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Malonyl‐CoA serves as the main building block for the biosynthesis of many important polyketides, as well as fatty acid–derived compounds, such as biofuel. Escherichia coli, Corynebacterium gultamicum, and Saccharomyces cerevisiae have recently been engineered for the biosynthesis of such compounds. However, the developed processes and strains often have insufficient productivity. In the current study, we used enzyme‐engineering approach to improve the binding of acetyl‐CoA with ACC. We generated different mutations, and the impact was calculated, which reported that three mutations, that is, S343A, T347W, and S350W, significantly improve the substrate binding. Molecular docking investigation revealed an altered binding network compared to the wild type. In mutants, additional interactions stabilize the binding of the inner tail of acetyl‐CoA. Using molecular simulation, the stability, compactness, hydrogen bonding, and protein motions were estimated, revealing different dynamic properties owned by the mutants only but not by the wild type. The findings were further validated by using the binding‐free energy (BFE) method, which revealed these mutations as favorable substitutions. The total BFE was reported to be −52.66 ± 0.11 kcal/mol for the wild type, −55.87 ± 0.16 kcal/mol for the S343A mutant, −60.52 ± 0.25 kcal/mol for T347W mutant, and −59.64 ± 0.25 kcal/mol for the S350W mutant. This shows that the binding of the substrate is increased due to the induced mutations and strongly corroborates with the docking results. In sum, this study provides information regarding the essential hotspot residues for the substrate binding and can be used for application in industrial processes. [ABSTRACT FROM AUTHOR]

Plasmalogens are a subclass of glycerophospholipids that have a vinyl-ether bond at the sn -1 position and are thought to have several physiological functions. The creation of non-natural plasmalogens with functional groups is desired for the establishment of the prevention of diseases caused by the depletion of plasmalogens. Phospholipase D (PLD) has both hydrolysis and transphosphatidylation activities. In particular, PLD from Streptomyces antibioticus has been investigated extensively due to its high transphosphatidylation activity. However, it has been difficult to stably express recombinant PLD in Escherichia coli and to express it as a soluble protein. In this study, we used the E. coli strain, SoluBL21™, and achieved stable PLD expression from the T7 promoter and increased soluble fraction in the cell. We also improved the purification method of PLD using His-tag at the C terminus. We obtained PLD with ∼730 mU mg−1 protein of specific activity, and the yield was ∼420 mU l−1 culture, corresponding to 76 mU per gram of wet cells. Finally, we synthesized a non-natural plasmalogen with 1,4-cyclohexanediol bound to the phosphate group at the sn -3 position by transphosphatidylation of the purified PLD. This method will contribute to the expansion of the chemical structure library of non-natural plasmalogens. [ABSTRACT FROM AUTHOR]

Secondary metabolic gene clusters widely exist in the genomes of Streptomyces but mostly remain silent. To awaken this hidden reservoir of natural products, various strategies concerning secondary metabolic pathways are applied. Here, we describe that butenolide signaling molecule deficiency and glucose addition can interdependently activate the expression of silent oviedomycin biosynthetic gene clusters in Streptomyces ansochromogenes and Streptomyces antibioticus. Since oviedomycin is a promising anti-tumor lead compound, in order to improve its yield, we use the cluster-situated genes (ovmF , ovmG , ovmI and ovmH) encoding the enzymes for acyl carrier protein modification and precursor biosynthesis, and the discrete precursor biosynthetic genes (pyk2 , gap1 and accA2) involved in glycolysis to assemble two gene modules (pFGIH and pPGA). Their co-overexpression in ΔsabA (a disruption mutant of sabA encoding SAB synthase) has superimposed effect on the yield of oviedomycin, which can be further increased to 59-fold in the presence of galactose as optimal carbon source. This is the most unambiguous evidence that butenolide signaling system can synergize with the optimization of primary metabolism to regulate the expression of secondary metabolic gene clusters, providing efficient strategies for mining natural products of Streptomyces. [Display omitted] • SAB signaling system in synergy with glucose activated oviedomycin biosynthesis. • Glycolysis-related and cluster-situated genes were chosen to form gene modules. • Overexpression of PGA-FGIH gene modules effectively increased oviedomycin yield. • Galactose had a superimposed effect with PGA-FGIH on oviedomycin production. [ABSTRACT FROM AUTHOR]

Hemiactinomycin ( 1 ), an intermediate derivative of actinomycin biosynthesis, together with three known actinomycins ( 2 - 4 ) , were isolated from the ethanolic extract of Streptomyces antibioticus H41-55 fermentation mycelium by using various column chromatography. The structure of the derivative was established by extensive spectroscopic analysis, including HRESIMS, 1D, and 2D NMR spectroscopy. In addition, the anti-inflammatory activities of all the isolates were tested. The derivative ( 1 ) showed inhibiting NO release activities in LPS-induced RAW 264.7 macrophages with the IC 50 values of 15.41 ± 0.66 μM.

