Enoyl Reductase Domain Research Articles

Antibiotic Skeletal Diversification via Differential Enoylreductase Recruitment and Module Iteration in trans-Acyltransferase Polyketide Synthases.

Microorganisms are remarkable chemists capable of assembling complex molecular architectures that penetrate cells and bind biomolecular targets with exquisite selectivity. Consequently, microbial natural products have wide-ranging applications in medicine and agriculture. How the "blind watchmaker" of evolution creates skeletal diversity is a key question in natural products research. Comparative analysis of biosynthetic pathways to structurally related metabolites is an insightful approach to addressing this. Here, we report comparative biosynthetic investigations of gladiolin, a polyketide antibiotic from Burkholderia gladioli with promising activity against multidrug-resistant Mycobacterium tuberculosis, and etnangien, a structurally related antibiotic produced by Sorangium cellulosum. Although these metabolites have very similar macrolide cores, their C21 side chains differ significantly in both length and degree of saturation. Surprisingly, the trans-acyltransferase polyketide synthases (PKSs) that assemble these antibiotics are almost identical, raising intriguing questions about mechanisms underlying structural diversification in this important class of biosynthetic assembly line. In vitro reconstitution of key biosynthetic transformations using simplified substrate analogues, combined with gene deletion and complementation experiments, enabled us to elucidate the origin of all the structural differences in the C21 side chains of gladiolin and etnangien. The more saturated gladiolin side chain arises from a cis-acting enoylreductase (ER) domain in module 1 and in trans recruitment of a standalone ER to module 5 of the PKS. Remarkably, module 5 of the gladiolin PKS is intrinsically iterative in the absence of the standalone ER, accounting for the longer side chain in etnangien. These findings have important implications for biosynthetic engineering approaches to the creation of novel polyketide skeletons.

Identification of triazenyl indoles as inhibitors of fungal fatty acid biosynthesis with broad-spectrum activity

Rising drug resistance among pathogenic fungi, paired with a limited antifungal arsenal, poses an increasing threat to human health. To identify antifungal compounds, we screened the RIKEN natural product depository against representative isolates of four major human fungal pathogens. This screen identified NPD6433, a triazenyl indole with broad-spectrum activity against all screening strains, as well as the filamentous mold Aspergillus fumigatus. Mechanistic studies indicated that NPD6433 targets the enoyl reductase domain of fatty acid synthase 1 (Fas1), covalently inhibiting its flavin mononucleotide-dependent NADPH-oxidation activity and arresting essential fatty acid biosynthesis. Robust Fas1 inhibition kills Candida albicans, while sublethal inhibition impairs diverse virulence traits. At well-tolerated exposures, NPD6433 extended the lifespan of nematodes infected with azole-resistant C.albicans. Overall, identification of NPD6433 provides a tool with which to explore lipid homeostasis as a therapeutic target in pathogenic fungi and reveals a mechanism by which Fas1 function can be inhibited.

Structure of a modular polyketide synthase reducing region

The chemical scaffolds of numerous therapeutics are polyketide natural products, many formed by bacterial modular polyketide synthases (PKS). The large and flexible dimeric PKS modules have distinct extension and reducing regions. Structures are known for all individual enzyme domains and several extension regions. Here, we report the structure of the full reducing region from a modular PKS, the ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) domains of module 5 of the juvenimicin PKS. The modular PKS-reducing region has a different architecture than the homologous fatty acid synthase (FAS) and iterative PKS systems in its arrangement of domains and dimer interface. The structure reveals a critical role for linker peptides in the domain interfaces, leading to discovery of key differences in KR domains dependent on module composition. Finally, our studies provide insight into the mechanism underlying modular PKS intermediate shuttling by carrier protein (ACP) domains.

Expanding the Utility of Bioinformatic Data for the Full Stereostructural Assignments of Marinolides A and B, 24- and 26-Membered Macrolactones Produced by a Chemically Exceptional Marine-Derived Bacterium.

