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Pigments and other secondary metabolites originating from marine microbes have been a promising natural colorants and drugs for multifaceted applications. However, marine actinobacteria producing such natural molecules are least investigated in terms of their taxonomy, chemical diversity and applications in biomedical, textile, and food industries. In this study, sioxanthin pigment-producing Gram-positive actinobacteria, Micromonospora sp. strain SH-82 was isolated from a marine sponge, Scopalina hapalia, and its whole genome was analyzed. Strain SH-82is a prolific producer of diverse chemical molecules as it produced more compounds on A1 medium with different culture conditions. The genome size of SH-82 is 6.24 Mb (6,246,890 bp) carrying 23 identified biosynthetic gene clusters. A total of 5415 CDS, 60 tRNA, 9 rRNA, and 1 tmRNA are identified from SH-82 genome. The GC content (%) of whole genome was 71.6%. Strain SH-82 harbors genes encoding type I, type II, and type III polyketide synthases. Based on the multi-locus sequence analysis and fatty acid methyl ester (FAME) composition, strain SH-82 is confirmed as a novel species. The genetic information of Micromonospora sp. SH-82 has been deposited to NCBI under the BioProject ID PRJNA1087320, with corresponding identifiers in the Sequence Read Archive (SRA) as SAMN40439676 and the Genome accession as CP148049., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The structure of the sidechain crosslinked Tyr-Leu-Trp peptide produced by the biarylitide crosslinking cytochrome P450 Blt from Micromonospora sp. MW-13 has been reanalysed by a series of NMR, computational and isotope labelling experiments and shown to contain a C-N rather than a C-O bond. Additional in vivo experiments using such a modified peptide show there is a general tolerance of biarylitide crosslinking P450 enzymes for histidine to tryptophan mutations within their minimal peptide substrate sequences despite the lack of such residues noted in natural biarylitide gene clusters. This work further highlights the impressive ability of P450s from biarylitide biosynthesis pathways to act as biocatalysts for the formation of a range of sidechain crosslinked tripeptides., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Among the actinomycetes in the rare genera, Micromonospora is of great interest since it has been shown to produce novel therapeutic compounds. Particular emphasis is now on its isolation from plants since its population from soil has been extensively explored. The strain CR3 was isolated as an endophyte from the roots of Hieracium canadense, and it was identified as Micromonospora chokoriensis through 16S gene sequencing and phylogenetic analysis. The in-vitro analysis of its extract revealed it to be active against the clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and Candida tropicalis (15 mm). No bioactivity was observed against Gram-negative bacteria, Escherichia coli ATCC 25922, and Klebsiella pneumoniae ATCC 706003. The Micromonospora chokoriensis CR3 extract was also analyzed through the HPLC-DAD-UV-VIS resident database, and it gave a maximum match factor of 997.334 with the specialized metabolite BagremycinA (BagA). The in-silico analysis indicated that BagA strongly interacted with the active site residues of the sterol 14-α demethylase and thymidylate kinase enzymes, with the lowest binding energies of - 9.7 and - 8.3 kcal/mol, respectively. Furthermore, the normal mode analysis indicated that the interaction between these proteins and BagA was stable. The DFT quantum chemical properties depicted BagA to be reasonably reactive with a HOMO-LUMO gap of (ΔE) of 4.390 eV. BagA also passed the drug-likeness test with a synthetic accessibility score of 2.06, whereas Protox-II classified it as a class V toxicity compound with high LD 50 of 2644 mg/kg. The current study reports an endophytic actinomycete, M. chokoriensis, associated with H. canadense producing the bioactive metabolite BagA with promising antimicrobial activity, which can be further modified and developed into a safe antimicrobial drug., (© 2024. The Author(s).)

Importance: Mismanagement of PET plastic waste significantly threatens human and environmental health. Together with the relentless increase in plastic production, plastic pollution is an issue of rising concern. In response to this challenge, scientists are investigating eco-friendly approaches, such as bioprocessing and microbial factories, to sustainably manage the growing quantity of plastic waste in our ecosystem. Industrial applicability of enzymes capable of degrading PET is limited by numerous factors, including their scarcity in nature. The objective of this study is to enhance our understanding of this group of enzymes by identifying and characterizing novel enzymes that can facilitate the breakdown of PET waste. This data will expand the enzymatic repertoire and provide valuable insights into the prerequisites for successful PET degradation., Competing Interests: A patent application has been filed on this work by the authors.

The intense use of tellurium (Te) in industrial applications, along with the improper disposal of Te-derivatives, is causing their accumulation in the environment, where oxyanion tellurite (TeO 3 2 - ) is the most soluble, bioavailable, and toxic Te-species. On the other hand, tellurium is a rare metalloid element whose natural supply will end shortly with possible economic and technological effects. Thus, Te-containing waste represents the source from which Te should be recycled and recovered. Among the explored strategies, the microbial TeO 3 2 - biotransformation into less toxic Te-species is the most appropriate concerning the circular economy. Actinomycetes are ideal candidates in environmental biotechnology. However, their exploration in TeO 3 2- biotransformation is scarce due to limited knowledge regarding oxyanion microbial processing. Here, this gap was filled by investigating the cell tolerance, adaptation, and response to TeO 3 2- of a Micromonospora strain isolated from a metal(loid)-rich environment. To this aim, an integrated biological, physical-chemical, and statistical approach combining physiological and biochemical assays with confocal or scanning electron (SEM) microscopy and Fourier-transform infrared spectroscopy in attenuated total reflectance mode (ATR-FTIR) was designed. Micromonospora cells exposed to TeO 3 2- under different physiological states revealed a series of striking cell responses, such as cell morphology changes, extracellular polymeric substance production, cell membrane damages and modifications, oxidative stress burst, protein aggregation and phosphorylation, and superoxide dismutase induction. These results highlight this Micromonospora strain as an asset for biotechnological purposes.

In this study, a single-component high-yielding Micromonospora echinospora strain 49-92S-KL01 was constructed by deleting methyltransferase-encoding genes genK and genL. In 5-L fermentation trials, gentamicin C1a titers in the mutant strain were 3.22-fold higher than that in the parental strain (211 U/mL vs. 50 U/mL). The glycolysis pathway and tricarboxylic acid cycle fluxes were reduced by 26.8% and 26.6%, respectively, compared to the parental strain according to the metabolic flux analysis during the stationary phase, resulting in lower levels of energy supplements required for the cellular maintenance. Meanwhile, a significant enhancement in precursor (paromamine) accumulation and availability was observed in 49-92S-KL01 compared to parental strain. These results indicate that genK and genL significantly affect the synthesis of gentamicin C1a. In addition, this study provides a more rational strategy for gentamicin C1a production., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Micromonospora , one of the most important actinomycetes genera, is well-known as the treasure trove of bioactive secondary metabolites (SMs). Herein, together with an in-depth genomic analysis of the reported Micromonospora strains, all SMs from this genus are comprehensively summarized, containing structural features, bioactive properties, and mode of actions as well as their biosynthetic and chemical synthesis pathways. The perspective enables a detailed view of Micromonospora -derived SMs, which will enrich the chemical diversity of natural products and inspire new drug discovery in the pharmaceutical industry.

