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Structure of the aglycone core of pimaricin. Mycosamine (M) is attached to the hydroxyl at C-15 in the pimaricin molecule. The 12 C-C bonds formed by the polyketide synthase are boxed and numbered in italics. Bold lines indicate the building units. Lactonization of the acyl chain between C-1 and C-25 results in the formation of the pimaricinolide ring.
Source publication
The biosynthetic gene cluster for the 26-membered ring of the polyene macrolide pimaricin extends for about 110 kilobase pairs
of contiguous DNA in the genome of Streptomyces natalensis. Two sets of polyketide synthase (PKS) genes are separated by a group of small polyketide-functionalizing genes. Two of the
polyketide synthase genes, pimS0 and pim...
Citations
... Antibiotics 2023, 12, 119 ...
The biosynthesis of polyene macrolides, which are natural products produced by soil actinomycetes, have been extensively explored, and recent studies have focused on the effects of post–polyketide synthase (PKS) modifications to polyene macrolides on toxicity, water solubility, and antifungal activity. For example, there are interactions between glycosyl, carboxyl, and hydroxyl or epoxy groups generated in the post-PKS modification steps; salt bridges will be formed between carboxylate and ammonium on the mycosamine; and water bridges will be formed between hydroxy and hydroxyl on mycosamine. These interactions will affect their water solubility and substrate-recognition specificity. This review summarizes research related to these post-PKS modification groups and discusses some genetic engineering operation problems and solutions that may be encountered when modifying these post-PKS modification groups. In addition, this review provides a basis for the structural research of polyene macrolide antibiotics and contributes to comprehensive and systematic knowledge, and it may thus encourage researchers to develop novel antifungal drugs with higher therapeutic indexes and medical values.
... Samples for quantification of actinorhodin and undecylprodigiosin production were obtained from 20 mL sucrose-free R5A [28] cultures grown at 30 • C and 200 rpm in 100 mL flasks inoculated with 10 7 spores/mL, or with mycelium equivalent to 26.3 mg protein/mL in the bald strains. The bald phenotype was analysed in TBO [29], SFM and MM supplemented with 20% mannitol [26]. When indicated, plates were amended with 50 µg/mL methionine. ...
Streptomyces DNA replication starts with the DnaA binding to the origin of replication. Differently to most bacteria, cytokinesis only occurs during sporulation. Cytokinesis is modulated by the divisome, an orderly succession of proteins initiated by FtsZ. Here, we characterised SCO2102, a protein harbouring a DnaA II protein–protein interaction domain highly conserved in Streptomyces. The ΔSCO2102 knockout shows highly delayed sporulation. SCO2102-mCherry frequently co-localises with FtsZ-eGFP during sporulation and greatly reduces FtsZ-eGFP Z-ladder formation, suggesting a role of SCO2102 in sporulation. SCO2102 localises up-stream of SCO2103, a methylenetetrahydrofolate reductase involved in methionine and dTMP synthesis. SCO2102/SCO2103 expression is highly regulated, involving two promoters and a conditional transcription terminator. The ΔSCO2103 knockout shows reduced DNA synthesis and a non-sporulating phenotype. SCO2102-mCherry co-localises with SCO2103-eGFP during sporulation, and SCO2102 is essential for the SCO2103 positioning at sporulating hyphae, since SCO2103-eGFP fluorescent spots are absent in the ΔSCO2102 knockout. We propose a model in which SCO2102 positions SCO2103 in sporulating hyphae, facilitating nucleotide biosynthesis for chromosomal replication. To the best of our knowledge, SCO2102 is the first protein harbouring a DnaA II domain specifically found during sporulation, whereas SCO2103 is the first methylenetetrahydrofolate reductase found to be essential for Streptomyces sporulation.
... In the present study, to identify secondary metabolites other than STZ produced by strain NBRC14001, we elucidated the genome sequence of the strain and detected a Type I polyketide synthase (PKS) gene cluster resembling a natamycin (NTM) BGC [12] in its genome using an antiSMASH analysis. Based on this information, we herein revealed that S. achromogenes subsp. ...
... After assembly and annotation, we elucidated the genome sequence of strain NBRC14001 consisting of 8,897,387 bp by PacBio RSII sequencing (genome sequence has not yet been deposited in public database). The antiSMASH analysis [3] of the genome sequence revealed that a type I PKS gene cluster (sac_7029~sac_7047), which exhibits moderately high similarity to a NTM BGC [12], is present in the genome. Fourteen out of 21 genes in Microorganisms 2022, 10, 37 4 of 11 NTM BGC exhibit similarities to genes involved in type I PKS cluster of strain NBRC14001 (that is, 67% of genes in NTM BGC show similarities to genes in the type I PKS cluster of NBRC14001). ...