Marinolides A and B, two new 24- and 26-membered bacterial macrolactones, were isolated from the marine-derived actinobacterium AJS-327 and their stereostructures initially assigned by bioinformatic data analysis. Macrolactones typically possess complex stereochemistry, the assignments of which have been one of the most difficult undertakings in natural products chemistry, and in most cases, the use of X-ray diffraction methods and total synthesis have been the major methods of assigning their absolute configurations. More recently, however, it has become apparent that the integration of bioinformatic data is growing in utility to assign absolute configurations. Genome mining and bioinformatic analysis identified the 97 kb mld biosynthetic cluster harboring seven type I polyketide synthases. A detailed bioinformatic investigation of the ketoreductase and enoylreductase domains within the multimodular polyketide synthases, coupled with NMR and X-ray diffraction data, allowed for the absolute configurations of marinolides A and B to be determined. While using bioinformatics to assign the relative and absolute configurations of natural products has high potential, this method must be coupled with full NMR-based analysis to both confirm bioinformatic assignments as well as any additional modifications that occur during biosynthesis.

Insights into azalomycin F assembly-line contribute to evolution-guided polyketide synthase engineering and identification of intermodular recognition

Modular polyketide synthase (PKS) is an ingenious core machine that catalyzes abundant polyketides in nature. Exploring interactions among modules in PKS is very important for understanding the overall biosynthetic process and for engineering PKS assembly-lines. Here, we show that intermodular recognition between the enoylreductase domain ER1/2 inside module 1/2 and the ketosynthase domain KS3 inside module 3 is required for the cross-module enoylreduction in azalomycin F (AZL) biosynthesis. We also show that KS4 of module 4 acts as a gatekeeper facilitating cross-module enoylreduction. Additionally, evidence is provided that module 3 and module 6 in the AZL PKS are evolutionarily homologous, which makes evolution-oriented PKS engineering possible. These results reveal intermodular recognition, furthering understanding of the mechanism of the PKS assembly-line, thus providing different insights into PKS engineering. This also reveals that gene duplication/conversion and subsequent combinations may be a neofunctionalization process in modular PKS assembly-lines, hence providing a different case for supporting the investigation of modular PKS evolution.

Pharmacological fatty acid synthase inhibitors differently affect the malignant phenotype of oral cancer cells.

ObjectiveFatty acid synthase levels are associated with aggressiveness, prognosis, and risk of metastasis in oral squamous cell carcinomas. This enzyme contains seven catalytic domains and its inhibition by synthetic or natural drugs has antineoplastic properties such as C75, which is a synthetic inhibitor of the β- ketoacyl synthase domain, the antibiotic triclosan, ligand of the enoyl reductase domain, and the antiobesity drug orlistat, which inhibits the thioesterase domain. Here, we sought to investigate and compare the in vitro effects of C75, triclosan, and orlistat on malignant phenotypes of the cell line SCC-9: proliferation, cell cycle, apoptosis, adhesion, migration, and invasion. DesignHalf-maximal inhibitory concentration (IC50) was determined using cell viability assays. Cell death and cell cycle progression were analyzed by Annexin V-PE/7-ADD-PerCP labeling and propidium iodide staining, respectively. Cell migration and invasion were assayed by transwells assays and cell adhesion using collagen and fibronectin. ResultsC75 showed the lowest IC50 and higher inhibition of lipid droplets at low concentrations and reduced cell motility. Triclosan showed the intermediate IC50 value, excellent reduction of lipid bodies at the IC50 when compared with C75 and orlistat. Also, triclosan reduced cell cycle progression, adhesion, migration, and invasion of SCC-9 and induced the highest levels of apoptosis. Orlistat promoted cell cycle arrest, but showed the lowest induction of apoptosis and did not affected invasion and adhesion of SCC-9. ConclusionAltogether, despite the particular effects of the analyzed fatty acid synthase inhibitors, triclosan showed to better interfere in tumorigenic phenotypes of SCC-9 cells.