Glycosyltransferase (GT)-specific degenerate PCR screening followed by in silico sequence analyses of the target clone was used to isolate a member of family1 GT-encoding genes from the established fosmid libraries of soil actinomycetes Micromonospora echinospora ATCC 27932. A recombinant MeUGT1 was heterologously expressed as a His-tagged protein in E. coli , and its enzymatic reaction with semi-synthetic phenoxodiol isoflavene (as a glycosyl acceptor) and uridine diphosphate-glucose (as a glycosyl donor) created two different glycol-attached products, thus revealing that MeUGT1 functions as an isoflavonoid glycosyltransferase with regional flexibility. Chromatographic separation of product glycosides followed by the instrumental analyses, clearly confirmed these previously unprecedented glycosides as phenoxodiol-4'-α- O -glucoside and phenoxodiol-7-α- O -glucoside, respectively. The antioxidant activities of the above glycosides are almost the same as that of parental phenoxodiol, whereas their anti-proliferative activities are all superior to that of cisplatin (the most common platinum chemotherapy drug) against two human carcinoma cells, ovarian SKOV-3 and prostate DU-145. In addition, they are more water-soluble than their parental aglycone, as well as remaining intractable to the simulated in vitro digestion test, hence demonstrating the pharmacological potential for the enhanced bio-accessibility of phenoxodiol glycosides. This is the first report on the microbial enzymatic biosynthesis of phenoxodiol glucosides.

A group of peptide metabolites ( 1 - 4 ), designated as mintaimycins, were isolated from Micromonospora sp. C-3509. The planar structures of mintaimycins were determined by combination of mass spectrometry, 1D and 2D NMR spectroscopy, and the stereochemistry of mintaimycins were partially resolved by Marfey's or Mosher's method. Mintaimycins featured a central β -methylphenylalanine or phenylalanine linked at its amino group with 5-methyl-2-hexenoic acid, and at its carboxyl group with 5-hydroxy-norleucine or leucine that combined a derivative of hexanoic acid or 4-methylpentanoic acid. Mintaimycin A 1 ( 1 ), the principal component, was found to exhibit the biological activity of inducing pre-adipocyte differentiation of 3T3-L1 fibroblast cells at 10.0 μmol/L.

Dynemicin is an enediyne natural product from Micromonospora chersina ATCC53710. Access to the biosynthetic gene cluster of dynemicin has enabled the in vitro study of gene products within the cluster to decipher their roles in assembling this unique molecule. This paper reports the crystal structure of DynF, the gene product of one of the genes within the biosynthetic gene cluster of dynemicin. DynF is revealed to be a dimeric eight-stranded β-barrel structure with palmitic acid bound within a cavity. The presence of palmitic acid suggests that DynF may be involved in binding the precursor polyene heptaene, which is central to the synthesis of the ten-membered ring of the enediyne core., (open access.)

An acyldipeptide, micromonosporamide A, was isolated from the fermentation broth of Micromonospora sp. MM609M-173N6 by bioassay-guided fractionation using a glutamine compensation assay. The planar structure was elucidated on the basis of comprehensive one- and two-dimensional nuclear magnetic resonance and high-resolution mass spectrometry. The relative and absolute configuration of the entire molecule were determined using a combined approach, involving chromatographic analysis by liquid chromatography-mass spectrometry, advanced Marfey's method, and total synthesis. Micromonosporamide A exhibited glutamine-dependent antiproliferative activity.

Strain CAP181 T , an endophytic actinobacterium, was isolated from a surface sterilized root sample of a native pine tree, Flinders University, Adelaide, South Australia. Chemotaxonomic data including cell wall components, major fatty acids, and major menaquinones confirmed the affiliation of strain CAP181 T to the genus Micromonospora. This strain was Gram stain positive with well-developed substrate mycelia to form a single spore with hairy surface. The phylogenetic tree showed that M. coerulea NBRC 13504  T is the closest phylogenetic neighbour, sharing 99.2% 16S rRNA gene similarity and the next closest neighbor is M. chaiyaphumensis DSM 45246  T (98.7%). Genome mining of this strain revealed genes encoding to enzymes relating to nitrogen fixation and bioremediation. Based on genotypic and phenotypic studies including DNA-DNA hybridization data, strain CAP181 T was different from any of the closely related species with valid names. The name proposed for the new species is Micromonospora veneta sp. nov. The type strain is CAP181 T (= DSM 109713  T  = NRRL B-65535  T )., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Micromonospora craniellae LHW63014 T is a novel marine Micromonospora, isolated from a Craniella species sponge collected in the South China Sea. In this study, we report the complete genome sequence of M. craniellae LHW63014 T , which is comprised of a circular chromosome of 6,839,926 bp with the G + C content of 70.9 mol%. The complete genome contained 6572 protein-coding genes, 48 tRNA genes, and 9 rRNA genes. Genomic annotations revealed that 79.09% of the protein-coding genes were assigned to the COG database, among which, the abundant genes were predicted to be involved in transcription, replication, recombination and repair, and amino acid transport and metabolism. Secondary metabolites prediction using antiSMASH revealed that 22 biosynthetic gene clusters (BGC) of secondary metabolites were located in the genome of M. craniellae LHW63014 T , 19 of which showed low similarity (<50%) to known BGCs and 5 of which showed the closest homology with BGCs encoding metal ion-chelating agents, indicating the immense potential of M. craniellae LHW63014 T to produce a wide variety of novel antibiotics, especially for metal ion-chelating agents., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A Micromonospora strain, isolate MT25 T , was recovered from a sediment collected from the Challenger Deep of the Mariana Trench using a selective isolation procedure. The isolate produced two major metabolites, n -acetylglutaminyl glutamine amide and desferrioxamine B, the chemical structures of which were determined using 1D and 2D-NMR, including 1 H- 15 N HSQC and 1 H- 15 N HMBC 2D-NMR, as well as high resolution MS. A whole genome sequence of the strain showed the presence of ten natural product-biosynthetic gene clusters, including one responsible for the biosynthesis of desferrioxamine B. Whilst 16S rRNA gene sequence analyses showed that the isolate was most closely related to the type strain of Micromonospora chalcea , a whole genome sequence analysis revealed it to be most closely related to Micromonospora tulbaghiae 45142 T . The two strains were distinguished using a combination of genomic and phenotypic features. Based on these data, it is proposed that strain MT25 T (NCIMB 15245 T , TISTR 2834 T ) be classified as Micromonospora provocatoris sp. nov. Analysis of the genome sequence of strain MT25 T (genome size 6.1 Mbp) revealed genes predicted to responsible for its adaptation to extreme environmental conditions that prevail in deep-sea sediments.

