under the MIT license. The code related to the figures and analyses presented in the manuscript is available at https://github.com/ay-amityadav/HiFiBGC_analyses.

Biocatalytic Tetrapeptide Macrocyclization by Cryptic Penicillin-binding Protein-type Thioesterases.

Cyclic tetrapeptides (CTPs) are a diverse class of natural products with a broad range of biological activities. However, they are extremely challenging to synthesize due to the ring strain associated with their small ring size. While chemical methods have been developed to access CTPs, they generally require the presence of certain amino acids, limiting their substrate scopes. Herein, we report the first bioinformatics guided discovery of a thioesterase from a cryptic biosynthetic gene cluster for peptide cyclization. Specifically, we hypothesized that predicted Penicillin-binding type thioesterases (PBP-TEs) from cryptic nonribosomal peptide synthetase gene clusters containing four adenylation domains would catalyze tetrapeptide cyclization. We found that one of the predicted PBP-TEs, WP516, efficiently cyclizes a wide variety of tetrapeptide substrates. To date, it is only the second stand-alone enzyme capable of cyclizing tetrapeptides, and its substrate scope greatly surpasses that of the only other reported tetrapeptide cyclase Ulm16. AlphaFold modeling and covalent docking were used to rationalize the broad substrate scope of WP516 in comparison to other PBP-TEs. Overall, the bioinformatics guided workflow outlined in this paper, and the discovery of WP516, represent promising tools for the biocatalytic production of head-to-tail CTPs, as well as a more general strategy for discovery of enzymes for peptide cyclization.

Genome-based analysis of biosynthetic potential from antimycotic Streptomyces rochei strain A144.

Streptomyces rochei is a species of Streptomyces with a diverse range of biological activities. S. rochei strain A144 was isolated from desert soils and exhibits antagonistic activity against several plant pathogenic fungi. The genome of S. rochei A144 was sequenced and revealed the presence of one linear chromosome and one plasmid. The chromosome length was found to be 8,085,429bp, with a GC content of 72.62%, while the Plas1 length was 177,399bp, with a GC content of 69.08%. Comparative genomics was employed to analyse the S. rochei group. There is a high degree of collinearity between the genomes of S. rochei strains. Based on pan-genome analysis, S. rochei has 10,315 gene families, including 4051 core and 2322 unique genes. AntiSMASH was used to identify the gene clusters for secondary metabolites, identifying 33 secondary metabolite genes on the A144 genome. Among them, 18 clusters were found to be >70% identical to known biosynthetic gene clusters (BGCs), indicating that A144 has the potential to synthesize secondary metabolites. The majority of the BGCs were found to be conserved within the S. rochei group, including those encoding polyketide synthases (PKS), terpenes, non-ribosomal peptide synthetases (NRPS), other ribosomally synthesised and post-translationally modified peptides (RiPP), nicotianamine-iron transporters, lanthipeptides, and a few other types. The S. rochei group can be a potential genetic source of useful secondary metabolites with applications in medicine and biotechnology.

Pyrrolizwillin, ein Einzigartiges Bakterielles Alkaloid, Gebildet durch eine Kombination aus Nichtribosomaler Peptidsynthetase und nicht‐Enzymatischer Dimerisierung

AbstractPyrrolizidinalkaloide (PAs) sind eine strukturell vielfältige Gruppe spezialisierter heterocyclischer Metabolite, die durch eine Hexahydro‐1H‐pyrrolizin‐Kernstruktur charakterisiert werden. PAs werden über zwei unterschiedliche Biosynthesewege gebildet. In Pflanzen werden sie durch eine Homospermidinsynthase synthetisiert, während sie in Bakterien durch die kombinierte Wirkung einer nichtribosomalen Peptidsynthetase und einer Baeyer–Villiger‐Monooxygenase entstehen. Während die toxischen Eigenschaften pflanzlicher PAs und ihr Vorkommen in tierischen und menschlichen Lebensmitteln umfassend untersucht wurden, sind die biologischen Funktionen und die Biosynthese komplexerer bakterieller PAs noch nicht gut verstanden. Hier berichten wir über die Identifizierung und Charakterisierung eines bakteriellen Biosynthese‐Genclusters aus Xenorhabdus hominickii, xhpA‐G, das für die Produktion des PA‐Pseudodimers Pyrrolizwillin verantwortlich ist. Die Analyse von Promotor‐Austauschmutanten aus X. hominickii zusammen mit der heterologen Expression von xhpA‐G in E. coli ergab eine Reihe von Derivaten und Zwischenprodukten, von denen zwei chemisch synthetisiert wurden. Diese Informationen wurden genutzt, um einen detaillierten Biosyntheseweg zu Pyrrolizwillin zu postulieren. Darüber hinaus haben wir die Hydrolase XhpG, das Schlüsselenzym bei der Umwandlung des Zwischenprodukts Pyrrolizixenamid zu Pyrrolizwillin, mittels Röntgenkristallographie und Kleinwinkel‐Röntgenstreuung (SAXS) charakterisiert.