Biosynthesis of Fungal Drimane‐Type Sesquiterpene Esters

AbstractDrimane‐type sesquiterpenes exhibit various biological activities and are widely present in eukaryotes. Here, we completely elucidated the biosynthetic pathway of the drimane‐type sesquiterpene esters isolated from Aspergillus calidoustus and we discovered that it involves a drimenol cyclase having the same catalytic function previously only reported in plants. Moreover, since many fungal drimenol derivatives possess a γ‐butyrolactone ring, we clarified the functions of the cluster‐associated cytochrome P450 and FAD‐binding oxidoreductase discovering that these two enzymes are solely responsible for the formation of those structures. Furthermore, swapping of the enoyl reductase domain in the identified polyketide synthase led to the production of metabolites containing various polyketide chains with different levels of saturation. These findings have deepened our understanding of how fungi synthesize drimane‐type sesquiterpenes and the corresponding esters.

Biosynthesis of Fungal Drimane-Type Sesquiterpene Esters.

Drimane‐type sesquiterpenes exhibit various biological activities and are widely present in eukaryotes. Here, we completely elucidated the biosynthetic pathway of the drimane‐type sesquiterpene esters isolated from Aspergillus calidoustus and we discovered that it involves a drimenol cyclase having the same catalytic function previously only reported in plants. Moreover, since many fungal drimenol derivatives possess a γ‐butyrolactone ring, we clarified the functions of the cluster‐associated cytochrome P450 and FAD‐binding oxidoreductase discovering that these two enzymes are solely responsible for the formation of those structures. Furthermore, swapping of the enoyl reductase domain in the identified polyketide synthase led to the production of metabolites containing various polyketide chains with different levels of saturation. These findings have deepened our understanding of how fungi synthesize drimane‐type sesquiterpenes and the corresponding esters.

Function of ORFC of the polyketide synthase gene cluster on fatty acid accumulation in Schizochytrium limacinum SR21

BackgroundAs a potential source of polyunsaturated fatty acids (PUFA), Schizochytrium sp. has been widely used in industry for PUFA production. Polyketide synthase (PKS) cluster is supposed to be the primary way of PUFA synthesis in Schizochytrium sp. As one of three open reading frames (ORF) in the PKS cluster, ORFC plays an essential role in fatty acid biosynthesis. However, the function of domains in ORFC in the fatty acid synthesis of Schizochytrium sp. remained unclear.ResultsIn this study, heterologous expression and overexpression were carried out to study the role of ORFC and its domains in fatty acid accumulation. Firstly, ORFC was heterologously expressed in yeast which increased the PUFA content significantly. Then, the dehydratase (DH) and enoyl reductase (ER) domains located on ORFC were overexpressed in Schizochytrium limacinum SR21, respectively. Fatty acids profile analysis showed that the contents of PUFA and saturated fatty acid were increased in the DH and ER overexpression strains, respectively. This indicated that the DH and ER domains played distinct roles in lipid accumulation. Metabolic and transcriptomic analysis revealed that the pentose phosphate pathway and triacylglycerol biosynthesis were enhanced, while the tricarboxylic acid cycle and fatty acids oxidation were weakened in DH-overexpression strain. However, the opposite effect was found in the ER-overexpression strain.ConclusionTherefore, ORFC was required for the biosynthesis of fatty acid. The DH domain played a crucial role in PUFA synthesis, whereas the ER domain might be related to saturated fatty acids (SFA) synthesis in Schizochytrium limacinum SR21. This research explored the role of ORFC in the PKS gene cluster in Schizochytrium limacinum and provided potential genetic modification strategies for improving lipid production and regulating PUFA and SFA content.