Chlorothricin (CHL), produced by Streptomyces antibioticus DSM 40725 (wild-type strain, WT), belongs to a growing family of spirotetronate antibiotics that have biological activities inhibiting pyruvate carboxylase and malate dehydrogenase. ChlF2, a cluster-situated SARP regulator, can activate the transcription of chlJ, chlC3, chlC6, chlE1, chlM, and chlL to control CHL biosynthesis. Co-expression of chlF2 and chlK encoding type II thioesterase in WT strain under the control of Pkan led to high production of chlorothricin by 840% in comparison with that of WT. Since the inhibitory activity of CHL against several Gram-positive bacteria is higher than des-CHL, combinatorial strategies were applied to promote the conversion of des-CHL to CHL. Over-expression of chlB4, encoding a halogenase, combining with the supplementation of sodium chloride led to further 41% increase of CHL production compared to that of F2OE, a chlF2 over-expression strain. These findings provide new insights into the fine-tuned regulation of spirotetronate family of antibiotics and the construction of high-yield engineered strains.

Streptomyces antibioticus strain ess_amA8 was isolated from the insect Tapinoma simrothi , and was tested for the production of compounds that inhibit the growth of microorganisms. The cultural and physiological descriptions of the strain revealed that it belonged to the Streptomyces genus. The 16S rRNA gene from the Streptomyces isolate was sequenced, and its length was 1156 bp and it was submitted to Genbank (KF996507). According to molecular and morphological studies, the isolated strain was ultimately identified as strain ess_amA8 of Streptomyces antibioticus. The key compound was extracted from the strain ess_amA8, which was active in vitro , against a wide range of microorganisms, such as Staphylococcus aureus ATCC6538, Bacillus subtilis ATCC 6633, Pseudomonas aeruginosa ATCC 9027, Escherichia coli ATCC-7839, Candida albicans , Fusarium moniliforme , Aspergillus niger , and Aspergillus flavus. The physico-chemical property of the purified antimicrobial compound analyzed and its proposed empirical formula was C 27 H 25 O 13. According to the IUPAC name, this compound was identified as 5,7-diethoxy-2-(3-methoxy-4-methylphenyl)-3-[(2R,3S,4R,5R,6S)-3,4,5,6-tetrahydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl] oxy-4H-chromen-4-one, and was a quercetin 3-O-glucoside derivative. In summary, the present study highlights the fact that the purified quercetin 3-O-glucoside antimicrobial derivative, first report to this Streptomyces sp., and it is a flavanone glycoside. [ABSTRACT FROM AUTHOR]

Ivana Crnovčić,1 Christian Rückert,2 Siamak Semsary,1 Manuel Lang,1 Jörn Kalinowski,2 Ullrich Keller1 1Institut für Chemie, Technische Universität Berlin, Berlin-Charlottenburg, 2Technology Platform Genomics, Center for Biotechnology, Bielefeld University, Bielefeld, Germany Abstract: Sequencing the actinomycin (acm) biosynthetic gene cluster of Streptomyces antibioticus IMRU 3720, which produces actinomycin X (Acm X), revealed 20 genes organized into a highly similar framework as in the bi-armed acm C biosynthetic gene cluster of Streptomyces chrysomallus but without an attached additional extra arm of orthologues as in the latter. Curiously, the extra arm of the S. chrysomallus gene cluster turned out to perfectly match the single arm of the S. antibioticus gene cluster in the same order of orthologues including the the presence of two pseudogenes, scacmM and scacmN, encoding a cytochrome P450 and its ferredoxin, respectively. Orthologues of the latter genes were both missing in the principal arm of the S. chrysomallus acm C gene cluster. All orthologues of the extra arm showed a G +C-contents different from that of their counterparts in the principal arm. Moreover, the similarities of translation products from the extra arm were all higher to the corresponding translation products of orthologue genes from the S. antibioticus acm X gene cluster than to those encoded by the principal arm of their own gene cluster. This suggests that the duplicated structure of the S. chrysomallus acm C biosynthetic gene cluster evolved from previous fusion between two one-armed acm gene clusters each from a different genetic background. However, while scacmM and scacmN in the extra arm of the S. chrysomallus acm C gene cluster are mutated and therefore are non-functional, their orthologues saacmM and saacmN in the S. antibioticus acm C gene cluster show no defects seemingly encoding active enzymes with functions specific for Acm X biosynthesis. Both acm biosynthetic gene clusters lack a kynurenine-3-monooxygenase gene necessary for biosynthesis of 3-hydroxy-4-methylanthranilic acid, the building block of the Acm chromophore, which suggests participation of a genome-encoded relevant monooxygenase during Acm biosynthesis in both S. chrysomallus and S. antibioticus. Keywords: actinomycin, actinomycin halves, biosynthesis, Streptomyces chrysomallus, ­Streptomyces anulatus Streptomyces antibioticus, genomes, 3-hydroxy-4-methylanthranilic acid (4-MHA), evolution of biosynthetic gene cluster, genetic transmission of biosynthetic gene cluster, actinomycin C, actinomycin X

