A male-derived nonribosomal peptide pheromone controls female schistosome development.

Schistosomes cause morbidity and death throughout the developing world due to the massive numbers of eggs female worms deposit into the blood of their host. Studies dating back to the 1920s show that female schistosomes rely on constant physical contact with a male worm both to become and remain sexually mature; however, the molecular details governing this process remain elusive. Here, we uncover a nonribosomal peptide synthetase that is induced in male worms upon pairing with a female and find that it is essential for the ability of male worms to stimulate female development. We demonstrate that this enzyme generates ß-alanyl-tryptamine that is released by paired male worms. Furthermore, synthetic ß-alanyl-tryptamine can replace male worms to stimulate female sexual development and egg laying. These data reveal that peptide-based pheromone signaling controls female schistosome sexual maturation, suggesting avenues for therapeutic intervention and uncovering a role for nonribosomal peptides as metazoan signaling molecules.

The structure of the monobactam-producing thioesterase domain of SulM forms a unique complex with the upstream carrier protein domain.

Nonribosomal peptide synthetases (NRPSs) are responsible for the production of important biologically active peptides. The large, multidomain NRPSs operate through an assembly line strategy in which the growing peptide is tethered to carrier domains that deliver the intermediates to neighboring catalytic domains. While most NRPS domains catalyze standard chemistry of amino acid activation, peptide bond formation, and product release, some canonical NRPS catalytic domains promote unexpected chemistry. The paradigm monobactam antibiotic sulfazecin is produced through the activity of a terminal thioesterase domain of SulM, which catalyzes an unusual ß-lactam-forming reaction in which the nitrogen of the C-terminal N-sulfo-2,3-diaminopropionate residue attacks its thioester tether to release the monobactam product. We have determined the structure of the thioesterase domain as both a free-standing domain and a didomain complex with the upstream holo peptidyl-carrier domain. The position of variant lid helices results in an active site pocket that is quite constrained, a feature that is likely necessary to orient the substrate properly for ß-lactam formation. Modeling of a sulfazecin tripeptide into the active site identifies a plausible binding mode identifying potential interactions for the sulfamate and the peptide backbone with Arg2849 and Asn2819, respectively. The overall structure is similar to the ß-lactone-forming thioesterase domain that is responsible for similar ring closure in the production of obafluorin. We further use these insights to enable bioinformatic analysis to identify additional, uncharacterized ß-lactam-forming biosynthetic gene clusters by genome mining.

Tumor Homing Chimeric Peptide Rhomboids to Improve Photodynamic Performance by Inhibiting Therapy-Upregulated Cyclooxygenase-2.

Negative therapeutic feedback of inflammation would extensively attenuate the antitumor effect of photodynamic therapy (PDT). In this work, tumor homing chimeric peptide rhomboids (designated as NP-Mel) are fabricated to improve photodynamic performance by inhibiting PDT-upregulated cyclooxygenase-2 (COX-2). The hydrophobic photosensitizer of protoporphyrin IX (PpIX) and palmitic acid are conjugated onto the neuropilin receptors (NRPs) targeting peptide motif (CGNKRTR) to obtain tumor homing chimeric peptide (Palmitic-K(PpIX)CGNKRTR), which can encapsulate the COX-2 inhibitor of meloxicam. The well dispersed NP-Mel not only improves the drug stability and reactive oxygen species (ROS) production ability, but also increase the breast cancer targeted drug delivery to intensify the PDT effect. In vitro and in vivo studies verify that NP-Mel will decrease the secretion of prostaglandin E2 (PGE2) after PDT treatment, inducing the downregulation of IL-6 and TNF-α expressions to suppress PDT induced inflammation. Ultimately, an improved PDT performance of NP-Mel is achieved without inducing obvious systemic toxicity, which might inspire the development of sophisticated nanomedicine in consideration of the feedback induced therapeutic resistance.

Biosynthesis of novel desferrioxamine derivatives requires unprecedented crosstalk between separate NRPS-independent siderophore pathways.

