Cellular pharmacokinetic of methotrexate and its modulation by folylpolyglutamate synthetase and γ-glutamyl hydrolase in tumor cells.

BACKGROUND AND PURPOSE: Clinical studies showed that prolonged infusion of methotrexate (MTX) leads to more severe adverse reactions than short infusion of MTX at the same dose. We hypothesized that it is the saturation of folate polyglutamate synthetase (FPGS) at high MTX concentration that limits the intracellular synthesis rate of methotrexate polyglutamate (MTX-PG). Due to a similar accumulation rate, a longer infusion duration may increase the concentration of MTX-PG and, result in more serious adverse reactions. In this study, we validated this hypothesis. EXPERIMENTAL APPROACH: A549, BEL-7402 and MHCC97H cell lines were treated with MTX at gradient concentrations. Liquid chromatograph-mass spectrometer (UPLC-MS/MS) was used to quantify the intracellular concentration of MTX-PG and the abundance of FPGS and γ-glutamyl hydrolase (GGH). High quality data were used to fit the cell pharmacokinetic model. KEY RESULTS: Both cell growth inhibition rate and intracellular MTX-PG concentration showed a nonlinear relationship with MTX concentration. The parameter Vmax in the model, which represents the synthesis rate of MTX-PG, showed a strong correlation with the abundance of intracellular FPGS. CONCLUSION AND IMPLICATIONS: According to the model fitting results, it was confirmed that the abundance of FPGS is a decisive factor limiting the synthesis rate of MTX-PG. The proposed hypothesis was verified in this study. In addition, based on the intracellular metabolism, a reasonable explanation was provided for the correlation between the severity of adverse reactions of MTX and infusion time. This study provides a new strategy for the individualized treatment and prediction of efficacy/side effects of MTX.

Biosynthetic gene clusters with biotechnological applications in novel Antarctic isolates from Actinomycetota.

Actinomycetota have been widely described as valuable sources for the acquisition of secondary metabolites. Most microbial metabolites are produced via metabolic pathways encoded by biosynthetic gene clusters (BGCs). Although many secondary metabolites are not essential for the survival of bacteria, they play an important role in their adaptation and interactions within microbial communities. This is how bacteria isolated from extreme environments such as Antarctica could facilitate the discovery of new BGCs with biotechnological potential. This study aimed to isolate rare Actinomycetota strains from Antarctic soil and sediment samples and identify their metabolic potential based on genome mining and exploration of biosynthetic gene clusters. To this end, the strains were sequenced using Illumina and Oxford Nanopore Technologies platforms. The assemblies were annotated and subjected to phylogenetic analysis. Finally, the BGCs present in each genome were identified using the antiSMASH tool, and the biosynthetic diversity of the Micrococcaceae family was evaluated. Taxonomic annotation revealed that seven strains were new and two were previously reported in the NCBI database. Additionally, BGCs encoding type III polyketide synthases (T3PKS), beta-lactones, siderophores, and non-ribosomal peptide synthetases (NRPS) have been identified, among others. In addition, the sequence similarity network showed a predominant type of BGCs in the family Micrococcaceae, and some genera were distinctly grouped. The BGCs identified in the isolated strains could be associated with applications such as antimicrobials, anticancer agents, and plant growth promoters, among others, positioning them as excellent candidates for future biotechnological applications and innovations. KEY POINTS: • Novel Antarctic rare Actinomycetota strains were isolated from soil and sediments • Genome-based taxonomic affiliation revealed seven potentially novel species • Genome mining showed metabolic potential for novel natural products.

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.

Exploring the Biological Pathways of Siderophores and Their Multidisciplinary Applications: A Comprehensive Review.