Collagenases are proteases able to degrade native and denatured collagen, with broad applications such as leather, food, and pharmaceutical industries. The aim of this research was to purify and characterize a collagenase from Streptomyces antibioticus . In the present work, the coffee ground substrate provided conditions to obtaining high collagenase activity (377.5 U/mL) using anion-exchange DEAE-Sephadex G50 chromatographic protocol. SDS-PAGE revealed the metallo-collagenase with a single band of 41.28 kDa and was able to hydrolyzed type I and type V collagen producing bioactive peptides that delayed the coagulation time. The enzyme activity showed stability across a range of pH (6.0-11) and temperature (30-55 °C) with optima at pH 7.0 and 60 °C, respectively. Activators include Mg +2 , Ca +2 , Na + , K + , while full inhibition was given by other tested metalloproteinase inhibitors. Kinetic parameters (K m of 27.14 mg/mol, V max of 714.29 mg/mol/min, K cat of 79.9 s -1 and K cat /K m of 2.95 mL/mg/s) and thermodynamic parameters (E a of 65.224 kJ/mol, ΔH of 62.75 kJ/mol, ΔS of 1.96 J/mol, ΔG of 62.16 kJ/mol, ΔG E-S of 8.18 kJ/mol and ΔG E-T of -2.64 kJ/mol) were also defined. Coffee grounds showed to be an interesting source to obtaining a collagenase able to produce bioactive peptides with anticoagulant activity.

Tyrosinase is a monooxygenase that catalyzes both the hydroxylation of p-hydroxyphenyl moieties to o-catechols and the oxidation of o-catechols to o-quinones. Apart from its critical functionality in melanogenesis and the synthesis of various neurotransmitters, this enzyme is also used in a variety of biotechnological applications, most notably mediating covalent cross-linking between polymers containing p-hydroxyphenyl groups, forming a hydrogel. Tyrosinases from the genus Streptomyces are usually secreted as a complex with their caddie protein. In this study, we report an increased secretion efficiency observed when the Streptomyces antibioticus tyrosinase gene melC2 was introduced into Pseudomonas fluorescens along with its caddie protein gene melC1, which has the DNA sequence for the Tat (twinarginine translocation) signal. [ABSTRACT FROM AUTHOR]

An actinomycete strain 200-09, isolated from a soil sample collected from the coast of Hawaii, USA, was identified as Streptomyces antibioticus on the basis of its morphological, physiological and biochemical characteristics as well as 16S rDNA analysis. A new antimycin-type antibiotic, kitamycin C (1), together with kitamycin A (2), kitamycin B (3), urauchmycin B (4), deisovaleryblastomycin (5) was isolated from a cultured broth of strain 200-09. The structure of the new compound was determined by spectroscopic data, including HR-ESI-MS and NMR. All the compounds exhibited antifungal activities against Candida albicans with MIC of about 25.0 μg mL−1. [ABSTRACT FROM AUTHOR]

An actinomycete strain (H12-15) isolated from a sea sediment in a mangrove district was identified as Streptomyces antibioticus on the basis of 16S rDNA gene sequence analysis as well as the investigation of its morphological, physiological, and biochemical characteristics. Two novel benzamido nonacyclic dilactones, namely neoantimycins A (1) and B (2), together with the known antimycins A1ab (3a,b), A2a (4), and A9 (5), were isolated from the culture broth of this strain. Compounds 1 and 2 are the first natural modified ATNs with an unusual benzamide unit. The structures of these new compounds, including their absolute configuration, were established on the basis of HRMS, NMR spectroscopic data, and quantum chemical ECD calculations. Their cytotoxicities against human breast adenocarcinoma cell line MCF-7, the human glioblastoma cell line SF-268, and the human lung cancer cell line NCI-H460 were also tested. All compounds exhibited mild cytotoxic activity. However, Compounds 1 and 2 showed no activity against C. albicans at the test concentration of 1 mg/mL via paper disc diffusion, while the known antimycins showed obvious antifungal activity. [ABSTRACT FROM AUTHOR]