Iron is essential to many biological processes but its poor solubility in aerobic environments restricts its bioavailability. To overcome this limitation, bacteria have evolved a variety of strategies, including the production and secretion of iron-chelating siderophores. Here, we describe the discovery of four series of siderophores from Streptomyces ambofaciens ATCC23877, three of which are unprecedented. MS/MS-based molecular networking revealed that one of these series corresponds to acylated desferrioxamines (acyl-DFOs) recently identified from S. coelicolor. The remaining sets include tetra- and penta-hydroxamate acyl-DFO derivatives, all of which incorporate a previously undescribed building block. Stable isotope labeling and gene deletion experiments provide evidence that biosynthesis of the acyl-DFO congeners requires unprecedented crosstalk between two separate non-ribosomal peptide synthetase (NRPS)-independent siderophore (NIS) pathways in the producing organism. Although the biological role(s) of these new derivatives remain to be elucidated, they may confer advantages in terms of metal chelation in the competitive soil environment due to the additional bidentate hydroxamic functional groups. The metabolites may also find application in various fields including biotechnology, bioremediation, and immuno-PET imaging.IMPORTANCEIron-chelating siderophores play important roles for their bacterial producers in the environment, but they have also found application in human medicine both in iron chelation therapy to prevent iron overload and in diagnostic imaging, as well as in biotechnology, including as agents for biocontrol of pathogens and bioremediation. In this study, we report the discovery of three novel series of related siderophores, whose biosynthesis depends on the interplay between two NRPS-independent (NIS) pathways in the producing organism S. ambofaciens-the first example to our knowledge of such functional cross-talk. We further reveal that two of these series correspond to acyl-desferrioxamines which incorporate four or five hydroxamate units. Although the biological importance of these novel derivatives is unknown, the increased chelating capacity of these metabolites may find utility in diagnostic imaging (for instance, 89Zr-based immuno-PET imaging) and other applications of metal chelators.

Strategies for improving fengycin production: a review.

Fengycin is an important member of the lipopeptide family with a wide range of applications in the agricultural, food, medical and cosmetic industries. However, its commercial application is severely hindered by low productivity and high cost. Therefore, numerous studies have been devoted to improving the production of fengycin. We summarize these studies in this review with the aim of providing a reference and guidance for future researchers. This review begins with an overview of the synthesis mechanism of fengycin via the non-ribosomal peptide synthetases (NRPS), and then delves into the strategies for improving the fengycin production in recent years. These strategies mainly include fermentation optimization and metabolic engineering, and the metabolic engineering encompasses enhancement of precursor supply, application of regulatory factors, promoter engineering, and application of genome-engineering (genome shuffling and genome-scale metabolic network model). Finally, we conclude this review with a prospect of fengycin production.

Bacillus velezensis BE2 controls wheat and barley diseases by direct antagonism and induced systemic resistance.

Wheat and barley rank among the main crops cultivated on a global scale, providing the essential nutritional foundation for both humans and animals. Nevertheless, these crops are vulnerable to several fungal diseases, such as Septoria tritici blotch and net blotch, which significantly reduce yields by adversely affecting leaves and grain quality. To mitigate the effect of these diseases, chemical fungicides have proven to be genuinely effective; however, they impose a serious environmental burden. Currently, biocontrol agents have attracted attention as a sustainable alternative to fungicides, offering an eco-friendly option. The study aimed to assess the efficacy of Bacillus velezensis BE2 in reducing disease symptoms caused by Zymoseptoria tritici and Pyrenophora teres. This bacterium exhibited significant antagonistic effects in vitro by suppressing fungal development when pathogens and the beneficial strain were in direct confrontation. These findings were subsequently confirmed through microscopic analysis, which illustrated the strain's capacity to inhibit spore germination and mycelial growth in both pathogens. Additionally, the study analysed the cell-free supernatant of the bacterium using UPLC-MS (ultra-performance liquid chromatography-mass spectrometry). The results revealed that strain BE2 produces, among other metabolites, different families of cyclic lipopeptides that may be involved in biocontrol. Furthermore, the beneficial effects of strain BE2 in planta were assessed by quantifying the fungal DNA content directly at the leaf level after bacterization, using two different application methods (foliar and drenching). The results indicated that applying the beneficial bacterium at the root level significantly reduced pathogens pressure. Finally, gene expression analysis of different markers showed that BE2 application induced a priming effect within the first hours after infection. KEY POINTS: • BE2 managed Z. tritici and P. teres by direct antagonism and induced systemic resistance. • Strain BE2 produced seven metabolite families, including three cyclic lipopeptides. • Application of strain BE2 at the root level triggered plant defense mechanisms.