Proteomic analyses reveal mechanisms of action of biocontrol agents on ochratoxin A repression in Penicillium nordicum

Penicillium nordicum is among the main ochratoxin A (OTA) producers in dry-cured fermented sausages. Recent strategies to prevent its development on the surface of these products include the substitution of synthetic antifungals, such as potassium sorbate, by natural compounds. However, the mechanisms of action of the latter are still unclear. The Proteomic analysis is a useful tool to elucidate the basis of the antifungal compound effects on moulds. Therefore, the aim of this study was to evaluate the efficiency of biocontrol agents on the OTA repression in P. nordicum and its relation to proteome changes. Seven different combinations of rosemary leaves, rosemary essential oil, acorn shell extract and the yeast Debaryomyces hansenii have been tested as biocontrol agents against two ochratoxigenic P. nordicum strains on a dry-cured fermented sausage-based medium. The rosemary essential oil and the combination of rosemary leaves with D. hansenii provoked significant inhibition of OTA production in both moulds (p ≤ 0.05). The acorn shell extract and D. hansenii individually decreased OTA only in one of the mould strains. The Proteomic analyses revealed that rosemary essential oil reduced the abundance of proteins involved in the polyketide synthase enoylreductase domain in both moulds, which would explain the OTA reduction. However, the combination of rosemary leaves with D. hansenii showed inter-strain differences, reducing the abundance of proteins related to the cell wall integrity and to the purine pathway, depending on the tested mould. In conclusion, rosemary essential oil and the combination of rosemary leaves with D. hansenii can be proposed as efficient biocontrol agents against ochratoxigenic P. nordicum on dry-cured fermented sausages, although some inter-strain differences at proteome level were found regarding the mechanism of action involved in the reported OTA repression.

Engineering the acyltransferase domain of epothilone polyketide synthase to alter the substrate specificity

BackgroundPolyketide synthases (PKSs) include ketone synthase (KS), acyltransferase (AT) and acyl carrier protein (ACP) domains to catalyse the elongation of polyketide chains. Some PKSs also contain ketoreductase (KR), dehydratase (DH) and enoylreductase (ER) domains as modification domains. Insertion, deletion or substitution of the catalytic domains may lead to the production of novel polyketide derivatives or to the accumulation of desired products. Epothilones are 16-membered macrolides that have been used as anticancer drugs. The substrate promiscuity of the module 4 AT domain of the epothilone PKS (EPOAT4) results in production of epothilone mixtures; substitution of this domain may change the ratios of epothilones. In addition, there are two dormant domains in module 9 of the epothilone PKS. Removing these redundant domains to generate a simpler and more efficient assembly line is a desirable goal.ResultsThe substitution of module 4 drastically diminished the activity of epothilone PKS. However, with careful design of the KS-AT linker and the post-AT linker, replacing EPOAT4 with EPOAT2, EPOAT6, EPOAT7 or EPOAT8 (specifically incorporating methylmalonyl-CoA (MMCoA)) significantly increased the ratio of epothilone D (4) to epothilone C (3) (the highest ratio of 4:3 = 4.6:1), whereas the ratio of 4:3 in the parental strain Schlegelella brevitalea 104-1 was 1.4:1. We also obtained three strains by swapping EPOAT4 with EPOAT3, EPOAT5, or EPOAT9, which specifically incorporate malonyl-CoA (MCoA). These strains produced only epothilone C, and the yield was increased by a factor of 1.8 compared to that of parental strain 104-1. Furthermore, mutations of five residues in the AT domain identified Ser310 as the critical factor for MMCoA recognition in EPOAT4. Then, the mutation of His308 to valine or tyrosine combined with the mutation of Phe310 to serine further altered the product ratios. At the same time, we successfully deleted the inactive module 9 DH and ER domains and fused the ΨKR domain with the KR domain through an ~ 25-residue linker to generate a productive and simplified epothilone PKS.ConclusionsThese results suggested that the substitution and deletion of catalytic domains effectively produces desirable compounds and that selection of the linkers between domains is crucial for maintaining intact PKS catalytic activity.