Siderophores are a class of small molecules renowned for their high iron binding capacity, essential for all life forms requiring iron. This article provides a detailed review of the diverse classifications, and biosynthetic pathways of siderophores, with a particular emphasis on siderophores synthesized via nonribosomal peptide synthetase (NRPS) and non-NRPS pathways. We further explore the secretion mechanisms of siderophores in microbes and plants, and their role in regulating bioavailable iron levels. Beyond biological functions, the applications of siderophores in medicine, agriculture, and environmental sciences are extensively discussed. These applications include biological pest control, disease treatment, ecological pollution remediation, and heavy metal ion removal. Through a comprehensive analysis of the chemical properties and biological activities of siderophores, this paper demonstrates their wide prospects in scientific research and practical applications, while also highlighting current research gaps and potential future directions.

The Micacocidin Production-Related RSc1806 Deletion Alters the Quorum Sensing-Dependent Gene Regulation of Ralstonia pseudosolanacearum Strain OE1-1.

The soil-borne phytopathogenic gram-negative bacterium Ralstonia solanacearum species complex (RSSC) produces staphyloferrin B and micacocidin as siderophores that scavenge for trivalent iron (Fe3+) in the environment, depending on the intracellular divalent iron (Fe2+) concentration. The staphyloferrin B-deficient mutant reportedly retains its virulence, but the relationship between micacocidin and virulence remains unconfirmed. To elucidate the effect of micacocidin on RSSC virulence, we generated the micacocidin productivity-deficient mutant (ΔRSc1806) that lacks RSc1806, which encodes a putative polyketide synthase/non-ribosomal peptide synthetase, using the RSSC phylotype I Ralstonia pseudosolanacearum strain OE1-1. When incubated in the condition without Fe2+, ΔRSc1806 showed significantly lower Fe3+-scavenging activity, compared with OE1-1. Until 8 days after inoculation on tomato plants, ΔRSc1806 was not virulent, similar to the mutant (ΔphcA) missing phcA, which encodes the LysR-type transcriptional regulator PhcA that regulates the expression of the genes responsible for quorum sensing (QS)-dependent phenotypes including virulence. The transcriptome analysis revealed that RSc1806 deletion significantly altered the expression of more than 80% of the PhcA-regulated genes in the mutant grown in medium with or without Fe2+. Among the PhcA-regulated genes, the transcript levels of the genes whose expression was affected by the deletion of RSc1806 were strongly and positively correlated between the ΔRSc1806 and the phcA-deletion mutant. Furthermore, the deletion of RSc1806 significantly modified QS-dependent phenotypes, similar to the effects of the deletion of phcA. Collectively, our findings suggest that the deletion of micacocidin production-related RSc1806 alters the regulation of PhcA-regulated genes responsible for QS-dependent phenotypes including virulence as well as Fe3+-scavenging activity. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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.

Enzyme engineering lets us play with new building blocks in non-ribosomal peptide synthesis.

In a recent issue of Nature Chemical Biology, Folger et al. demonstrated a high-throughput approach for engineering peptide bond forming domains from non-ribosomal peptide synthesis. A non-ribosomal peptide synthetase module from surfactin biosynthesis was reprogrammed to accept a fatty acid substrate into peptide biosynthesis, thus illustrating the potential of this approach for generating novel bioactive peptides.

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.

Genome Mining of a Fungal Polyketide Synthase-Nonribosomal Peptide Synthetase Hybrid Megasynthetase Pathway to Synthesize a Phytotoxic N-Acyl Amino Acid.

Guided by the retrobiosynthesis hypothesis, we characterized a fungal polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) hybrid megasynthetase pathway to generate 2-trans-4-trans-2-methylsorbyl-d-leucine (1), a polyketide amino acid conjugate that inhibits Arabidopsis root growth. The biosynthesis of 1 includes a PKS-NRPS enzyme to assemble an N-acyl amino alcohol intermediate, which is further oxidized to an N-acyl amino acid (NAAA), demonstrating a new biosynthetic logic for synthesizing NAAAs and expanding the chemical space of products encoded by fungal PKS-NRPS clusters.

Non-ribosomal peptide synthetase (NRPS)-encoding products and their biosynthetic logics in Fusarium.