Streptomyces rimosus can produce antibacterial and antifungal antibiotics, which have important applications in medicine and agriculture. Seventy-nine microbial strains were employed to assay interaction between S. rimosus MY02 and different fungi, actinomyces, and bacteria when confront cultured on solid media. The results showed that the presence of a microorganism might affect the activity of another one. When S. rimosus MY02 confront cultured with other microorganisms, the inductive effect might be positive or negative. In this study, fungi showed to be effective elicitors, with a highest inductivity rate of 90.1%, and all of fungi showed positive induction behavior. Followed by bacteria with 59.6% of the tested bacterial strains showing positive inductivity, and the highest inductivity was 54.9%. Only six actinomyces (counting for 40% of the tested actinomyces strains) showed positive inductivity, and the highest induction rate of the strain NK413 was 34.1%. We also found that growth of most of bacteria or actinomyces which showed negative inductivity were similar or better than that of the strain MY02. However, the growth status of the strains was not positive related to inducing ability directly. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma (PDAC) is characterized by accelerated production and degradation of hyaluronan (HA), a major component of extracellular matrix involved in the malignant phenotype of cancer. In particular, increased hyaluronidase (HYAL) activity plays a critical role in cancer progression, at least in part, by producing low-molecular-weight- (LMW-) HA or small fragments of HA, suggesting HYAL as a target for cancer treatment. Hyaluromycin, a new member of the rubromycin family of antibiotics, was isolated from the culture extract of a marine-derived Streptomyces hyaluromycini as a HYAL inhibitor. We investigated the antitumor effects of hyaluromycin in PDAC cells. We examined the effects of hyaluromycin on the proliferation and migration of PDAC cells. To elucidate the mechanisms underlying the effect of hyaluromycin on PDAC cells, we examined the concentration of LMW-HA in the conditioned media after treating PDAC cells with hyaluromycin. We demonstrate that hyaluromycin inhibits proliferation and migration of PDAC cells. We also found that these antitumor effects of hyaluromycin were associated with a decreased concentration of LMW-HA and a decreased phosphorylation of ribosomal protein S6. Our results suggest that hyaluromycin is a promising new drug against this highly aggressive neoplasm. [ABSTRACT FROM AUTHOR]

Background OleP is a cyt P450 from Streptomyces antibioticus carrying out epoxigenation of the antibiotic oleandomycin during its biosynthesis. The timing of its reaction has not been fully clarified, doubts remain regarding its substrate and catalytic mechanism. Methods The crystal structure of OleP in complex with clotrimazole, an inhibitor of P450s used in therapy, was solved and the complex formation dynamics was characterized by equilibrium and kinetic binding studies and compared to ketoconazole, another azole differing for the N1-substituent. Results Clotrimazole coordinates the heme and occupies the active site. Most of the residues interacting with clotrimazole are conserved and involved in substrate binding in MycG, the P450 epoxigenase with the highest homology with OleP. Kinetic characterization of inhibitor binding revealed OleP to follow a simple bimolecular reaction, without detectable intermediates. Conclusions Clotrimazole-bound OleP adopts an open form, held by a π-π stacking chain that fastens helices F and G and the FG loop. Affinity is affected by the interactions of the N1 substituent within the active site, given the one order of magnitude difference of the off-rate constants between clotrimazole and ketoconazole. Based on structural similarities with MycG, we propose a binding mode for both oleandomycin intermediates, that are the candidate substrates of OleP. General significance Among P450 epoxigenases OleP is the only one that introduces an epoxide on a non-activated C–C bond. The data here presented are necessary to understand the rare chemistry carried out by OleP, to engineer it and to design more selective and potent P450-targeted drugs. [ABSTRACT FROM AUTHOR]

The glutarimide-containing polyketides represent a fascinating class of natural products that exhibit a multitude of biological activities. We have recently cloned and sequenced the biosynthetic gene clusters for three members of the glutarimide-containing polyketides-iso-migrastatin (iso-MGS) from Streptomyces platensis NRRL 18993, lactimidomycin (LTM) from Streptomyces amphibiosporus ATCC 53964, and cycloheximide (CHX) from Streptomyces sp. YIM56141. Comparative analysis of the three clusters identified mgsA and chxA, from the mgs and chx gene clusters, respectively, that were predicted to encode the PimR-like Streptomyces antibiotic regulatory proteins (SARPs) but failed to reveal any regulatory gene from the ltm gene cluster. Overexpression of mgsA or chxA in S. platensis NRRL 18993, Streptomyces sp. YIM56141 or SB11024, and a recombinant strain of Streptomyces coelicolor M145 carrying the intact mgs gene cluster has no significant effect on iso-MGS or CHX production, suggesting that MgsA or ChxA regulation may not be rate-limiting for iso-MGS and CHX production in these producers. In contrast, overexpression of mgsA or chxA in S. amphibiosporus ATCC 53964 resulted in a significant increase in LTM production, with LTM titer reaching 106 mg/L, which is five-fold higher than that of the wild-type strain. These results support MgsA and ChxA as members of the SARP family of positive regulators for the iso-MGS and CHX biosynthetic machinery and demonstrate the feasibility to improve glutarimide-containing polyketide production in Streptomyces strains by exploiting common regulators. [ABSTRACT FROM AUTHOR]

The article discusses a study on the use of actinomycete streptomyces antibioticus in the nitrite formation from organic nitrogen to support the bacterial growth cell growth and potential involvement of nitirc oxide. It states the synchronization in the production of nitrite and ammonium with rapid cell growth depicted during the incubation. It also provides an overview on the results during the occurrence of novel nitrogen metabolic pathway in streptomyces antibioticus.