Everninomicins are orthoester oligosaccharide antibiotics with potent activity against multidrug-resistant bacterial pathogens. Everninomicins act by disrupting ribosomal assembly in a distinct region in comparison to clinically prescribed drugs. We employed microporous intergeneric conjugation with Escherichia coli to manipulate Micromonospora for targeted gene-replacement studies of multiple putative methyltransferases across the octasaccharide scaffold of everninomicin effecting the A 1 , C, F, and H rings. Analyses of gene-replacement and genetic complementation mutants established the mutability of the everninomicin scaffold through the generation of 12 previously unreported analogues and, together with previous results, permitted assignment of the ten methyltransferases required for everninomicin biosynthesis. The in vitro activity of A 1 - and H-ring-modifying methyltransferases demonstrated the ability to catalyze late-stage modification of the scaffold on an A 1 -ring phenol and H-ring C-4' hydroxy moiety. Together these results establish the potential of the everninomicin scaffold for modification through mutagenesis and in vitro modification of advanced biosynthetic intermediates., (© 2020 Wiley-VCH GmbH.)

The biosynthesis of the three structural subclasses of enediyne antitumor antibiotics remains largely unknown beyond a common C 16 -hexaene precursor. For the anthraquinone-fused subtype, however, an unexpected iodoanthracene γ-thiolactone was established to be a mid-pathway intermediate to dynemicin A. Having deleted a putative flavin-dependent oxidoreductase from the dynemicin biosynthetic gene cluster, we can now report four metabolites that incorporate the iodoanthracene and reveal the formation of the C-N bond linking the anthraquinone and enediyne halves emblematic of this structural subclass. The coupling of an aryl iodide and an amine is familiar from organometallic chemistry, but has little or no precedent in natural product biosynthesis. These metabolites suggest further that enediyne formation occurs early in the overall biosynthesis, and that even earlier events might convert the C 16 -hexaene to a common C 15 intermediate that partitions to enediyne and anthraquinone building blocks for the heterodimerization., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Liquid chromatography coupled with high resolution mass spectrometry (LC-HRESMS)-assisted metabolomic profiling of two sponge-associated actinomycetes, Micromonospora sp. UR56 and Actinokineospora sp. EG49, revealed that the co-culture of these two actinomycetes induced the accumulation of metabolites that were not traced in their axenic cultures. Dereplication suggested that phenazine-derived compounds were the main induced metabolites. Hence, following large-scale co-fermentation, the major induced metabolites were isolated and structurally characterized as the already known dimethyl phenazine-1,6-dicarboxylate ( 1 ), phenazine-1,6-dicarboxylic acid mono methyl ester (phencomycin; 2 ), phenazine-1-carboxylic acid (tubermycin; 3 ), N-(2-hydroxyphenyl)-acetamide ( 9 ), and p -anisamide ( 10 ). Subsequently, the antibacterial, antibiofilm, and cytotoxic properties of these metabolites ( 1 - 3 , 9 , and 10 ) were determined in vitro. All the tested compounds except 9 showed high to moderate antibacterial and antibiofilm activities, whereas their cytotoxic effects were modest. Testing against Staphylococcus DNA gyrase-B and pyruvate kinase as possible molecular targets together with binding mode studies showed that compounds 1 - 3 could exert their bacterial inhibitory activities through the inhibition of both enzymes. Moreover, their structural differences, particularly the substitution at C-1 and C-6, played a crucial role in the determination of their inhibitory spectra and potency. In conclusion, the present study highlighted that microbial co-cultivation is an efficient tool for the discovery of new antimicrobial candidates and indicated phenazines as potential lead compounds for further development as antibiotic scaffold.

Marine Micromonospora was revealed to be a rather untapped and a rich source of chemically diverse and unique bioactive natural products. This review is aimed to make a comprehensive survey of secondary metabolites that were derived from marine Micromonospora including chemical diversity and biological activities. A total of 116 compounds from 41 marine Micromonospora species have been reported, covering the literatures from 1997 to 2019. These compounds contain several structural classes such as polyketides (PKS), nonribosomal peptides (NRPS), PKS-NRPS hybrids, terpenes and others, and they present cytotoxic, antibacterial, antiparasitic, chemopreventive or antioxidant activities., (© 2020 Wiley-VHCA AG, Zurich, Switzerland.)

A new bacterial strain, designated HM134 T , was isolated from a sample of soil collected from a Chinese mangrove Avicennia marina forest. Assessed by a polyphasic approach, the taxonomy of strain HM134 T was found to be associated with a range of phylogenetic and chemotaxonomic properties consistent with the genus Micromonospora. Phylogenetic analysis based on the 16s rRNA gene sequence indicated that strain HM134 T formed a distinct lineage with the most closely related species, including M. rifamycinica AM105 T , M. wenchangensis CCTCC AA 2012002 T and M. mangrovi 2803GPT1-18 T . The ANI values between strain HM134 T and the reference strains ranged from 82.6% to 95.2%, which was below the standard criteria for classifying strains as the same species (96.5%). Strain HM134 T and related species shared in silico dDDH similarities values below the recommended 70% cut-off for the delineation of species (range from 25.7-62.6%). The DNA G+C content of strain HM134 T was 73.2 mol%. Analysis of phylogenetic, genomic, phenotypic and chemotaxonomic characteristics revealed that strain HM134 T is considered to represent a novel species of the genus Micromonospora, for which the name M. zhangzhouensis sp. nov. is proposed. The extract of strain HM134 T was demonstrated to exhibit cytotoxic activity against the human cancer cell lines HepG2, HCT-116 and A549. Active substance presented in the fermentation broth of strain HM134 T was isolated by bioassay-guided analysis and purified afterwards. A new derivative of diterpenoid was identified through electrospray ionizing mass spectrometry (MS) and nuclear magnetic resonance (NMR). The compound showed different cytotoxic activities against cancer cells, with the highest cytotoxicity against HCT-116, corresponding to IC 50 value of 38.4 μg/mL.

Integrating MS-based metabolomics approaches, LC-MS-PCA and molecular networking enabled the targeted isolation of five new pyrrole-derived alkaloids, phallusialides A-E ( 1 - 5 ), from a marine-derived Micromonospora sp. bacterium. The structures of 1 - 5 were elucidated by analysis of their HRMS, MS/MS, and NMR spectroscopic data. The absolute configuration of phallusialide A ( 1 ) was determined on the basis of comparisons of experimental and theoretically calculated ECD spectra. Compounds 1 and 2 exhibited antibacterial activity against methicillin resistant S. aureus (MRSA) and E. coli , with MIC values of 32 and 64 μg/mL, respectively, whereas 3 - 5 showed no antibacterial activity even at 256 μg/mL, yielding important SAR insights for this class of compounds.