Fungal non-ribosomal peptide synthetase (NRPS)-encoding products play a paramount role in new drug discovery. Fusarium, one of the most common filamentous fungi, is well-known for its biosynthetic potential of NRPS-type compounds with diverse structural motifs and various biological properties. With the continuous improvement and extensive application of bioinformatic tools (e.g., anti-SMASH, NCBI, UniProt), more and more biosynthetic gene clusters (BGCs) of secondary metabolites (SMs) have been identified in Fusarium strains. However, the biosynthetic logics of these SMs have not yet been well investigated till now. With the aim to increase our knowledge of the biosynthetic logics of NPRS-encoding products in Fusarium, this review firstly provides an overview of research advances in elucidating their biosynthetic pathways.

Identification of the Cirratiomycin Biosynthesis Gene Cluster in Streptomyces Cirratus: Elucidation of the Biosynthetic Pathways for 2,3-Diaminobutyric Acid and Hydroxymethylserine.

Cirratiomycin, a heptapeptide with antibacterial activity, was isolated and characterized in 1981; however, its biosynthetic pathway has not been elucidated. It contains several interesting nonproteinogenic amino acids, such as (2S,3S)-2,3-diaminobutyric acid ((2S,3S)-DABA) and α-(hydroxymethyl)serine, as building blocks. Here, we report the identification of a cirratiomycin biosynthetic gene cluster in Streptomyces cirratus. Bioinformatic analysis revealed that several Streptomyces viridifaciens and Kitasatospora aureofaciens strains also have this cluster. One S. viridifaciens strain was confirmed to produce cirratiomycin. The biosynthetic gene cluster was shown to be responsible for cirratiomycin biosynthesis in S. cirratus in a gene inactivation experiment using CRISPR-cBEST. Interestingly, this cluster encodes a nonribosomal peptide synthetase (NRPS) composed of 12 proteins, including those with an unusual domain organization: a stand-alone adenylation domain, two stand-alone condensation domains, two type II thioesterases, and two NRPS modules that have no adenylation domain. Using heterologous expression and in vitro analysis of recombinant enzymes, we revealed the biosynthetic pathway of (2S,3S)-DABA: (2S,3S)-DABA is synthesized from l-threonine by four enzymes, CirR, CirS, CirQ, and CirB. In addition, CirH, a glycine/serine hydroxymethyltransferase homolog, was shown to synthesize α-(hydroxymethyl)serine from d-serine in vitro. These findings broaden our knowledge of nonproteinogenic amino acid biosynthesis.

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.

Controlling Substrate- and Stereospecificity of Condensation Domains in Nonribosomal Peptide Synthetases.

Nonribosomal peptide synthetases (NRPSs) are sophisticated molecular machines that biosynthesize peptide drugs. In attempts to generate new bioactive compounds, some parts of NRPSs have been successfully manipulated, but especially the influence of condensation (C-)domains on substrate specificity remains enigmatic and poorly controlled. To understand the influence of C-domains on substrate preference, we extensively evaluated the peptide formation of C-domain mutants in a bimodular NRPS system. Thus, we identified three key mutations that govern the preference for stereoconfiguration and side-chain identity. These mutations show similar effects in three different C-domains (GrsB1, TycB1, and SrfAC) when di- or pentapeptides are synthesized in vitro or in vivo. Strikingly, mutation E386L allows the stereopreference to be switched from d- to l-configured donor substrates. Our findings provide valuable insights into how cryptic specificity filters in C-domains can be re-engineered to clear roadblocks for NRPS engineering and enable the production of novel bioactive compounds.

High-throughput reprogramming of an NRPS condensation domain.