Abstract: Simocyclinone D8 (SD8) is an antibiotic produced by Streptomyces antibioticus that targets DNA gyrase. A previous structure of SD8 complexed with the N-terminal domain of the DNA gyrase A protein (GyrA) suggested that four SD8 molecules stabilized a tetramer of the protein; subsequent mass spectrometry experiments suggested that a protein dimer with two symmetry-related SD8s was more likely. This work describes the structures of a further truncated form of the GyrA N-terminal domain fragment with and without SD8 bound. The structure with SD8 has the two SD8 molecules bound within the same GyrA dimer. This new structure is entirely consistent with the mutations in GyrA that confer SD8 resistance and, by comparison with a new apo structure of the GyrA N-terminal domain, reveals the likely conformation changes that occur upon SD8 binding and the detailed mechanism of SD8 inhibition of gyrase. Isothermal titration calorimetry experiments are consistent with the crystallography results and further suggest that a previously observed complex between SD8 and GyrB is ~1000-fold weaker than the interaction with GyrA. [Copyright &y& Elsevier]

Although recent molecular techniques have greatly expanded our knowledge of microbial biogeography, the functional biogeography of soil microorganisms remains poorly understood. In this work, we explore geographic variation in Streptomyces phenotypes that are critical to species interactions. Specifically, we characterize Streptomyces from different locations from multiple continents for antibiotic inhibition, resistance, and resource use phenotypes. Streptomyces from different locations varied significantly in antibiotic inhibition, resistance, and resource use indicating that communities vary in functional potential. Among all isolates, there were substantial differences in antibiotic inhibition, resistance, and resource use within and among and within Streptomyces species. Moreover, Streptomyces with near-identical 16S rRNA gene sequences from different locations sometimes differed significantly in inhibition, resistance, and resource use phenotypes, suggesting that these phenotypes may be locally adapted. Thus, in addition to a likely role of environmental filtering, variation in Streptomyces inhibitory, resistance, and resource use phenotypes among locations is likely to be a consequence of local selection mediated by species interactions. [ABSTRACT FROM AUTHOR]

We present and evaluate the actinomycin-production assay, a bacterial test for detecting DNA-damaging agents as a primary step for detecting the antitumor activity of new synthesized, modified or isolated compounds. It’s a simple test based on the activity of the agents against actinomycin antibiotic production of Streptomyces antibioticus, bacteria whose value is monitored by the inhibition activity of E. coli and S. aureus bacterial growth using two standard anticancer drugs cisplatin and flurouracil. In this assay, production of actinomycin antibiotic is measured as a specific indication of the ability of an agents to directly or indirectly damage DNA and that are of potential interest in carcinogenesis and cancer chemotherapy. Because in vivo tumor test systems using mammalian cell cultures were slow and expensive, we tested a novel cytotoxicity test, simple, sensitive, inexpensive and amenable to large-scale screening using bacteria cultures, as well as, it will offer another practical application in addition to cytotoxicity screening, the assay is used for testing antibacterial of the test compounds.

Using insertional mutagenesis, we have disrupted the RNase III gene, rnc, of the actinomycin-producing streptomycete, Streptomyces antibioticus. Disruption was verified by Southern blotting. The resulting strain grows more vigorously than its parent on actinomycin production medium but produces significantly lower levels of actinomycin. Complementation of the rnc disruption with the wild-type rnc gene from S. antibioticus restored actinomycin production to nearly wild-type levels. Western blotting experiments demonstrated that the disruptant did not produce full-length or truncated forms of RNase III. Thus, as is the case in Streptomyces coelicolor, RNase III is required for antibiotic production in S. antibioticus. No differences in the chemical half-lives of bulk mRNA were observed in a comparison of the S. antibioticus rnc mutant and its parental strain. [ABSTRACT FROM AUTHOR]

The impact of individual and interactive behaviors of various fermentation process parameters on laccase and peroxidase-free tyrosinase produc-tion were investigated by isolated Streptomyces antibioticus RSP-T1. Six key bioprocess factors (medium pH, rpm, incubation time, sodium chloride concentration, com-plex nitrogen [yeast extract + peptone], and carbon source [maltose]) were selected based on using a one variable at a time methodology. All selected parameters had an impact at individual and interactive levels on tyrosinase produc-tion. Only 25% of the improved tyrosinase production was attributed to the optimized fermentation parameters selected. Regarding the nutritional parameters, the complex nitrogen/carbon source concentration (maltose and yeast extract + peptone) was found to have the most significance impact on overall tyrosinase enzyme production. Physiological growth factors (pH, rpm, and incubation time) played key roles at an interactive level. A maximum yield of 12.60 lU/mL tyrosinase production was achieved with optimized medium, adjusted to 7.5 pH, consisting of 0.75% maltose (w/v), 0.2% yeast extract (w/v), 0.2% peptone (w/v), and 1.25% sodium chloride (w/v) at 160 rpm in 24 hours. This study identified the optimum medium component concentrations for improved tyrosinase production by 5. antibioticus RSP-T1. This strain requires complex nitrogen sources (yeast extract and peptone) for increased product yield. Overall, a greater than 250% increase in tyrosinase production was achieved using this optimization approach, as compared to conventional methods. [ABSTRACT FROM AUTHOR]