Cross‐Module Enoylreduction in the Azalomycin F Polyketide Synthase

AbstractThe colinearity of canonical modular polyketide synthases, which creates a direct link between multienzyme structure and the chemical structure of the biosynthetic end‐product, has become a cornerstone of knowledge‐based genome mining. Herein, we report genetic and enzymatic evidence for the remarkable role of an enoylreductase in the polyketide synthase for azalomycin F biosynthesis. This internal enoylreductase domain, previously identified as acting only in the second of two chain extension cycles on an initial iterative module, is shown to also catalyze enoylreduction in trans within the next module. The mechanism for this rare deviation from colinearity appears to involve direct cross‐modular interaction of the reductase with the longer acyl chain, rather than back transfer of the substrate into the iterative module, suggesting an additional and surprising plasticity in natural PKS assembly‐line catalysis.

Cross-Module Enoylreduction in the Azalomycin F Polyketide Synthase.

The colinearity of canonical modular polyketide synthases, which creates a direct link between multienzyme structure and the chemical structure of the biosynthetic end-product, has become a cornerstone of knowledge-based genome mining. Herein, we report genetic and enzymatic evidence for the remarkable role of an enoylreductase in the polyketide synthase for azalomycin F biosynthesis. This internal enoylreductase domain, previously identified as acting only in the second of two chain extension cycles on an initial iterative module, is shown to also catalyze enoylreduction in trans within the next module. The mechanism for this rare deviation from colinearity appears to involve direct cross-modular interaction of the reductase with the longer acyl chain, rather than back transfer of the substrate into the iterative module, suggesting an additional and surprising plasticity in natural PKS assembly-line catalysis.

Steric occlusion regulates proximal interactions of acyl carrier protein domain in fungal fatty acid synthase

The acyl carrier protein (ACP) domain shuttles substrates and reaction intermediates in type I fungal fatty acid synthases via transient protein-protein interactions. Here, using electron cryo-microscopy (cryoEM), we report the structure of a fungal FAS stalled at the dehydration reaction, which precedes the final enoyl reduction in the fatty acid biosynthesis cycle. This conformation revealed multiple contact sites between ACP and the dehydratase (DH) and enoyl reductase (ER) domains that occluded the ACP binding to the adjacent ER domain. Our data suggests a minimal path from the DH to the ER reaction site that requires minute changes in the coordinates of the structured N- and C- termini of the ACP domain.

Transcriptomic analysis of polyketide synthases in a highly ciguatoxic dinoflagellate, Gambierdiscus polynesiensisand low toxicity Gambierdiscus pacificus, from French Polynesia.

Marine dinoflagellates produce a diversity of polyketide toxins that are accumulated in marine food webs and are responsible for a variety of seafood poisonings. Reef-associated dinoflagellates of the genus Gambierdiscus produce toxins responsible for ciguatera poisoning (CP), which causes over 50,000 cases of illness annually worldwide. The biosynthetic machinery for dinoflagellate polyketides remains poorly understood. Recent transcriptomic and genomic sequencing projects have revealed the presence of Type I modular polyketide synthases in dinoflagellates, as well as a plethora of single domain transcripts with Type I sequence homology. The current transcriptome analysis compares polyketide synthase (PKS) gene transcripts expressed in two species of Gambierdiscus from French Polynesia: a highly toxic ciguatoxin producer, G. polynesiensis, versus a non-ciguatoxic species G. pacificus, each assembled from approximately 180 million Illumina 125 nt reads using Trinity, and compares their PKS content with previously published data from other Gambierdiscus species and more distantly related dinoflagellates. Both modular and single-domain PKS transcripts were present. Single domain β-ketoacyl synthase (KS) transcripts were highly amplified in both species (98 in G. polynesiensis, 99 in G. pacificus), with smaller numbers of standalone acyl transferase (AT), ketoacyl reductase (KR), dehydratase (DH), enoyl reductase (ER), and thioesterase (TE) domains. G. polynesiensis expressed both a larger number of multidomain PKSs, and larger numbers of modules per transcript, than the non-ciguatoxic G. pacificus. The largest PKS transcript in G. polynesiensis encoded a 10,516 aa, 7 module protein, predicted to synthesize part of the polyether backbone. Transcripts and gene models representing portions of this PKS are present in other species, suggesting that its function may be performed in those species by multiple interacting proteins. This study contributes to the building consensus that dinoflagellates utilize a combination of Type I modular and single domain PKS proteins, in an as yet undefined manner, to synthesize polyketides.