Engineered biosynthetic assembly lines could revolutionize the sustainable production of bioactive natural product analogs. Although yeast display is a proven, powerful tool for altering the substrate specificity of gatekeeper adenylation domains in nonribosomal peptide synthetases (NRPSs), comparable strategies for other components of these megaenzymes have not been described. Here we report a high-throughput approach for engineering condensation (C) domains responsible for peptide elongation. We show that a 120-kDa NRPS module, displayed in functional form on yeast, can productively interact with an upstream module, provided in solution, to produce amide products tethered to the yeast surface. Using this system to screen a large C-domain library, we reprogrammed a surfactin synthetase module to accept a fatty acid donor, increasing catalytic efficiency for this noncanonical substrate >40-fold. Because C domains can function as selectivity filters in NRPSs, this methodology should facilitate the precision engineering of these molecular assembly lines.

Heterologous Biosynthesis of the Sterol O-Acyltransferase Inhibitor Helvamide Unveils an α-Ketoglutarate-Dependent Cross-Linking Oxygenase.

We have identified the biosynthetic gene cluster (hvm) for the sterol O-acyltransferase inhibitor helvamide (1) from the genome of Aspergillus rugulosus MST-FP2007. Heterologous expression of hvm in A. nidulans produced a previously unreported analog helvamide B (5). An α-ketoglutarate-dependent oxygenase Hvm1 was shown to catalyze intramolecular cyclization of 1 to yield 5. The biosynthetic branch to the related hancockiamides and helvamides was found to be controlled by the substrate selectivity of monomodular nonribosomal peptide synthetases.

Poly-γ-glutamylation of biomolecules.

Poly-γ-glutamate tails are a distinctive feature of archaeal, bacterial, and eukaryotic cofactors, including the folates and F420. Despite decades of research, key mechanistic questions remain as to how enzymes successively add glutamates to poly-γ-glutamate chains while maintaining cofactor specificity. Here, we show how poly-γ-glutamylation of folate and F420 by folylpolyglutamate synthases and γ-glutamyl ligases, non-homologous enzymes, occurs via processive addition of L-glutamate onto growing γ-glutamyl chain termini. We further reveal structural snapshots of the archaeal γ-glutamyl ligase (CofE) in action, crucially including a bulged-chain product that shows how the cofactor is retained while successive glutamates are added to the chain terminus. This bulging substrate model of processive poly-γ-glutamylation by terminal extension is arguably ubiquitous in such biopolymerisation reactions, including addition to folates, and demonstrates convergent evolution in diverse species from archaea to humans.

The Lrp transcriptional factor of an entomopathogenic bacterium, Xenorhabdus hominickii, activates non-ribosomal peptide synthetases to suppress insect immunity.

Two bacterial genera, Xenorhabdus and Photorhabdus, are mutually symbiotic to the entomopathogenic nematodes, Steinernema and Heterorhabditis, respectively. The infective juveniles deliver the symbiotic bacteria to the hemocoel of target insects, in which the bacteria proliferate and help the development of the host nematode. The successful parasitism of the nematode-bacterial complex depends on host immunosuppression by the bacteria via their secondary metabolites. Leucine-responsive regulatory protein (Lrp) is a global bacterial transcriptional factor that plays a crucial role in parasitism. However, its regulatory targets to suppress insect immunity are not clearly understood. This study investigated the bacterial genes regulated by Lrp and the subsequent production of secondary metabolites in Xenorhabdus hominickii. Lrp expression occurred at the early infection stage of the bacteria in a target insect, Spodoptera exigua. A preliminary in silico screening indicated that 3.7% genes among 4075 predicted genes encoded in X. hominickii had the Lrp-response element on their promoters, including two non-ribosomal peptide synthetases (NRPSs). Eight NRPS (NRPS1-NRPS8) genes were predicted in the bacterial genome, in which six NRPS (NRPS3-NRPS8) expressions were positively correlated with Lrp expression in the infected larvae of S. exigua. Exchange of the Lrp promoter with an inducible promoter altered the production of the secondary metabolites and the NRPS expression levels. The immunosuppressive activities of X. hominickii were dependent on the Lrp expression level. The metabolites produced by Lrp expression included the eicosanoid-biosynthesis inhibitors and hemolytic factors. A cyclic dipeptide (=cPF) was produced by the bacteria at high Lrp expression and inhibited the phospholipase A2 activity of S. exigua in a competitive inhibitory manner. These results suggest that Lrp is a global transcriptional factor of X. hominickii and plays a crucial role in insect immunosuppression by modulating NRPS expression.