Unraveling the metabolic potential of biocontrol fungi through omics data: a key to enhancing large-scaleapplication strategies.

Biological control of pests and pathogens has attracted much attention due to its green, safe and effective characteristics. However, it faces the dilemma of insignificant effects in large-scale applications. Therefore, an in-depth exploration of the metabolic potential of biocontrol fungi based on big omics data is crucial for a comprehensive and systematic understanding of the specific modes of action operated by various biocontrol fungi. This article analyzes the preferences for extracellular carbon and nitrogen source degradation, secondary metabolites (nonribosomal peptides, polyketide synthases) and their product characteristics and the conversion relationship between extracellular primary metabolism and intracellular secondary metabolism for eight different filamentous fungi with characteristics appropriate for the biological control of bacterial pathogens and phytopathogenic nematodes. Further clarification is provided that Paecilomyces lilacinus, encoding a large number of hydrolase enzymes capable of degrading pathogen protection barrier, can be directly applied in the field as a predatory biocontrol fungus, whereas Trichoderma, as an antibiosis-active biocontrol control fungus, can form dominant strains on preferred substrates and produce a large number of secondary metabolites to achieve antibacterial effects. By clarifying the levels of biological control achievable by different biocontrol fungi, we provide a theoretical foundation for their application to cropping habitats.

NRPS-like ATRR in Plant-Parasitic Nematodes Involved in Glycine Betaine Metabolism to Promote Parasitism.

Plant-parasitic nematodes (PPNs) are among the most serious phytopathogens and cause widespread and serious damage in major crops. In this study, using a genome mining method, we identified nonribosomal peptide synthetase (NRPS)-like enzymes in genomes of plant-parasitic nematodes, which are conserved with two consecutive reducing domains at the N-terminus (A-T-R1-R2) and homologous to fungal NRPS-like ATRR. We experimentally investigated the roles of the NRPS-like enzyme (MiATRR) in nematode (Meloidogyne incognita) parasitism. Heterologous expression of Miatrr in Saccharomyces cerevisiae can overcome the growth inhibition caused by high concentrations of glycine betaine. RT-qPCR detection shows that Miatrr is significantly upregulated at the early parasitic life stage (J2s in plants) of M. incognita. Host-derived Miatrr RNA interference (RNAi) in Arabidopsis thaliana can significantly decrease the number of galls and egg masses of M. incognita, as well as retard development and reduce the body size of the nematode. Although exogenous glycine betaine and choline have no obvious impact on the survival of free-living M. incognita J2s (pre-parasitic J2s), they impact the performance of the nematode in planta, especially in Miatrr-RNAi plants. Following application of exogenous glycine betaine and choline in the rhizosphere soil of A. thaliana, the numbers of galls and egg masses were obviously reduced by glycine betaine but increased by choline. Based on the knowledge about the function of fungal NRPS-like ATRR and the roles of glycine betaine in host plants and nematodes, we suggest that MiATRR is involved in nematode-plant interaction by acting as a glycine betaine reductase, converting glycine betaine to choline. This may be a universal strategy in plant-parasitic nematodes utilizing NRPS-like ATRR to promote their parasitism on host plants.