DNA sequencing of a large collection of bacterial genomes reveals a wealth of orphan biosynthetic gene clusters (BGCs) with no identifiable products. BGC silencing, for those orphan clusters that are truly silent, rather than those whose products have simply evaded detection and cluster correlation, is postulated to result from transcriptional inactivation of these clusters under standard laboratory conditions. Here, we employ a multi-omics approach to demonstrate how interspecies interactions modulate the keyicin producing kyc cluster at the transcriptome level in cocultures of kyc-bearing Micromonospora sp. and a Rhodococcus sp. We further correlate coculture dependent changes in keyicin production to changes in transcriptomic and proteomic profiles and show that these changes are attributable to small molecule signaling consistent with a quorum sensing pathway. In piecing together the various elements underlying keyicin production in coculture, this study highlights how omics technologies can expedite future efforts to understand and exploit silent BGCs.

Polyketides and peptides obtained from actinobacteria are important therapeutic compounds which include front line antibiotics and anticancer drugs. Many screening programs are directed towards isolation of bioactive compounds from these organisms but the chances of finding novel antimicrobial leads among common actinobacteria are fast dwindling. As a result, the focus has shifted to the members of less exploited genera of rare actinobacteria. Three isolates, MMS8, MMS16 and KCR3 found to be potent polyketide and peptide producers were identified by 16S rRNA gene sequencing and their sequences deposited in the GenBank under the accession numbers MG407702, MG372012 and MG430204 respectively. MMS8 identified as Micromonospora auratinigra, yielded one potent compound determined to be chloroanthraquinone with an minimum inhibitory concentration (MIC) of 8 µg/ml against Bacillus subtilis and an IC 50 value of 10 µg/ml and 4 μg/ml against HeLa and IMR cell lines respectively. This is the first report of the production of chloroanthraquinone by M. auratinigra. MMS16, identified as a member of the family Micromonosporaceae, yielded a potent compound MMS16B analyzed to be a novel bafilomycin analogue. The MIC of the compound was found to be 7 μg/ml against B.subtilis and IC 50 value against HeLa and IMR was observed to be 9 μg/ml and 14 μg/ml respectively. MMS16B was also found to exhibit anti-quorum sensing (AQS) activity at sublethal concentrations. KCR3 identified as Kocuria kristinae yielded a novel antimicrobial peptide with antibacterial, antifungal and AQS activity. To the best of our knowledge, no antimicrobial activity has ever been reported from K. kristinae.

Deactivation of aminoglycosides by their modifying enzymes, including a number of aminoglycoside O -phosphotransferases, is the most ubiquitous resistance mechanism in aminoglycoside-resistant pathogens. Nonetheless, in a couple of biosynthetic pathways for gentamicins, fortimicins, and istamycins, phosphorylation of aminoglycosides seems to be a unique and initial step for the creation of a natural defensive structural feature such as a 3',4'- dideoxy scaffold. Our aim was to elucidate the biochemical details on the beginning of these C3',4'-dideoxygenation biosynthetic steps for aminoglycosides. The biosynthesis of istamycins must surely involve these 3',4'-didehydroxylation steps, but much less has been reported in terms of characterization of istamycin biosynthetic genes, especially about the phosphotransferase-encoding gene. In the disruption and complementation experiments pointing to a putative gene, istP , in the genome of wild-type Streptomyces tenjimariensis , the function of the istP gene was proved here to be a phosphotransferase. Next, an in-frame deletion of a known phosphotransferase-encoding gene forP from the genome of wild-type Micromonospora olivasterospora resulted in the appearance of a hitherto unidentified fortimicin shunt product, namely 3- O -methyl-FOR-KK1, whereas complementation of forP restored the natural fortimicin metabolite profiles. The bilateral complementation of an istP gene (or forP ) in the ΔforP mutant ( or ΔistP mutant strain) successfully restored the biosynthesis of 3',4'- dideoxy fortimicins and istamycins , thus clearly indicating that they are interchangeable launchers of the biosynthesis of 3',4'-dideoxy types of 1,4-diaminocyclitol antibiotics.

Diazobenzofluorene-containing atypical angucyclines exhibit promising biological activities. Here we report the inactivation of an amidotransferase-encoding gene flsN3 in Micromonospora rosaria SCSIO N160, a producer of fluostatins. Bioinformatics analysis indicated that FlsN3 was involved in the diazo formation. Chemical investigation of the flsN3 -inactivation mutant resulted in the isolation of a variety of angucycline aromatic polyketides, including four racemic aminobenzo[ b ]fluorenes stealthins D⁻G ( 9 ⁻ 12 ) harboring a stealthin C-like core skeleton with an acetone or butanone-like side chain. Their structures were elucidated on the basis of nuclear magnetic resonance (NMR) spectroscopic data and X-ray diffraction analysis. A plausible mechanism for the formation of stealthins D⁻G ( 9 ⁻ 12 ) was proposed. These results suggested a functional role of FlsN3 in the formation/modification of N⁻N bond-containing fluostatins.

Gentamicin B (GB), a valuable starting material for the preparation of the semisynthetic aminoglycoside antibiotic isepamicin, is produced in trace amounts by the wild-type Micromonospora echinospora. Though the biosynthetic pathway to GB has remained obscure for decades, we have now identified three hidden pathways to GB production via seven hitherto unknown intermediates in M. echinospora. The narrow substrate specificity of a key glycosyltransferase and the C6'-amination enzymes, in combination with the weak and unsynchronized gene expression of the 2'-deamination enzymes, limits GB production in M. echinospora. The crystal structure of the aminotransferase involved in C6'-amination explains its substrate specificity. Some of the new intermediates displayed similar premature termination codon readthrough activity but with reduced toxicity compared to the natural aminoglycoside G418. This work not only led to the discovery of unknown biosynthetic routes to GB, but also demonstrated the potential to mine new aminoglycosides from nature for drug discovery.

A number of strategies have been developed to mine novel natural products based on biosynthetic gene clusters and there have been dozens of successful cases facilitated by the development of genomic sequencing. During our study on biosynthesis of the antitumor polyketide kosinostatin (KST), we found that the genome of Micromonospora sp. strain TP-A0468, the producer of KST, contains other potential polyketide gene clusters, with no encoded products detected. Deletion of kst cluster led to abolishment of KST and the enrichment of several new compounds, which were isolated and characterized as 16-demethylrifamycins (referred to here as compounds 3 to 6). Transcriptional analysis demonstrated that the expression of the essential genes related to the biosynthesis of compounds 3 to 6 was comparable to the level in the wild-type and in the kst cluster deletion strain. This indicates that the accumulation of these compounds was due to the redirection of metabolic flux rather than transcriptional activation. Genetic disruption, chemical complementation, and bioinformatic analysis revealed that the production of compounds 3 to 6 was accomplished by cross talk between the two distantly placed polyketide gene clusters pks3 and M- rif This finding not only enriches the analogue pool and the biosynthetic diversity of rifamycins but also provides an auxiliary strategy for natural product discovery through genome mining in polyketide-producing microorganisms. IMPORTANCE Natural products are essential in the development of novel clinically used drugs. Discovering new natural products and modifying known compounds are still the two main ways to generate new candidates. Here, we have discovered several rifamycins with varied skeleton structures by redirecting the metabolic flux from the predominant polyketide biosynthetic pathway to the rifamycin pathway in the marine actinomycetes species Micromonospora sp. strain TP-A0468. Rifamycins are indispensable chemotherapeutics in the treatment of various diseases such as tuberculosis, leprosy, and AIDS-related mycobacterial infections. This study exemplifies a useful method for the discovery of cryptic natural products in genome-sequenced microbes. Moreover, the 16-demethylrifamycins and their genetically manipulable producer provide a new opportunity in the construction of novel rifamycin derivates to aid in the defense against the ever-growing drug resistance of Mycobacterium tuberculosis ., (Copyright © 2019 American Society for Microbiology.)