Recent progress in the reprogramming of nonribosomal peptide synthetases.

Nonribosomal peptide synthetases (NRPSs) biosynthesize nonribosomal peptide (NRP) natural products, which belong to the most promising resources for drug discovery and development because of their wide range of therapeutic applications. The results of genetic, biochemical, and bioinformatics analyses have enhanced our understanding of the mechanisms of the NRPS machinery. A major goal in NRP biosynthesis is to reprogram the NRPS machinery to enable the biosynthetic production of designed peptides. Reprogramming strategies for the NRPS machinery have progressed considerably in recent years, thereby increasing the yields and generating modified peptides. Here, the recent progress in NRPS reprogramming and its application in peptide synthesis are described.

Characterization and catalytic investigation of fungal single-module nonribosomal peptide synthetase in terpene-amino acid meroterpenoid biosynthesis.

Hybrid natural products are compounds that originate from diverse biosynthetic pathways and undergo a conjugation process, which enables them to expand their chemical diversity and biological functionality. Terpene-amino acid meroterpenoids have garnered increasing attention in recent years, driven by the discovery of noteworthy examples such as the anthelmintic CJ-12662, the insecticidal paeciloxazine, and aculene A (1). In the biosynthesis of terpene-amino acid natural products, single-module nonribosomal peptide synthetases (NRPSs) have been identified to be involved in the esterification step, catalyzing the fusion of modified terpene and amino acid components. Despite prior investigations into these NRPSs through gene deletion or in vivo experiments, the enzymatic basis and mechanistic insights underlying this family of single-module NRPSs remain unclear. In this study, we performed biochemical characterization of AneB by in vitro characterization, molecular docking, and site-directed mutagenesis. The enzyme reaction analyses, performed with L-proline and daucane/nordaucane sesquiterpene substrates, revealed that AneB specifically esterifies the C10-OH of aculenes with L-proline. Notably, in contrast to ThmA in CJ-12662 biosynthesis, which exclusively recognizes oxygenated amorpha-4,11-diene sesquiterpenes for L-tryptophan transfer, AneB demonstrates broad substrate selectivity, including oxygenated amorpha-4,11-diene and 2-phenylethanol, resulting in the production of diverse unnatural prolyl compounds. Furthermore, site-directed mutagenesis experiments indicated the involvement of H794 and D798 in the esterification catalyzed by AneB. Lastly, domain swapping between AneB and ThmA unveiled that the A‒T domains of ThmA can be effectively harnessed by the C domain of AneB for L-tryptophan transfer, thus highlighting the potential of the C domain of AneB for generating various terpene-amino acid meroterpenoid derivatives. ONE-SENTENCE SUMMARY: The enzymatic basis and mechanistic insights into AneB, a single-module NRPS, highlight its capacity to generate various terpene-amino acid meroterpenoid derivatives.

High-Throughput Engineering of Nonribosomal Extension Modules.

Nonribosomal peptides constitute an important class of natural products that display a wide range of bioactivities. They are biosynthesized by large assembly lines called nonribosomal peptide synthetases (NRPSs). Engineering NRPS modules represents an attractive strategy for generating customized synthetases for the production of peptide variants with improved properties. Here, we explored the yeast display of NRPS elongation and termination modules as a high-throughput screening platform for assaying adenylation domain activity and altering substrate specificity. Depending on the module, display of A-T bidomains or C-A-T tridomains, which also include an upstream condensation domain, proved to be most effective. Reprograming a tyrocidine synthetase elongation module to accept 4-propargyloxy-phenylalanine, a noncanonical amino acid that is not activated by the native protein, illustrates the utility of this approach for altering NRPS specificity at internal sites.