... After assembly and annotation, we elucidated the genome sequence of strain NBRC14001 consisting of 8,897,387 bp by PacBio RSII sequencing (genome sequence has not yet been deposited in public database). The antiSMASH analysis [3] of the genome sequence revealed that a type I PKS gene cluster (sac_7029 ~ sac_7047), which exhibits moderately high similarity to a NTM BGC [12], is present in the genome. Fourteen out of 21 genes in NTM BGC exhibit similarities to genes involved in type I PKS cluster of strain NBRC14001 (that is, 67% of genes in NTM BGC show similarities to genes in the type I PKS cluster of NBRC14001). ...
Information on microbial genome sequences is a powerful resource for accessing natural products with significant activities. We herein report the unveiling of lucensomycin production by Streptomyces achromogenes subsp. streptozoticus NBRC14001 based on the genome sequence of the strain. The genome sequence of strain NBRC14001 revealed the presence of a type I polyketide synthase gene cluster with similarities to a biosynthetic gene cluster for natamycin, which is a polyene macrolide antibiotic that exhibits antifungal activity. Therefore, we investigated whether strain NBRC14001 produces antifungal compound(s) and revealed that an extract from the strain inhibited the growth of Candida albicans. A HPLC analysis of a purified compound exhibiting antifungal activity against C. albicans showed that the compound differed from natamycin. Based on HR-ESI-MS spectrometry and a PubChem database search, the compound was predicted to be lucensomycin, which is a tetraene macrolide antibiotic, and this prediction was supported by the results of a MS/MS analysis. Furthermore, the type I polyketide synthase gene cluster in strain NBRC14001 corresponded well to lucesomycin biosynthetic gene cluster (lcm) in S. cyanogenus, which was very recently reported. Therefore, we concluded that the antifungal compound produced by strain NBRC14001 is lucensomycin.
... These compounds have been a focus of scientific interest since their discovery in the 1950s, and, thus far, more than 200 members have been discovered (3). Some polyene macrolides, such as amphotericin B (4), nystatin (5), candicidin (6,7), and natamycin (8), have been used as antifungal drugs or preservative agents. In addition, researchers have revealed the biosynthesis and fungicidal mechanism of polyene macrolides. ...
A group of polyene macrolides mainly composed of two constituents was isolated from the fermentation broth of Streptomyces roseoflavus Men-myco-93-63, which was isolated from soil where potato scabs were repressed naturally. One of this macrolides was roflamycoin, which was first reported in 1968, and the other was a novel compound named Men-myco-A, which had one methylene unit more than roflamycoin. Together, they were designated RM. This group of antibiotics exhibited broad-spectrum antifungal activities in vitro against 17 plant pathogenic fungi with 50% effective concentration (EC 50 ) values of 2.05-7.09 μg/mL and 90% effective concentration (EC 90 ) values of 4.32-54.45 μg/mL, which indicates their potential use in plant disease control. Furthermore, their biosynthetic gene cluster was identified, and the associated biosynthetic assembly line was proposed based on a module and domain analysis of polyketide synthases (PKSs) and supported by findings from gene inactivation experiments.
Importance
Streptomyces roseoflavus Men-myco-93-63 is a biocontrol strain that has been studied in our laboratory for many years and exhibits a good inhibitory effect in many crop diseases. Therefore, the identification of antimicrobial metabolites is necessary and our main objective. In this work, chemical, bioinformatic and molecular biological methods were combined to identify the structures and biosynthesis of the active metabolites. This work provides a new alternative agent for the biological control of plant diseases and is helpful for improving both the properties and yield of the antibiotics via genetic engineering.
... cyanogenus genome (see above) readily led us to recognize that one BGC shared significant similarity to the pimaricin (also known as natamycin) BGC (pim cluster) [44][45][46] . As Lcm and pimaricin are structural homologs 41 , we suggested that activation of this particular BGC results in Lcm accumulation by S. cyanogenus ΔlanI7 pGM4181 + and pGM4181d + . ...
Actinobacteria are among the most prolific sources of medically and agriculturally important compounds, derived from their biosynthetic gene clusters (BGCs) for specialized (secondary) pathways of metabolism. Genomics witnesses that the majority of actinobacterial BGCs are silent, most likely due to their low or zero transcription. Much effort is put into the search for approaches towards activation of silent BGCs, as this is believed to revitalize the discovery of novel natural products. We hypothesized that the global transcriptional factor AdpA, due to its highly degenerate operator sequence, could be used to upregulate the expression of silent BGCs. Using Streptomyces cyanogenus S136 as a test case, we showed that plasmids expressing either full-length adpA or its DNA-binding domain led to significant changes in the metabolome. These were evident as changes in the accumulation of colored compounds, bioactivity, as well as the emergence of a new pattern of secondary metabolites as revealed by HPLC-ESI-mass spectrometry. We further focused on the most abundant secondary metabolite and identified it as the polyene antibiotic lucensomycin. Finally, we uncovered the entire gene cluster for lucensomycin biosynthesis ( lcm ), that remained elusive for five decades until now, and outlined an evidence-based scenario for its adpA -mediated activation.