Expanding the Substrate Selectivity of the Fimsbactin Biosynthetic Adenylation Domain, FbsH.

Nonribosomal peptide synthetases (NRPSs) produce diverse natural products including siderophores, chelating agents that many pathogenic bacteria produce to survive in low iron conditions. Engineering NRPSs to produce diverse siderophore analogs could lead to the generation of novel antibiotics and imaging agents that take advantage of this unique iron uptake system in bacteria. The highly pathogenic and antibiotic-resistant bacteria Acinetobacter baumannii produces fimsbactin, an unusual branched siderophore with iron-binding catechol groups bound to a serine or threonine side chain. To explore the substrate promiscuity of the assembly line enzymes, we report a structure-guided investigation of the stand-alone aryl adenylation enzyme FbsH. We report structures bound to its native substrate 2,3-dihydroxybenzoic acid (DHB) as well as an inhibitor that mimics the adenylate intermediate. We produced enzyme variants with an expanded binding pocket that are more tolerant for analogs containing a DHB C4 modification. Wild-type and mutant enzymes were then used in an in vitro reconstitution analysis to assess the production of analogs of the final product as well as several early stage intermediates. This analysis shows that some altered substrates progress down the fimsbactin assembly line to the downstream domains. However, analogs from alternate building blocks are produced at lower levels, indicating that selectivity exists in the downstream catalytic domains. These findings expand the substrate scope of producing condensation products between serine and aryl acids and identify the bottlenecks for chemoenzymatic production of fimsbactin analogs.

Aeruginosin 525 (AER525) from Cyanobacterium Aphanizomenon Sp. (KUCC C2): A New Serine Proteases Inhibitor.

Aeruginosins (AERs) are one of the most common classes of cyanobacterial peptides synthesised through a hybrid non-ribosomal peptide synthase/polyketide synthase pathway. They have been found in Microcystis, Nodularia spumigena, Oscillatoria/Plantothrix, and Nostoc. The presence of AER in Aphanizomenon isolated from the Curonian Lagoon was reported for the first time in our previous work. Here, the structure of aeruginosin 525 (AER525), isolated from Aphanizomenon sp. KUCC C2, was characterised based on high-resolution mass spectrometry. This new AER variant shows potent activity against thrombin. It also inhibits trypsin and carboxypeptidase A but has no effect on elastase and chymotrypsin. In terms of the N-terminal residue and biological activity, AER525 displaces some similarity to dysinosins, which belongs to the most potent inhibitors of thrombin among AERs. The findings underline the potential of AER525 as a new anticoagulant agent.

Overexpression of PtNRPS1 enhances diatom-mediated bioremediation of salicylate pollution

The accumulation of the emerging pollutant salicylic acid (SA) in the environment has gained much attention. In this study, overexpression of the non-ribosomal peptide synthase (NRPS) gene, PtNRPS1 in the marine diatom Phaeodactylum tricornutum (PtNRPS1-OE) increased resistance to SA pollutants. It was assumed that the enhanced tolerance was due to the high binding affinity between recombinant PtNRPS1 (rNRPS1) and SA pollutants. Moreover, tandem mass spectrometry analysis determined the amino acids that participated in the covalently binding of SA. The removal efficiency of SA pollutants by PtNRPS1-OE cells was found to be markedly elevated. The mechanism underlying the removal of SA and 5-substituted SA (5-sSA) was proposed, following the co-localization analysis of rNRPS1 and SA. The purpose of this study was not about using PtNRPS1 as an enzyme to catalyze the synthesis of metabolite. Rather, it explored the possibility of using PtNRPS1 to remove pollutants, which further improves practical feasibility of microalgae-mediated bioremediation.