Unearthing a Cryptic Biosynthetic Gene Cluster for the Piperazic Acid-Bearing Depsipeptide Diperamycin in the Ant-Dweller Streptomyces sp. CS113.

Piperazic acid is a cyclic nonproteinogenic amino acid that contains a hydrazine N-N bond formed by a piperazate synthase (KtzT-like). This amino acid, found in bioactive natural products synthesized by non-ribosomal peptide synthetases (NRPSs), confers conformational constraint to peptides, an important feature for their biological activities. Genome mining of Streptomyces strains has been revealed as a strategy to identify biosynthetic gene clusters (BGCs) for potentially active compounds. Moreover, the isolation of new strains from underexplored habitats or associated with other organisms has allowed to uncover new BGCs for unknown compounds. The in-house "Carlos Sialer (CS)" strain collection consists of seventy-one Streptomyces strains isolated from the cuticle of leaf-cutting ants of the tribe Attini. Genomes from twelve of these strains have been sequenced and mined using bioinformatics tools, highlighting their potential to encode secondary metabolites. In this work, we have screened in silico those genomes, using KtzT as a hook to identify BGCs encoding piperazic acid-containing compounds. This resulted in uncovering the new BGC dpn in Streptomyces sp. CS113, which encodes the biosynthesis of the hybrid polyketide-depsipeptide diperamycin. Analysis of the diperamycin polyketide synthase (PKS) and NRPS reveals their functional similarity to those from the aurantimycin A biosynthetic pathway. Experimental proof linking the dpn BGC to its encoded compound was achieved by determining the growth conditions for the expression of the cluster and by inactivating the NRPS encoding gene dpnS2 and the piperazate synthase gene dpnZ. The identity of diperamycin was confirmed by High-Resolution Mass Spectrometry (HRMS) and Nuclear Magnetic Resonance (NMR) and by analysis of the domain composition of modules from the DpnP PKS and DpnS NRPS. The identification of the dpn BGC expands the number of BGCs that have been confirmed to encode the relatively scarcely represented BGCs for depsipeptides of the azinothricin family of compounds and will facilitate the generation of new-to-nature analogues by combinatorial biosynthesis.

Chalkophomycin Biosynthesis Revealing Unique Enzyme Architecture for a Hybrid Nonribosomal Peptide Synthetase and Polyketide Synthase.

Chalkophomycin is a novel chalkophore with antibiotic activities isolated from Streptomyces sp. CB00271, while its potential in studying cellular copper homeostasis makes it an important probe and drug lead. The constellation of N-hydroxylpyrrole, 2H-oxazoline, diazeniumdiolate, and methoxypyrrolinone functional groups into one compact molecular architecture capable of coordinating cupric ions draws interest to unprecedented enzymology responsible for chalkophomycin biosynthesis. To elucidate the biosynthetic machinery for chalkophomycin production, the chm biosynthetic gene cluster from S. sp. CB00271 was identified, and its involvement in chalkophomycin biosynthesis was confirmed by gene replacement. The chm cluster was localized to a ~31 kb DNA region, consisting of 19 open reading frames that encode five nonribosomal peptide synthetases (ChmHIJLO), one modular polyketide synthase (ChmP), six tailoring enzymes (ChmFGMNQR), two regulatory proteins (ChmAB), and four resistance proteins (ChmA'CDE). A model for chalkophomycin biosynthesis is proposed based on functional assignments from sequence analysis and structure modelling, and is further supported by analogy to over 100 chm-type gene clusters in public databases. Our studies thus set the stage to fully investigate chalkophomycin biosynthesis and to engineer chalkophomycin analogues through a synthetic biology approach.

Carrier Protein Interaction with Competing Adenylation and Epimerization Domains in a Nonribosomal Peptide Synthetase Analyzed by FRET.