Vaccine-Specific Immune Responses against Mycobacterium ulcerans Infection in a Low-Dose Murine Challenge Model.

The neglected tropical disease Buruli ulcer (BU) is an infection of subcutaneous tissue with Mycobacterium ulcerans There is no effective vaccine. Here, we assessed an experimental prime-boost vaccine in a low-dose murine tail infection model. We used the enoyl reductase (ER) domain of the M. ulcerans mycolactone polyketide synthases electrostatically coupled with a previously described Toll-like receptor 2 (TLR-2) agonist-based lipopeptide adjuvant, R4Pam2Cys. Mice were vaccinated and then challenged via tail inoculation with 14 to 20 CFU of a bioluminescent strain of M. ulcerans Mice receiving either the experimental ER vaccine or Mycobacterium bovis bacillus Calmette-Guérin (BCG) were equally protected, with both groups faring significantly better than nonvaccinated animals (P < 0.05). To explore potential correlates of protection, a suite of 29 immune parameters were assessed in the mice at the end of the experimental period. Multivariate statistical approaches were used to interrogate the immune response data to develop disease-prognostic models. High levels of interleukin 2 (IL-2) and low gamma interferon (IFN-γ) produced in the spleen best predicted control of infection across all vaccine groups. Univariate logistic regression revealed vaccine-specific profiles of protection. High titers of ER-specific IgG serum antibodies together with IL-2 and IL-4 in the draining lymph node (DLN) were associated with protection induced by the ER vaccine. In contrast, high titers of IL-6, tumor necrosis factor alpha (TNF-α), IFN-γ, and IL-10 in the DLN and low IFN-γ titers in the spleen were associated with protection following BCG vaccination. This study suggests that an effective BU vaccine must induce localized, tissue-specific immune profiles with controlled inflammatory responses at the site of infection.

Stimulated Biosynthesis of an C10-Deoxy Heptaene NPP B2 via Regulatory Genes Overexpression in Pseudonocardia autotrophica.

Polyene macrolides, such as nystatin A1, amphotericin B, and NPP A1, belong to a large family of valuable antifungal polyketide compounds that are typically produced by soil actinomycetes. Previously, NPP B1, a novel NPP A1 derivative harboring a heptaene core structure, was generated by introducing two amino acid substitutions in the putative NADPH-binding motif of the enoyl reductase domain in module 5 of the NPP A1 polyketide synthase in Pseudonocardia autotrophica. This derivative showed superior antifungal activity to NPP A1. In this study, another novel derivative called NPP B2 was developed, which lacks a hydroxyl group at the C10 position by site-specific gene disruption of the P450 hydroxylase NppL. To stimulate the extremely low expression of the NPP B2 biosynthetic pathway genes, the 32-kb NPP-specific regulatory gene cluster was overexpressed via site-specific chromosomal integration. The extra copy of the six NPP-specific regulatory genes led to a significant increase in the NPP B2 yield from 0.19 to 7.67 mg/L, which is the highest level of NPP B2 production ever achieved by the P. autotrophica strain. Subsequent in vitro antifungal activity and toxicity studies indicated that NPP B2 exhibited similar antifungal activity but significantly lower hemolytic toxicity than NPP B1. These results suggest that an NPP biosynthetic pathway refactoring and overexpression of its pathway-specific regulatory genes is an efficient approach to stimulating the production of an extremely low-level metabolite, such as NPP B2 in a pathway-engineered rare actinomycete strain.