Biotransformation of the highly substituted pyridine derivative 2-amino-4-methyl-3-nitropyridine by Cunninghamella elegans ATCC 26269 yielded three products each with a molecular weight of 169 Da which were identified as 2-amino-5-hydroxy-4-methyl-3-nitropyridine, 2-amino-4-hydroxymethyl-3-nitropyridine, and 2-amino-4-methyl-3-nitropyridine-1-oxide. Biotransformation by Streptomyces antibioticus ATCC 14890 gave two different products each with a molecular weight of 169 Da; one was acid labile and converted to the other stable product under acidic conditions. The structure of the stable product was established as 2-amino-4-methyl-3-nitro-6(1 H)-pyridinone, and that of the less stable product was assigned as its tautomer 2-amino-6-hydroxy-4-methyl-3-nitropyridine. Four of the five biotransformation products are new compounds. Several strains of Aspergillus also converted the same substrate to the lactam 2-amino-4-methyl-3-nitro-6(1 H)-pyridinone. Microbial hydroxylation by C. elegans was found to be inhibited by sulfate ion. In order to improve the yield and productivity of the 5-hydroxylation reaction by C. elegans, critical process parameters were determined and Design of Experiments (DOE) analyses were performed. Biotransformation by C. elegans was scaled up to 15-l fermentors providing 2-amino-5-hydroxy-4-methyl-3-nitropyridine at ca. 13 % yield in multi-gram levels. A simple isolation process not requiring chromatography was developed to provide purified 2-amino-5-hydroxy-4-methyl-3-nitropyridine of excellent quality. [ABSTRACT FROM AUTHOR]

A novel isolate belonging to the genus Streptomyces, strain SL-4, was isolated from soil sample collected from a sanitary landfill, New Delhi, India. The taxonomic status of this isolate was studied by polyphasic approach including morphological, physiological and chemo-taxonomic characterization. Spore chains of SL-4 were open loops, hooks or extended spirals of wide diameter (retinaculiperti). The cell wall peptidoglycan of the isolate SL-4 contained L,L-diaminopimelic acid, suggesting that the strain has a cell wall of chemotype-I. The polar lipid profile of the isolate was of Type II, with phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides. The 16SrRNA gene sequence similarity between SL-4 and its phylogenetic relatives Streptomyces atrovirens NRRLB 16357 (DQ026672), S. albogriseolus NRRLB 1305 (AJ494865), S viridodiastaticus NBRC 13106 (AB184317), S. caelestis NRRL 2418 (X80824), S. flavoviridis NBRC 12772 (AB184842), S. pilosus NBRC 12807 (AB184161) and S. longispororuber NBRC 13488 (AB184440) was 99.65, 99.65, 99.64, 99.23, 99.15, 99.14 and 99.13 % respectively. Subsequent DNA-DNA hybridization experiments with the test strain and its clade members showed 55.27, 44.27, 36.86, and 15.65 % relatedness between SL-4 and its relatives S. atrovirens, S. albogriseolus, S. viridodiastaticus and S. longispororuber respectively. The genotypic and phenotypic data was analyzed to verify possibility of the isolate SL-4 representing novel member of the genus Streptomyces, for which the name S. antibioticalis is being proposed. The type strain is SL-4 (=CCM 7434=MTCC 8588). [ABSTRACT FROM AUTHOR]

Summary: Phenazine-type metabolites arise from either phenazine-1-carboxylic acid (PCA) or phenazine-1,6-dicarboxylic acid (PDC). Although the biosynthesis of PCA has been studied extensively, PDC assembly remains unclear. Esmeraldins and saphenamycin, the PDC originated products, are antimicrobial and antitumor metabolites isolated from Streptomyces antibioticus Tü 2706. Herein, the esmeraldin biosynthetic gene cluster was identified on a dispensable giant plasmid. Twenty-four putative esm genes were characterized by bioinformatics, mutagenesis, genetic complementation, and functional protein expressions. Unlike enzymes involved in PCA biosynthesis, EsmA1 and EsmA2 together decisively promoted the PDC yield. The resulting PDC underwent a series of conversions to give 6-acetylphenazine-1-carboxylic acid, saphenic acid, and saphenamycin through a unique one-carbon extension by EsmB1–B5, a keto reduction by EsmC, and an esterification by EsmD1–D3, the atypical polyketide sythases, respectively. Two transcriptional regulators, EsmT1 and EsmT2, are required for esmeraldin production. [ABSTRACT FROM AUTHOR]