In multi-domain nonribosomal peptide synthetases (NRPSs) the order of domains and their catalytic specificities dictate the structure of the peptide product. Peptidyl-carrier proteins (PCPs) bind activated amino acids and channel elongating peptidyl intermediates along the protein template. To this end, fine-tuned interactions with the catalytic domains and large-scale PCP translocations are necessary. Despite crystal structure snapshots of several PCP-domain interactions, the conformational dynamics under catalytic conditions in solution remain poorly understood. We report a FRET reporter of gramicidin S synthetase 1 (GrsA; with A-PCP-E domains) to study for the first time the interaction between PCP and adenylation (A) domain in the presence of an epimerization (E) domain, a competing downstream partner for the PCP. Bulk FRET measurements showed that upon PCP aminoacylation a conformational shift towards PCP binding to the A domain occurs, indicating the E domain acts on its PCP substrate out of a disfavored conformational equilibrium. Furthermore, the A domain was found to preferably bind the D-Phe-S-Ppant-PCP stereoisomer, suggesting it helps in establishing the stereoisomeric mixture in favor of the D-aminoacyl moiety. These observations surprisingly show that the conformational logic can deviate from the order of domains and thus reveal new principles in the multi-domain interplay of NRPSs.

Isofunctional but Structurally Different Methyltransferases for Dithiolopyrrolone Diversification.

Dithiolopyrrolone (DTP) natural products are produced by several different bacteria and have potent antibacterial, antifungal and anticancer activities. While the amide of their DTP core can be methylated to fine-tune bioactivity, the enzyme responsible for the amide N-methylation has remained elusive in most taxa. Here, we identified the amide methyltransferase XrdM that is responsible for xenorhabdin (XRD) methylation in Xenorhabdus doucetiae but encoded outside of the XRD gene cluster. XrdM turned out to be isofunctional with the recently reported methyltransferase DtpM, that is involved in the biosynthesis of the DTP thiolutin, although its X-ray structure is unrelated to that of DtpM. To investigate the structural basis for ligand binding in both enzymes, we used X-ray crystallography, modeling, site-directed mutagenesis, and kinetic activity assays. Our study expands the limited knowledge of post-non-ribosomal peptide synthetase (NRPS) amide methylation in DTP biosynthesis and reveals an example of convergent evolution of two structurally completely different enzymes for the same reaction in different organisms.

Discovery and Biosynthesis of Ureidopeptide Natural Products Macrocyclized via Indole N-acylation in Marine Microbulbifer spp. Bacteria.

Commensal bacteria associated with marine invertebrates are underappreciated sources of chemically novel natural products. Using mass spectrometry, we had previously detected the presence of peptidic natural products in obligate marine bacteria of the genus Microbulbifer cultured from marine sponges. In this report, the isolation and structural characterization of a panel of ureidohexapeptide natural products, termed the bulbiferamides, from Microbulbifer strains is reported wherein the tryptophan side chain indole participates in a macrocyclizing peptide bond formation. Genome sequencing identifies biosynthetic gene clusters encoding production of the bulbiferamides and implicates the involvement of a thioesterase in the indolic macrocycle formation. The structural diversity and widespread presence of bulbiferamides in commensal microbiomes of marine invertebrates point toward a possible ecological role for these natural products.

Analysing Megasynthetase Mutants at High Throughput Using Droplet Microfluidics.

Nonribosomal peptide synthetases (NRPSs) are giant enzymatic assembly lines that deliver many pharmaceutically valuable natural products, including antibiotics. As the search for new antibiotics motivates attempts to redesign nonribosomal metabolic pathways, more robust and rapid sorting and screening platforms are needed. Here, we establish a microfluidic platform that reliably detects production of the model nonribosomal peptide gramicidin S. The detection is based on calcein-filled sensor liposomes yielding increased fluorescence upon permeabilization. From a library of NRPS mutants, the sorting platform enriches the gramicidin S producer 14.5-fold, decreases internal stop codons 250-fold, and generates enrichment factors correlating with enzyme activity. Screening for NRPS activity with a reliable non-binary sensor will enable more sophisticated structure-activity studies and new engineering applications in the future.