Functional expression of a highly-reducing polyketide synthase of Emericella variecolor IFM42010, an asteltoxin-producing strain, resulted in production of two polyenoic β-ketolactones with opposite stereochemistry.

The asteltoxin-producing fungus Emericella variecolor IFM42010 possesses 22 highly-reducing polyketide synthase (HR-PKS) genes. Of these, an HR-PKS with a methyltransferase domain but lacking an enoylreductase domain could be involved in the biosynthesis of asteltoxin and related compounds. From six such candidate HR-PKS genes, Ev460pks was analyzed by gene disruption in E. variecolor and heterologous expression in Aspergillus oryzae. The Ev460pks-disrupted strain retained asteltoxin production ability, indicating that Ev460pks is not involved in asteltoxin biosynthesis. The A. oryzae transformant harboring the Ev460pks gene produced compounds 1 and 2, along with several unidentified products possibly decomposed from 2. Spectroscopic analyses revealed that 1 was a 4-methyl-β-ketolactone with a methylheptatriene side-chain at the C-5 position, and 2 was also a 4-methyl-β-ketolactone, bearing a dimethyltetradecahexaene side-chain at the same position. The relative configuration at C-4 in compounds 1 and 2 was opposite.

Electron cryomicroscopy observation of acyl carrier protein translocation in type I fungal fatty acid synthase

During fatty acid biosynthesis, acyl carrier proteins (ACPs) from type I fungal fatty acid synthase (FAS) shuttle substrates and intermediates within a reaction chamber that hosts multiple spatially-fixed catalytic centers. A major challenge in understanding the mechanism of ACP-mediated substrate shuttling is experimental observation of its transient interaction landscape within the reaction chamber. Here, we have shown that ACP spatial distribution is sensitive to the presence of substrates in a catalytically inhibited state, which enables high-resolution investigation of the ACP-dependent conformational transitions within the enoyl reductase (ER) reaction site. In two fungal FASs with distinct ACP localization, the shuttling domain is targeted to the ketoacyl-synthase (KS) domain and away from other catalytic centers, such as acetyl-transferase (AT) and ER domains by steric blockage of the KS active site followed by addition of substrates. These studies strongly suggest that acylation of phosphopantetheine arm of ACP may be an integral part of the substrate shuttling mechanism in type I fungal FAS.

Nystatin-like Pseudonocardia polyene B1, a novel disaccharide-containing antifungal heptaene antibiotic

Polyene macrolides such as nystatin A1 and amphotericin B belong to a large family of very valuable antifungal polyketide compounds typically produced by soil actinomycetes. Recently, nystatin-like Pseudonocardia polyene (NPP) A1 has been identified as a unique disaccharide-containing tetraene antifungal macrolide produced by Pseudonocardia autotrophica. Despite its significantly increased water solubility and decreased hemolytic activity, its antifungal activity remains limited compared with that of nystatin A1. In this study, we developed NPP B1, a novel NPP A1 derivative harboring a heptaene core structure, by introducing two amino acid substitutions in the putative NADPH-binding motif of the enoyl reductase domain in module 5 of the NPP A1 polyketide synthase NppC. The low level NPP B1 production yield was successfully improved by eliminating the native plasmid encoding a polyketide biosynthetic gene cluster present in P. autotrophica. In vitro and in vivo antifungal activity and toxicity studies indicated that NPP B1 exhibited comparable antifungal activity against Candida albicans and was less toxic than the most potent heptaene antifungal, amphotericin B. Moreover, NPP B1 showed improved pharmacokinetic parameters compared to those of amphotericin B, suggesting that NPP B1 could be a promising candidate for development into a pharmacokinetically improved and less-toxic polyene antifungal antibiotic.