... However, the activity of lucensomycin against B. cinerea causing postharvest gray mold and its disease control efficacy have not been evaluated thus far. Polyene macrolides containing several conjugated double bond in macrocyclic lactone ring are one of the major group of antifungal agent (Aparicio et al., 1999;Ghannoum and Rice, 1999). Since the first antifungal polyene nystatin has been isolated from S. noursei in the 1950, more than 200 compounds belonging to polyenes including amphotericin B, filipin, natamycin, nystatin and rapamycin have been isolated from Streptomyces spp. ...
Microbial secondary metabolites have been a valuable source of antifungal compounds for the fungicide industry to develop plant disease control agents. In order to find natural antifungal compounds useful for the control of Botrytis cinerea causing postharvest gray mold, we screened microbial culture extracts and found the extract of Streptomyces plumbeus strain CA5 which was isolated from the soil sample of Chuncheon Province. The cell extract of CA5 strain markedly reduced the disease incidence of gray mold on grapes to a value of 22.2 % while the non-treated control showed 100 % disease incidence. The active ingredient (CA5A) was identified from the cell extract of CA5 strain using a variety of chromatographic methods and spectroscopic analyses. The structure of CA5A was determined to be the polyene macrolide lucensomycin. Lucensomycin showed inhibitory effects against the spores of B. cinerea at a concentration as low as 1 mg L⁻¹ and completely inhibited gray mold development on grapes at a concentration of 100 mg L⁻¹.
... Over the last several decades, complete polyene biosynthetic gene clusters from nystatin, amphotericin, pimaricin, and candicidin have been isolated and characterized (Aparicio et al., 1999(Aparicio et al., , 2003Zotchev et al., 2000;Caffrey et al., 2001). Polyene compounds are biosynthesized typically by a giant enzyme complex called polyketide synthase (PKS), followed by further modification of the core macrolide ring by post-PKS modification enzymes, including P450 hydroxylases and glycosyltransferases (Kim et al., 2015). ...
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.
... As described in detail in the ESI, † the NMR data suggest that meijiemycin contains a linear carbon backbone and a perosamine sugar ( Fig. 1E and Table S3, ESI †). Comparing to the three antifungal polyenepolyol drugs natamycin, 6 amphotericin B, 7 nystatin, 8 meijiemycin has a larger and linear molecular architecture and a perosamine instead of a mycosamine sugar moiety (Fig. S7, ESI †). The relative configurations of meijiemycin (1) were determined based on NOE correlation and J-based analyses and 13 C NMR universal databases (see details in Fig. S2-S6, ESI †). ...
Genome-guided discovery of a novel linear polyene-polyol that induces ergosterol aggregation and inhibits hyphal growth.
... According to the genome information of the four S t re p t o m y c e s s t r a i n s , S . c h a t t a n o o g e n s i s L 1 0 , S. gilvosporeus F607, S. lydicus A02, and S. natalensis ATCC 27448, each harbors an intact natamycin biosynthetic gene cluster and all four strains can produce natamycin (Aparicio et al. 1999;Du et al. 2009;Wu et al. 2017;Zong et al. 2018). As shown in Fig. 1a, the natamycin biosynthetic gene cluster is located at different positions on different chromosomes. ...
As antibiotics are always toxic to the antibiotic-producing strains themselves, most Streptomyces strains have evolved several self-resistance mechanisms, among which the antibiotic efflux system is understood best and is commonly found. Among the efflux systems, the ATP-binding cassette (ABC) transporter superfamily and the major facilitator superfamily (MFS) are two important transporter families. In this work, the ABC transporters and the MFS transporters from the four reported natamycin-producing Streptomyces strains have been investigated in order to clarify whether these Streptomyces strains share similar efflux strategies for natamycin metabolism. Fifty-one groups of homologous exporter genes were identified as shared by four strains. Differential transcriptional analysis between the natamycin-producing strain Streptomyces chattanoogensis L10 and its ΔscnS0 mutant, which produces no natamycin, reveals that the expression levels of 25 of the above groups of genes were observably changed. The production of natamycin declined over 30% after solely knocking out several of these 25 groups of genes in S. chattanoogensis L10. This indicates that these transporters participate in the efflux of molecules related to natamycin biosynthesis. Our study is the first to demonstrate that the exporters participating in a particular antibiotic metabolism can be excavated and identified quickly by the strategy of genome mining and homologous comparison in the antibiotic-producing strains, leading to deeper understanding of the complex self-resistance mechanisms in Streptomycetes.
... This is a contrast to the more common mycosamine sugar found in many macrocyclic polyene-polyols such as natamycin, amphotericin B, and nystatin. [16][17][18] The NOE correlations arisen from the 1, 3-diaxial interaction between H-1′ and both H-3′ and H-5′ are indicative of the β-type glycosidic linkage, as well as the αorientations of 3′-OH and 6′-Me. The H-2′ was concluded to be equatorially β-oriented based on a weak coupling (near 0 Hz) observed between H-1′ and H-2′. ...