Hydroxy Acid Activation in Fungal Non-Ribosomal Peptide Synthesis Assessed by Site-Directed Mutagenesis.

The fungal cyclodepsipeptides (CDPs) enniatin, beauvericin, bassianolide, and PF1022 consist of alternating N-methylated l-amino and d-hydroxy acids. They are synthesized by non-ribosomal peptide synthetases (NRPS). The amino acid and hydroxy acid substrates are activated by adenylation (A) domains. Although various A domains have been characterized thus giving insights into the mechanism of substrate conversion, little is known about the utilization of hydroxy acids in NRPSs. Therefore, we used homology modelling and molecular docking of the A1 domain of enniatin synthetase (EnSyn) to gain insights into the mechanism of hydroxy acid activation. We introduced point mutations into the active site and used a photometric assay to study the substrate activation. The results suggest that the hydroxy acid is selected by interaction with backbone carbonyls rather than by a specific side chain. These insights enhance the understanding of non-amino acid substrate activation and could contribute to the engineering of depsipeptide synthetases.

Biomimetic engineering of nonribosomal peptide synthesis.

Nonribosomal peptides (NRPs) have gained attention due to their diverse biological activities and potential applications in medicine and agriculture. The natural diversity of NRPs is a result of evolutionary processes that have occurred over millions of years. Recent studies have shed light on the mechanisms by which nonribosomal peptide synthetases (NRPSs) evolve, including gene duplication, recombination, and horizontal transfer. Mimicking natural evolution could be a useful strategy for engineering NRPSs to produce novel compounds with desired properties. Furthermore, the emergence of antibiotic-resistant bacteria has highlighted the urgent need for new drugs, and NRPs represent a promising avenue for drug discovery. This review discusses the engineering potential of NRPSs in light of their evolutionary history.

The non-ribosomal peptide synthetase-independent siderophore (NIS) rhizobactin produced by Caballeronia mineralivorans PML1(12) confers the ability to weather minerals.

To mobilize nutrients entrapped into minerals and rocks, heterotrophic bacteria living in nutrient-poor environments have developed different mechanisms based mainly on acidolysis and chelation. However, the genetic bases of these mechanisms remain unidentified. To fill this gap, we considered the model strain Caballeronia mineralivorans PML1(12) known to be effective at weathering. Based on its transcriptomics and proteomics responses in Fe-depleted conditions, we pointed a cluster of genes differentially expressed and putatively involved in the production of siderophores. In this study, we report the characterization of this gene region coding for the production of a non-ribosomal peptide synthetase-independent siderophore (NIS). Targeted mutagenesis associated with functional assays and liquid chromatography coupled to high-resolution tandem mass spectrometry demonstrated the production of a single siderophore, identified as rhizobactin. This siderophore represents the first NIS containing malic acid in its structure. The evidence for the implication of rhizobactin in mineral weathering was demonstrated during a hematite dissolution assay. This study provides the first demonstration of the synthesis of a NIS in the genus Caballeronia and its involvement in mineral weathering. Our conclusions reinforce the idea that strain PML1(12) is particularly well adapted to nutrient-poor environments. IMPORTANCE This work deciphers the molecular and genetic bases used by strain PML1(12) of Caballeronia mineralivorans to mobilize iron and weather minerals. Through the combination of bioinformatics, chemical, and phylogenetic analyses, we characterized the siderophore produced by strain PML1(12) and the related genes. This siderophore was identified as rhizobactin and classified as a non-ribosomal peptide synthetase-independent siderophore (NIS). Contrary to the previously identified NIS synthetases that form siderophores containing citric acid, α-ketoglutarate, or succinic acid, our analyses revealed that rhizobactin contains malic acid in its structure, representing, therefore, the first identified NIS with such an acid and probably a new NIS category. Last, this work demonstrates for the first time the effectiveness at weathering minerals of a siderophore of the NIS family. Our findings offer relevant information for different fields of research, such as environmental genomics, microbiology, chemistry, and soil sciences.