Aimed to produce thermostable phosphatidylinositol (PI)-synthesizing phospholipase D (PLD), we initiated site-directed combinatorial mutagenesis followed by high-throughput screening. Previous site-directed combinatorial mutagenesis of wild-type Streptomyces PLD produced a mutant, DYR (W187D/Y191Y/Y385R) with PI-synthesizing ability. Deriving PI as a product of transphosphatidylation between phosphatidylcholine and myo-inositol, with myo-inositol in excess at high-temperature reaction conditions can increase yield due to enhanced solubility of this substrate. Thus, we improved DYR's thermostability by introduction of random mutations into selected amino acid positions having high B-factor. Screening of the libraries under restricted conditions yielded single-point mutants, specifically D40H, T291Y and R329G. Combinations of these point mutations yielded double (D40H/T291Y, D40H/R329G and T291Y/R329G) and triple (D40H/T291Y/R329G) mutants. PI synthesis at elevated temperatures pointed at D40H/T291Y as the most efficient enzyme. Circular dichroism analysis revealed D40H/T291Y to have increased melting temperature and postponed onset of thermal unfolding compared with DYR. Thermal tolerance study at 65°C confirmed D40H/T291Y's thermostability as its half-inactivation time was 8.7 min longer compared with DYR. This mutant had significantly less root-mean-square deviation change compared with DYR and showed no change in root-mean-square fluctuation when temperature shifts from 40 to 60°C, as determined by molecular dynamics analysis. Acquired different degrees of thermostability were also observed for several other DYR mutants. [ABSTRACT FROM PUBLISHER]

An oleandomycin glycosyltransferase (OleD GT) gene from Streptomyces antibioticus was functionally expressed in Escherichia coli BL21 (DE3) with various molecular chaperones. The purified recombinant OleD GT catalyzed glycosylation of various flavonoids: apigenin, chrysin, daidzein, genistein, kaempferol, luteolin, 4-methylumbelliferone, naringenin, quercetin and resveratrol with UDP-glucose. 4.6 μg OleD GT was readily immobilized onto 1 mg hybrid nanoparticles of FeO/silica/NiO on the basis of the affinity between His-tag and NiO nanoparticles with retention of 90% activity. In batch reaction, more than 90% naringenin (20 μM) was converted to its glycoside in 5 h. The immobilized OleD GT was efficiently reused for seven times whilst maintaining >60% of the residual activity in repeated glycosylation of naringenin. [ABSTRACT FROM AUTHOR]

Abstract: Tyrosinase (tyr) purified from Pseudomonas putida F6, Streptomyces antibioticus, and Agaricus bisporus (mushroom) oxidises 3 regioisomers of fluorophenol as well as 3,4-difluorophenol (3,4FP). The catalytic efficiency (k cat/K m) of tyrosinase towards any one substrate is different for each enzyme source. Oftentimes a large difference in affinity (K m), and turnover (k cat) is observed for different enzyme sources with the same substrate. The best catalytic efficiency towards a fluorinated substrate was observed for P. putida F6 tyr with 4-fluorophenol (4FP). The presence of a second fluorine on the aromatic ring (3,4FP) resulted in a decrease in the catalytic efficiency of all three enzymes compared to values for 4FP. However, the K m value for 3,4FP decreased for P. putida F6 tyr indicating a higher affinity for P. putida F6 tyr for 3,4FP compared to 4FP. Furthermore the k cat value for 3,4FP increased for mushroom tyr in comparison to the value for 4FP indicating a higher maximum turnover of 3,4FP compared to 4FP for mushroom tyr. All three sources of tyr exhibited lower catalytic efficiencies for 3-fluorophenol (3FP) and 2-fluorophenol (2FP) compared to 4FP. However, the K m value for 2FP was lower than that for 3FP for both S. antibioticus and mushroom tyr indicating a higher affinity for 2FP over 3FP. [Copyright &y& Elsevier]

Abstract: Streptomyces antibioticus tyrosinase was kinetically characterized after purification by PEG-8000/phosphate phase partitioning and ammonium sulfate fractionation using tert-butylcathechol (TBC) and dopamine. The enzyme showed an optimal pH at 6.5 and a K M of 1.2mM and 8.4mM, respectively. The effect of several modulators was studied on this Gram-positive bacterium tyrosinase. In addition, previously undescribed characterization of apparent inhibition and activation of a bacterial tyrosinase using different kinds of cyclodextrins was carried out. When a hydrophobic substrate of S. antibioticus tyrosinase, in this case, tert-butylcatechol was used, a marked substrate sequestrant effect was observed in the presence of hydroxypropyl-β-cyclodextrins (OH-β-CDs) and gamma cyclodextrins (γ-CDs). This sequestrant effect was due to the complexation of TBC into the CD cavity. Moreover, the effect of some hydrophobic inhibitors in the presence of OH-β-CDs and γ-CDs was studied using dopamine, a hydrophilic substrate of S. antibioticus tyrosinase. Increasing concentrations of CDs in the presence of inhibitors like hexestrol or hinokitiol, were able to reactivate the inhibited enzyme to reach the non-inhibited level, as a result of the complexation of these inhibitory compounds in the hydrophobic core of the CDs. This dual effect of CDs as apparent inhibitor and activator has never before described being observed in bacteria. [Copyright &y& Elsevier]