A Gram-stain positive actinomycete, strain AZ1-19 T , isolated from roots of Azadirachta indica A. Juss. var. siamensis Valeton, collected from Chachoengsao province, Thailand, was characterised taxonomically by using a polyphasic approach. Strain AZ1-19 T was found to have characteristics consistent with those of members of the genus Micromonospora. The cell wall peptidoglycan of the strain was found to contain meso-diaminopimelic acid. The predominant phospholipids were identified as diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. The characteristic whole-cell sugars were identified as glucose, xylose, galactose and mannose. The major menaquinones were found to be MK-9(H 6 ), MK-10(H 6 ) and MK-10(H 8 ), and the major cellular fatty acids were identified as iso-C 16:0 , iso-C 15:0 , anteiso-C 15:0 and anteiso-C 17:0 . Comparative analysis of 16S rRNA gene sequences revealed that strain AZ1-19 T is closely related to Micromonospora costi CS1-12 T (98.75% similarity), Micromonospora avicenniae 268506 T (98.75%), Micromonospora haikouensis 232617 T (98.68%) and Micromonospora siamensis TT2-4 T (98.61%), whilst the corresponding phylogenetic analysis based on partial gyrase subunit B (gyrB) gene sequences indicated that strain AZ1-19 T forms a clade with M. avicenniae 268506 T with a high bootstrap value. The DNA G + C content was determined to be 69.8 mol%. Moreover, a combination of DNA-DNA relatedness values and some phenotypic and chemotaxonomic properties indicated that the strain could be distinguished from closely related species. Therefore, it is considered that strain AZ1-19 T represents a novel Micromonospora species for which the name Micromonospora azadirachtae sp. nov. is proposed. The type strain is AZ1-19 T (= KCTC 39941 T  = NBRC 112784 T  = JCM 32148 T  = TISTR 2559 T ).

Actinomycete bacteria isolated from freshwater environments are an unexplored source of natural products. Here we report the complete genome of the Great Lakes-derived Micromonospora sp. strain B006, revealing its potential for natural product biosynthesis. The 7-megabase pair chromosome of strain B006 was sequenced using Illumina and Oxford Nanopore technologies followed by Sanger sequencing to close remaining gaps. All identified biosynthetic gene clusters (BGCs) were manually curated. Five known BGCs were identified encoding desferrioxamine, alkyl- O-dihydrogeranylmethoxyhydroquinone, a spore pigment, sioxanthin, and diazepinomicin, which is currently in phase II clinical trials to treat Phelan-McDermid syndrome and co-morbid epilepsy. We report here that strain B006 is indeed a producer of diazepinomicin and at yields higher than previously reported. Moreover, 11 of the 16 identified BGCs are orphan, eight of which were transcriptionally active under the culture condition tested. Orphan BGCs include an enediyne polyketide synthase and an uncharacteristically large, 36-module polyketide synthase-nonribosomal peptide synthetase BGC. We developed a genetics system for Micromonospora sp. B006 that will contribute to deorphaning BGCs in the future. This study is one of the few attempts to report the biosynthetic capacity of a freshwater-derived actinomycete and highlights this resource as a potential reservoir for new natural products.

For decades, researchers have lacked the ability to rapidly correlate microbial identity with bacterial metabolism. Since specialized metabolites are critical to bacterial function and survival in the environment, we designed a data acquisition and bioinformatics technique (IDBac) that utilizes in situ matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze protein and specialized metabolite spectra recorded from single bacterial colonies picked from agar plates. We demonstrated the power of our approach by discriminating between two Bacillus subtilis strains in <30 min solely on the basis of their differential ability to produce cyclic peptide antibiotics surfactin and plipastatin, caused by a single frameshift mutation. Next, we used IDBac to detect subtle intraspecies differences in the production of metal scavenging acyl-desferrioxamines in a group of eight freshwater Micromonospora isolates that share >99% sequence similarity in the 16S rRNA gene. Finally, we used IDBac to simultaneously extract protein and specialized metabolite MS profiles from unidentified Lake Michigan sponge-associated bacteria isolated from an agar plate. In just 3 h, we created hierarchical protein MS groupings of 11 environmental isolates (10 MS replicates each, for a total of 110 spectra) that accurately mirrored phylogenetic groupings. We further distinguished isolates within these groupings, which share nearly identical 16S rRNA gene sequence identity, based on interspecies and intraspecies differences in specialized metabolite production. IDBac is an attempt to couple in situ MS analyses of protein content and specialized metabolite production to allow for facile discrimination of closely related bacterial colonies., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Gentamicin C complex from Micromonospora echinospora remains a globally important antibiotic, and there is revived interest in the semisynthesis of analogs that might show improved therapeutic properties. The complex consists of five components differing in their methylation pattern at one or more sites in the molecule. We show here, using specific gene deletion and chemical complementation, that the gentamicin pathway up to the branch point is defined by the selectivity of the methyltransferases GenN, GenD1, and GenK. Unexpectedly, they comprise a methylation network in which early intermediates are ectopically modified. Using whole-genome sequence, we have also discovered the terminal 6'- N -methyltransfer required to produce gentamicin C2b from C1a or gentamicin C1 from C2, an example of an essential biosynthetic enzyme being located not in the biosynthetic gene cluster but far removed on the chromosome. These findings fully account for the methylation pattern in gentamicins and open the way to production of individual gentamicins by fermentation, as starting materials for semisynthesis., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

There is a need to clarify relationships within the actinobacterial genus Micromonospora, the type genus of the family Micromonosporaceae, given its biotechnological and ecological importance. Here, draft genomes of 40 Micromonospora type strains and two non-type strains are made available through the Genomic Encyclopedia of Bacteria and Archaea project and used to generate a phylogenomic tree which showed they could be assigned to well supported phyletic lines that were not evident in corresponding trees based on single and concatenated sequences of conserved genes. DNA G+C ratios derived from genome sequences showed that corresponding data from species descriptions were imprecise. Emended descriptions include precise base composition data and approximate genome sizes of the type strains. antiSMASH analyses of the draft genomes show that micromonosporae have a previously unrealised potential to synthesize novel specialized metabolites. Close to one thousand biosynthetic gene clusters were detected, including NRPS, PKS, terpenes and siderophores clusters that were discontinuously distributed thereby opening up the prospect of prioritising gifted strains for natural product discovery. The distribution of key stress related genes provide an insight into how micromonosporae adapt to key environmental variables. Genes associated with plant interactions highlight the potential use of micromonosporae in agriculture and biotechnology.