Rapid Unambiguous Structure Elucidation of Streptnatamide A, a New Cyclic Peptide Isolated from A Marine-derived Streptomyces sp.

Cyclic peptides have been excellent source of drug leads. With the advances in discovery platforms, the pharmaceutical industry has a growing interest in cyclic peptides and has pushed several into clinical trials. However, structural complexity of cyclic peptides brings extreme challenges for structure elucidation efforts. Isotopic fine structure analysis, Nuclear magnetic resonance (NMR), and detailed tandem mass spectrometry rapidly provided peptide sequence for streptnatamide A, a cyclic peptide isolated from a marine-derived Streptomyces sp. Marfey's analysis determined the stereochemistry of all amino acids, enabling the unambiguous structure determination of this compound. A non-ribosomal peptide synthetase biosynthetic gene cluster (stp) was tentatively identified and annotated for streptnatamide A based on the in silico analysis of whole genome sequencing data. These analytical tools will be powerful tools to overcome the challenges for cyclic peptide structure elucidation and accelerate the development of bioactive cyclic peptides.

Stapled NRPS enhances the production of valinomycin in Escherichia coli.

Nonribosomal peptides (NRPs) are a large family of secondary metabolites with notable bioactivities, which distribute widely in natural resources across microbes and plants. To obtain these molecules, heterologous production of NRPs in robust surrogate hosts like Escherichia coli represent a feasible approach. However, reconstitution of the full biosynthetic pathway in a host often leads to low productivity, which is at least in part due to the low efficiency of enzyme interaction in vivo except for the well-known reasons of metabolic burden (e.g., expression of large NRP synthetases-NRPSs with molecular weights of >100 kDa) and cellular toxicity on host cells. To enhance the catalytic efficiency of large NRPSs in vivo, here we propose to staple NRPS enzymes by using short peptide/protein pairs (e.g., SpyTag/SpyCatcher) for enhanced NRP production. We achieve this goal by introducing a stapled NRPS system for the biosynthesis of the antibiotic NRP valinomycin in E. coli. The results indicate that stapled valinomycin synthetase (Vlm1 and Vlm2) enables higher product accumulation than those two free enzymes (e.g., the maximum improvement is nearly fourfold). After further optimization by strain and bioprocess engineering, the final valinomycin titer maximally reaches about 2800 µg/L, which is 73 times higher than the initial titer of 38 µg/L. We expect that stapling NRPS enzymes will be a promising catalytic strategy for high-level biosynthesis of NRP natural products.

Enzymatic reconstitution of salicylate formation in promysalin biosynthesis.

Promysalin is an amphipathic antibiotic isolated from Pseudomonas promysalinigenes (previously Pseudomonas putida RW10S1) which shows potent antibacterial activities against Gram-negative pathogens by inactivating succinate dehydrogenase. Based on the in-vivo studies, promysalin is hypothesized to be assembled from three building blocks: salicylic acid, proline, and myristic acid via a proposed but uncharacterized hybrid NRPS-PKS biosynthetic pathway. So far, no in-vitro biosynthetic studies have been reported for this promising antibiotic. Here, we report the first in-vitro reconstitution and biochemical characterization of two early enzymes on the pathway: PpgH, an isochorismate synthase (IS), and PpgG, an isochorismate pyruvate lyase (IPL) which are involved in the biosynthesis of salicylic acid, the polar fragment of promysalin. We also report a secondary chorismate mutase (CM) activity for PpgG. Based on our biochemical experiments, preliminary mechanistic proposals have been postulated for PpgH and PpgG. We believe this study will lay a strong foundation for elucidating the functions and mechanisms of other intriguing enzymes of the promysalin biosynthesis pathway, which may potentially unravel interesting enzyme chemistries and promote pathway engineering in the future.