Abstract: Streptomyces antibioticus tyrosinase was purified using PEG-8000/phosphate phase partitioning and ammonium sulfate fractionation between 0 and 60%, rendering a clear and stable enzyme compared with the black one that is usually obtained when only ammonium sulphate fractionation is used, and with a 93% of recovery. This enzyme shows both monophenolase (also termed cresolase) and diphenolase (catecholase) activities, confirming that it is a real tyrosinase. The monophenolase activity was characterized by a lag period, whose duration depends on the substrate concentration, the pH, and the presence of catalytic amounts of o-diphenol. The enzyme showed substrate inhibition for p-cresol, with a K M of 0.97mM and a K si of 23mM. By increasing the concentration of o-diphenols, it was possible to evaluate the enzyme activation constant, K act, which showed a value of 0.2μM. The diphenolase activity was kinetically characterized with 4-methylcathechol, showed an optimal pH at 6.5 and a K M of 1.3mM. [Copyright &y& Elsevier]

Abstract: A process of programmed cell death taking place late in the aerial mycelium was previously reported in surface cultures of Streptomyces antibioticus ATCC11891. In this study, we present evidence for the occurrence of a similar process taking place early in the vegetative mycelium of surface cultures of the same strain. Several indicators, such as cell wall and membrane disruption, DNA degradation and release of the cytoplasmic content into the exocellular medium, support the existence of active, highly regulated cell suicide involving specific enzymes. Calcium-dependent proteolytic activation of a precursor of nucleases and the nucleolytic formation of a ladder of chromosomal bands are conspicuous events associated with the initiation of the death process. [Copyright &y& Elsevier]

The four furanones (butenolides) 1–4, which had been isolated from the fermentation broth of Streptomyces antibioticus TU¨ 99 and in preliminary tests had been shown to be biologically active, were synthesized by reaction of the readily available furanones 9 or 16 with 2-methylpropanal or 2-methylbutanal. In addition, a series of analogs was prepared in a similar way from 16 using different aldehydes. The hitherto unknown absolute configurations of the natural products 1–4 as well as those of all the analogs prepared were determined with Mosher's NMR method and/or X-ray crystallography. Some of the compounds synthesized proved to be active in the quorum sensing system of Chromobacterium violaceum. [Copyright &y& Elsevier]

Streptomyces antibioticus Tü 6040 is the producer of simocyclinones, which belong to a novel family of angucyclinone antibiotics some of which show antitumor activities. Growth and antibiotic production is dependent on the medium composition, especially on the carbon and nitrogen source, and on the fermentation conditions. The best results with respect to antibiotic productivity were achieved using a chemically defined medium with glycerol and L-lysine as carbon and nitrogen source, respectively, in an airlift fermenter with minimised shear stress at low gas flow rates withour oxygen limitation. These conditions led to a homogeneous formation of pellets of 1–2 mm in diameter and guaranteed reproducible product yields of the main compound, simocyclinone D8, in the range of 300 mg/l. [ABSTRACT FROM AUTHOR]

Acetyl-CoA carboxylase (ACC) is crucial for polyketides biosynthesis and acts as an essential metabolic checkpoint. It is also an attractive drug target against obesity, cancer, microbial infections, and diabetes. However, the lack of knowledge, particularly sequence-structure function relationship to narrate ligand-enzyme binding, has hindered the progress of ACC-specific therapeutics and unnatural "natural" polyketides. Structural characterization of such enzymes will boost the opportunity to understand the substrate binding, designing new inhibitors and information regarding the molecular rules which control the substrate specificity of ACCs. To understand the substrate specificity, we determined the crystal structure of AccB (Carboxyl-transferase, CT) from Streptomyces antibioticus with a resolution of 2.3 Å and molecular modeling approaches were employed to unveil the molecular mechanism of acetyl-CoA recognition and processing. The CT domain of S. antibioticus shares a similar structural organization with the previous structures and the two steps reaction was confirmed by enzymatic assay. Furthermore, to reveal the key hotspots required for the substrate recognition and processing, in silico mutagenesis validated only three key residues (V223, Q346, and Q514) that help in the fixation of the substrate. Moreover, we also presented atomic level knowledge on the mechanism of the substrate binding, which unveiled the terminal loop (500-514) function as an opening and closing switch and pushes the substrate inside the cavity for stable binding. A significant decline in the hydrogen bonding half-life was observed upon the alanine substitution. Consequently, the presented structural data highlighted the potential key interacting residues for substrate recognition and will also help to re-design ACCs active site for proficient substrate specificity to produce diverse polyketides., (Copyright © 2022 Elsevier Ltd. All rights reserved.)