Advances in genomics and metabolomics have made clear in recent years that microbial biosynthetic capacities on Earth far exceed previous expectations. This is attributable, in part, to the realization that most microbial natural product (NP) producers harbor biosynthetic machineries not readily amenable to classical laboratory fermentation conditions. Such "cryptic" or dormant biosynthetic gene clusters (BGCs) encode for a vast assortment of potentially new antibiotics and, as such, have become extremely attractive targets for activation under controlled laboratory conditions. We report here that coculturing of a Rhodococcus sp. and a Micromonospora sp. affords keyicin, a new and otherwise unattainable bis-nitroglycosylated anthracycline whose mechanism of action (MOA) appears to deviate from those of other anthracyclines. The structure of keyicin was elucidated using high resolution MS and NMR technologies, as well as detailed molecular modeling studies. Sequencing of the keyicin BGC (within the Micromonospora genome) enabled both structural and genomic comparisons to other anthracycline-producing systems informing efforts to characterize keyicin. The new NP was found to be selectively active against Gram-positive bacteria including both Rhodococcus sp. and Mycobacterium sp. E. coli-based chemical genomics studies revealed that keyicin's MOA, in contrast to many other anthracyclines, does not invoke nucleic acid damage.

Gentamicins are heavily methylated, clinically valuable pseudotrisaccharide antibiotics produced by Micromonospora echinospora. GenN has been characterized as an S-adenosyl-l-methionine-dependent methyltransferase with low sequence similarity to other enzymes. It is responsible for the 3″-N-methylation of 3″-dehydro-3″-amino-gentamicin A2, an essential modification of ring III in the biosynthetic pathway to the gentamicin C complex. Purified recombinant GenN also efficiently catalyzes 3″-N-methylation of related aminoglycosides kanamycin B and tobramycin, which both contain an additional hydroxymethyl group at the C5″ position in ring III. We have obtained eight cocrystal structures of GenN, at a resolution of 2.2 Å or better, including the binary complex of GenN and S-adenosyl-l-homocysteine (SAH) and the ternary complexes of GenN, SAH, and several aminoglycosides. The GenN structure reveals several features not observed in any other N-methyltransferase that fit it for its role in gentamicin biosynthesis. These include a novel N-terminal domain that might be involved in protein:protein interaction with upstream enzymes of the gentamicin X2 biosynthesis and two long loops that are involved in aminoglycoside substrate recognition. In addition, the analysis of structures of GenN in complex with different ligands, supported by the results of active site mutagenesis, has allowed us to propose a catalytic mechanism and has revealed the structural basis for the surprising ability of native GenN to act on these alternative substrates.

Gentamicin B and gentamicin C1a are the direct precursor for Isepamicin and Etimicin synthesis, respectively. Although producing strains have been improved for many years, both gentamicin B titer and gentamicin C1a titer in the fermentation are still low. Because all gentamicin components are biosynthesized using UDP-N-acetyl-d-glucosamine (UDP-GlcNAc) and UDP-xylose as precursors, we tried to explore strategies for development of strains capable of directing greater fluxes of these precursors into production of gentamicins. The glycosyltransferases KanM1 and GenM2, which are responsible for UDP-GlcNAc and UDP-xylose transfer, respectively, were overexpressed in gentamicin B producing strain Micromonospora echinospora JK4. It was found that gentamicin B could be improved by up to 54% with improvement of KanM1 and GenM2 expression during appropriately glucose feeding. To prove this strategy is widely usable, the KanM1 and GenM2 were also overexpressed in gentamicin C1a producing strain, titers of gentamicin C1a improved by 45% when compared with titers of the starting strain. These results demonstrated overexpression the glycosyltransferases that transfer primary metabolites into secondary metabolites is workable for improvement of gentamicins production., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

The cytochrome P450 enzyme RosC catalyzes a two-step, hydroxylation and alcohol oxidation, oxidation reaction to form the C-20 formyl group in the biosynthesis of a 16-membered macrolide antibiotic rosamicin produced by Micromonospora rosaria IFO13697. RosC is presumed to be involved in the formation of 20-carboxyrosamicin because it has been isolated from the culture broth of M. rosaria. Here, we confirmed that RosC has catalytic activity, with E. coli expressing RosC converting rosamicin into 20-carboxyrosamicin. Therefore, it was revealed that RosC is a multifunctional P450 that catalyzes a three-step oxidation reaction that leads to the formation of the hydroxyl group, formyl group and carboxyl group at C-20 on the macrolactone ring in the rosamicin biosynthetic pathway. Moreover, the cytochrome P450 enzyme TylI, which is involved in formation of the formyl group of a 16-membered macrolide antibiotic tylosin produced by Streptomyces fradiae ATCC 19609, also converted rosamicin into 20-carboxyrosamicin., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The new ansa macrolide antibiotics 1 to 4 have been isolated from cultures of a Micromonospora sp. obtained from a marine sediment. Rifamycins 1 and 2 are the first natural ansa macrolides to have a 3-amino substituent. Sporalactams A (3) and B (4) are comprised of a heterocylic core 5 and a 14-membered ansa bridge that are both unprecedented. Sporalactam B (4) shows selective and potent inhibition of Mycobacterium tuberculosis.

Ranking among the most effective anticancer drugs, anthracyclines represent an important family of aromatic polyketides generated by type II polyketide synthases (PKSs). After formation of polyketide cores, the post-PKS tailoring modifications endow the scaffold with various structural diversities and biological activities. Here we demonstrate an unprecedented four-enzyme-participated hydroxyl regioisomerization process involved in the biosynthesis of kosinostatin. First, KstA15 and KstA16 function together to catalyze a cryptic hydroxylation of the 4-hydroxyl-anthraquinone core, yielding a 1,4-dihydroxyl product, which undergoes a chemically challenging asymmetric reduction-dearomatization subsequently acted by KstA11; then, KstA10 catalyzes a region-specific reduction concomitant with dehydration to afford the 1-hydroxyl anthraquinone. Remarkably, the shunt product identifications of both hydroxylation and reduction-dehydration reactions, the crystal structure of KstA11 with bound substrate and cofactor, and isotope incorporation experiments reveal mechanistic insights into the redox dearomatization and rearomatization steps. These findings provide a distinguished tailoring paradigm for type II PKS engineering.

Micromonospora genus produces >700 bioactive compounds of medical relevance. In spite of its ability to produce high number of bioactive compounds, no genome sequence is available with comprehensive secondary metabolite gene clusters analysis for anti-microbial producing Micromonospora strains. Thus, here we contribute the full genome sequence of Micromonospora sp. HK10 strain, which has high antibacterial activity against several important human pathogens like, Mycobacterium abscessus, Mycobacterium smegmatis, Bacillus subtillis, Staphylococcus aureus, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella and Escherichia coli. We have generated whole genome sequence data of Micromonospora sp. HK10 strain using Illumina NexSeq 500 sequencing platform (2×150bp paired end library) and assembled it de novo. The sequencing of HK10 genome enables identification of various genetic clusters associated with known- and probably unknown- antimicrobial compounds, which can pave the way for new antimicrobial scaffolds., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A Micromonospora strain, designated DS3186(T), isolated from sediment collected from the Black Sea off the Yason Peninsula, Ordu, Turkey, was examinated using a polyphasic approach. The strain was found to have chemotaxonomic, morphological and phylogenetic properties consistent with its clasification in the genus Micromonospora. A comparative 16S rRNA gene sequence analysis showed that the strain was closely related to the type strains of Micromonospora olivasterospora (99.0 %), Micromonospora equina (98.8 %), Micromonospora rhizosphaerae (98.8 %) and Micromonospora viridifaciens (98.8 %); low levels of DNA-DNA relatednes were found between the isolate and the M. olivasterospora and M. rhizosphaerae strains. Corresponding phylogenetic analysis based on partial gyrB gene sequences showed that strain DS3186(T) formed a subclade with the type strains of Micromonospora eburnea, M. equina, Micromonospora narathiwatensis and M. viridifaciens. Strain DS3186(T) was distinguished from its close phylogenetic neighbours using a combination of chemotaxonomic, morphological and physiological properties. Consequently, it is proposed that strain DS3186(T) represents a novel Micromonospora species for which the name Micromonospora yasonensis sp. nov. is proposed. The type strain is DS3186(T) (=DSM 45980(T) = KCTC 29433(T)).

Gentamicin consists primarily of four components, which have different patterns of methylation at C-6' position. The methyl groups have a significant impact on gentamicin antimicrobial activity. Sequence analysis predicted that GenN was a methyltransferase in the gentamicin biosynthetic pathway. To study the function of genN, it was disrupted in Micromonospora echinospora. The genN disruption strains produced 3″-N-demethyl-gentamicin C complex instead of the gentamicin C complex. In this study, 3″-N-demethyl gentamicin C1a was purified from the broth of disruption strain, and its structure was elucidated using MS and NMR. Besides 3″-N-demethyl products corresponding to gentamicin C1a, C2, and C2a, two 3″-N-demethyl products corresponding to gentamicin C1 were detected, which were concluded as C-6' epimers originating from decreased substrate specificity of 6'-N methyltransferase. To explore the effects of 3″-N-methyl on gentamicin antimicrobial activity, antimicrobial activity of these demethyl gentamicin analogues were tested in vitro. 3″-N-Demethyl gentamicin components have identical activity with corresponding components of gentamicin. The results of bioassays showed that the 3″-N-methyl group has little impact on gentamicin activity. However, these highly bioactive compounds afforded a unique opportunity for creating new and high potent aminoglycoside antibiotics., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

In this study, an efficient high-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-ion trap-tandem mass spectrometry (MS/MS) was developed for the identification of the biosynthetic congeners involved in the aminocyclitol aminoglycosidic fortimicin pathway from Micromonospora olivasterospora fermentation. The usage of both acid extraction (pH ∼2.5) followed by an cationic-exchanging SPE cleanup and pentafluoropropionic acid mediated ion-pairing chromatography with ESI-ion trap-MS/MS detection was determined to be sufficiently practical to profile the fortimicin (FOR) congeners produced in a culture broth. The limit of the quantification for the fortimicin A (FOR-A) standard spiked in the culture broth was ∼1.6 ng mL(-1). The average recovery rate was 93.6%, and the intra- and inter-day precisions were <5% with accuracy in the range from 87.1 to 94.2%. Moreover, the epimeric mixtures including FOR-KH, FOR-KR, and FOR-B were separately resolved through a macrocyclic glycopeptide (teicoplanin)-bonded chiral column. As a result, ten natural FOR pseudodisaccharide analogs were identified and semi-quantified in descending order as follows: FOR-A, FOR-B, DCM, FOR-KH plus FOR-KR, FOR-KK1, FOR-AP, FOR-KL1, FOR-AO, and FOR-FU-10. This is the first report on both the simultaneous characterization of diverse structurally closely related FORs derived from bacterial fermentation using HPLC-ESI-ion trap-MS/MS analysis and the chromatographic separation of the three FOR epimers.

Background: Isepamicin is a weakly toxic but highly active aminoglycoside antibiotic derivative of gentamicin B. Gentamicin B is a naturally occurring minor component isolated from Micromonospora echinospora. 2'-NH2-containing gentamicin C complex is a dominant compound produced by wild-type M. echinospora; by contrast, 2'-OH-containing gentamicin B is produced as a minor component. However, the biosynthetic pathway of gentamicin B remains unclear. Considering that gentamicin B shares a unique C2' hydroxyl group with kanamycin A, we aimed to design a new biosynthetic pathway of gentamicin B by combining twelve steps of gentamicin biosynthesis and two steps of kanamycin biosynthesis., Results: We blocked the biosynthetic pathway of byproducts and generated a strain overproducing JI-20A, which was used as a precursor of gentamicin B biosynthesis, by disrupting genK and genP. The amount of JI-20A produced in M. echinospora ∆K∆P reached 911 μg/ml, which was 14-fold higher than that of M. echinospora ∆P. The enzymes KanJ and KanK necessary to convert 2'-NH2 into 2'-OH from the kanamycin biosynthetic pathway were heterologously expressed in M. echinospora ΔKΔP to transform JI-20A into gentamicin B. The strain with kanJK under PermE* could produce 80 μg/ml of gentamicin B, which was tenfold higher than that of the wild-type strain. To enhance gentamicin B production, we employed different promoters and gene integration combinations. When a PhrdB promoter was used and kanJ and kanK were integrated in the genome through gene replacement, gentamicin B was generated as the major product with a maximum yield of 880 μg/ml., Conclusion: We constructed a new biosynthetic pathway of high-level gentamicin B production; in this pathway, most byproducts were removed. This method also provided novel insights into the biosynthesis of secondary metabolites via the combinatorial biosynthesis.

Maklamicin is a spirotetronate-class antibiotic produced by Micromonospora sp. NBRC 110955, and a polyketide assembly line and a glycerate utilization system are involved in its biosynthesis. One tailoring step in the biosynthesis is predicted to be post-polyketide synthase (PKS) modification, which seems to be catalysed by putative cytochrome P450 monooxygenases, MakC2 and/or MakC3. In this study, we characterized makC2 and makC3 in the biosynthesis of maklamicin and identified a new maklamicin analogue from a makC2 disruptant. Gene deletion of makC2 resulted in the complete loss of maklamicin production with concomitant accumulation of a new compound (29-deoxymaklamicin), while gene deletion of makC3 did not affect the maklamicin production, indicating that 29-deoxymaklamicin is an intermediate in the biosynthetic pathway of maklamicin and should serve as the substrate of MakC2. 29-Deoxymaklamicin showed strong-to-modest anti-microbial activity against gram-positive bacteria. The fact that Streptomyces avermitilis heterologously expressing makC2 successfully converted 29-deoxymaklamicin into maklamicin confirmed that MakC2 is the final-step hydroxylase in the formation of mature maklamicin., (Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Maklamicin, which is produced by the endophytic Micromonospora sp. NBRC 110955, is a spirotetronate-class antibiotic possessing anti-microbial activity against Gram-positive bacteria, and has several unique structural features different from other spirotetronates. Here we describe identification and characterization of the maklamicin biosynthetic (mak) gene cluster through draft genome sequencing, genomic library screening, and gene disruption. Sequence analysis revealed that a plausible maklamicin cluster resides in a 152 kb DNA region encoding 46 open reading frames, 24 of which can be assigned roles in the biosynthesis of polyketide backbone, spirotetronate or peripheral moieties, self-resistance and the regulation of maklamicin production. Disruption of the polyketide synthase (PKS) genes makA1 or makA4 resulted in a complete loss of maklamicin production, indicating that the type I modular PKS system is responsible for the biosynthesis of maklamicin. The mak gene cluster contained a set of biosynthetic genes for the formation of a tetronate moiety, which were found to be highly conserved in the gene clusters for spirotetronate antibiotics. Based on the estimated biosynthetic genes, we propose the biosynthetic pathway for maklamicin. Our findings provide not only insights on the biosynthetic mechanism of the unique structures in maklamicin, but also useful information to facilitate a comparative analysis of the spirotetronate biosynthetic pathways to expand the structural repertoire., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Sugar aminotransferases (SATs) are an important class of tailoring enzymes that catalyze the 5'-pyridoxal phosphate (PLP)-dependent stereo- and regiospecific installation of an amino group from an amino acid donor (typically L-Glu or L-Gln) to a corresponding ketosugar nucleotide acceptor. Herein we report the strategic structural study of two homologous C4 SATs (Micromonospora echinospora CalS13 and Escherichia coli WecE) that utilize identical substrates but differ in their stereochemistry of aminotransfer. This study reveals for the first time a new mode of SAT sugar nucleotide binding and, in conjunction with previously reported SAT structural studies, provides the basis from which to propose a universal model for SAT stereo- and regiochemical control of amine installation. Specifically, the universal model put forth highlights catalytic divergence to derive solely from distinctions within nucleotide sugar orientation upon binding within a relatively fixed SAT active site where the available ligand bound structures of the three out of four representative C3 and C4 SAT examples provide a basis for the overall model. Importantly, this study presents a new predictive model to support SAT functional annotation, biochemical study and rational engineering.

A screening of our actinomycete fraction library against the NCI-60 SKOV3 human tumor cell line led to the isolation of isopimara-2-one-3-ol-8,15-diene (1), lagumycin B (2), dehydrorabelomycin (3), phenanthroviridone (4), and WS-5995 A (5). These secondary metabolites were produced by a Micromonospora sp. isolated from sediment collected off the Cát Bà peninsula in the East Sea of Vietnam. Compound 1 is a novel Δ(8,9)-pimarane diterpene, representing one of approximately 20 actinomycete-produced diterpenes reported to date, while compound 2 is an angucycline antibiotic that has yet to receive formal characterization. The structures of 1 and 2 were elucidated by combined NMR and MS analysis and the absolute configuration of 1 was assigned by analysis of NOESY NMR and CD spectroscopic data. Compounds 2-5 exhibited varying degrees of cytotoxicity against a panel of cancerous and non-cancerous cell lines. Overall, this study highlights our collaborative efforts to discover novel biologically active molecules from the large, underexplored, and biodiversity-rich waters of Vietnam's East Sea.

Background: Calicheamicins (CAL) are enedyine natural products with potent antibiotic and cytotoxic activity, used in anticancer therapy. The O-methyltransferase CalO6 is proposed to catalyze methylation of the hydroxyl moiety at the C2 position of the orsellinic acid group of CAL., Results: Crystals of CalO6 diffracted non-isotropically, with the usable data extending to 3.4 Å. While no single method of crystal structure determination yielded a structure of CalO6, we were able to determine its structure by using molecular replacement-guided single wavelength anomalous dispersion by using diffraction data from native crystals of CalO6 and a highly non-isomorphous mercury derivative. The structure of CalO6 reveals the methyltransferase fold and dimeric organization characteristic of small molecule O-methyltransferases involved in secondary metabolism in bacteria and plants. Uncommonly, CalO6 was crystallized in the absence of S-adenosylmethionine (SAM; the methyl donor) or S-adenosylhomocysteine (SAH; its product)., Conclusions: Likely as a consequence of the dynamic nature of CalO6 in the absence of its cofactor, the central region of CalO6, which forms a helical lid-like structure near the active site in CalO6 and similar enzymes, is not observed in the electron density. We propose that this region controls the entry of SAM into and the exit of SAH from the active site of CalO6 and shapes the active site for substrate binding and catalysis.

A new HSQC-based (13)Cα CEST pulse scheme is proposed, which is suitable for uniformly (13)C- or (13)C, (15)N-labeled samples in either water or heavy water. Except for Thr and Ser residues, the sensitivity of this scheme for uniformly labeled samples is similar to that of the previous scheme for selectively (13)Cα-labeled samples with 100% isotope enrichment. The experiment is demonstrated on an acyl carrier protein domain. Our (13)Cα CEST data reveal that the minor state of the acyl carrier protein has high helical propensity. The new scheme will facilitate structural characterization of invisible minor states.

Calicheamicin γ1I (1) is an enediyne antitumor compound produced by Micromonospora echinospora spp. calichensis, and its biosynthetic gene cluster has been previously reported. Despite extensive analysis and biochemical study, several genes in the biosynthetic gene cluster of 1 remain functionally unassigned. Using a structural genomics approach and biochemical characterization, two proteins encoded by genes from the 1 biosynthetic gene cluster assigned as "unknowns", CalU16 and CalU19, were characterized. Structure analysis revealed that they possess the STeroidogenic Acute Regulatory protein related lipid Transfer (START) domain known mainly to bind and transport lipids and previously identified as the structural signature of the enediyne self-resistance protein CalC. Subsequent study revealed calU16 and calU19 to confer resistance to 1, and reminiscent of the prototype CalC, both CalU16 and CalU19 were cleaved by 1 in vitro. Through site-directed mutagenesis and mass spectrometry, we identified the site of cleavage in each protein and characterized their function in conferring resistance against 1. This report emphasizes the importance of structural genomics as a powerful tool for the functional annotation of unknown proteins.