Search

Proteins MiaA and MiaB catalyze sequential isopentenylation and methylthiolation, respectively, of adenosine residue in 37th position of tRNA XXA . The mia mutations were recently shown by us to affect secondary metabolism and morphology of Streptomyces. However, it remained unknown as to whether both or one of the aforementioned modifications is critical for colony development and antibiotic production. Here, we addressed this issue through analysis of Streptomyces albus J1074 strains carrying double miaAmiaB knockout or extra copy of miaB gene. The double mutant differed from wild-type and miaA-minus strains in severity of morphological defects, growth dynamics, and secondary metabolism. Introduction of extra copy of miaB gene into miaA mutant restored aerial mycelium formation to the latter on certain solid media. Hence, miaB gene might be involved in tRNA thiomethylation in the absence of miaA; either MiaA- or MiaB-mediated modification appears to be enough to support normal metabolic and morphological processes in Streptomyces.

Members of actinobacterial genus Streptomyces possess a sophisticated life cycle and are the deepest source of bioactive secondary metabolites. Although morphogenesis and secondary metabolism are subject to transcriptional co-regulation, streptomycetes employ an additional mechanism to initiate the aforementioned processes. This mechanism is based on delayed translation of rare leucyl codon UUA by the only cognate tRNA Leu (encoded by bldA). The bldA-based genetic switch is an extensively documented example of translational regulation in Streptomyces. Yet, after five decades since the discovery of bldA, factors that shape its function and peculiar conditionality remained elusive. Here we address the hypothesis that post-transcriptional tRNA modifications play a role in tRNA-based mechanisms of translational control in Streptomyces. Particularly, we studied two Streptomyces albus J1074 genes, XNR_1074 (miaA) and XNR_1078 (miaB), encoding tRNA (adenosine(37)-N6)-dimethylallyltransferase and tRNA (N6-isopentenyl adenosine(37)-C2)-methylthiotransferase respectively. These enzymes produce, in a sequential manner, a hypermodified ms UAA (encoded by bldA). The bldA-based genetic switch is an extensively documented example of translational regulation in Streptomyces. Yet, after five decades since the discovery of bldA, factors that shape its function and peculiar conditionality remained elusive. Here we address the hypothesis that post-transcriptional tRNA modifications play a role in tRNA-based mechanisms of translational control in Streptomyces. Particularly, we studied two Streptomyces albus J1074 genes, XNR_1074 (miaA) and XNR_1078 (miaB), encoding tRNA (adenosine(37)-N6)-dimethylallyltransferase and tRNA (N6-isopentenyl adenosine(37)-C2)-methylthiotransferase respectively. These enzymes produce, in a sequential manner, a hypermodified ms 2 i 6 A37 residue in most of the A36-A37-containing tRNAs. We show that miaB and especially miaA null mutant of S. albus possess altered morphogenesis and secondary metabolism. We provide genetic evidence that miaA deficiency impacts translational level of gene expression, most likely through impaired decoding of codons UXX and UUA in particular., (© 2019 John Wiley & Sons Ltd.)

The noursamycins A-F are chlorinated cyclic hexapeptides, which were identified and isolated from the strain Streptomyces noursei NTR-SR4 overexpressing a LuxR-like transcriptional activator. The molecules were structurally characterized by mass spectrometric analyses and 1D and 2D NMR spectroscopic techniques. The enzymatic machinery involved in the biosynthesis of these peptides is represented by a modular nonribosomal peptide synthetase (NRPS), and the corresponding gene cluster was identified in the S. noursei genome. The latter suggested the biosynthetic pathway for the noursamycins. Spectral networking analysis uncovered noursamycin derivatives that were later found to result from a relaxed substrate specificity of the A 3 and A 4 adenylation domains of the NRPS. The stereochemistry of the amino acid constituents of the noursamycins was resolved by chemical derivatization, subsequent enantiomer analytics by GC-EIMS, and in silico data analyses. Noursamycins A and B exhibited antibacterial activity against Gram-positive and Gram-negative bacteria, while no apparent cytotoxicity was observed.

Actinomycin peptide synthetase genes constitute two oppositely oriented transcriptional units, acmADR, and acmBC, separated by a non-coding intergenic region. Gene constructs of the intergenic region together with its adjoining gene acmA or acmB from the actinomycin biosynthetic gene cluster of Streptomyces chrysomallus were transferred into Streptomyces lividans TK64. Each construct expressed the respective synthetase indicating divergent promoters. Primer extension revealed for both directions -10 and -35 boxes similar to σ 70 -dependent promoters from Streptomyces and E. coli. No conspicuous regulatory sequences were detected. Accordingly, S. chrysomallus-grown in glucose-containing medium-produced the peptide synthetases AcmA and AcmB/C as well as actinomycin during logarithmic growth phase. Alignments with the corresponding intergenic region of the actinomycin biosynthetic gene cluster in Streptomyces antibioticus identified analogous -10 and -35 boxes of σ 70 consensus sequence. However, in S. antibioticus-cultivated in the same conditions-AcmA and AcmB/C were at maximum activity in late log phase and actinomycin formation peaked in stationary phase. The different patterns of formation of actinomycin and its peptide synthetases encoded by the highly homologous actinomycin biosynthetic gene clusters in S. chrysomallus and S. antibioticus suggest strain-specific control of biosynthesis in agreement with absence of pathway-specific regulatory genes., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The necrotrophic fungus Heterobasidion spp. is the causal agent of 'annosum root rot' of Norway spruce. In the presence of the rhizosphere bacterium Streptomyces AcH 505, enhanced colonization of Norway spruce roots with Heterobasidion abietinum 331 has previously been observed. By analyzing dual cultures of H. abietinum 331 and Streptomyces AcH 505 with HPLC, a fungal metabolite was identified that was increased in the presence of Streptomyces AcH 505. Likewise, challenge of H. abietum 331 with common antifungals produced by soil streptomycetes rendered the same effect. The structure of the compound, 5-formylsalicylic acid (5-FSA), was elucidated by HPLC-HR-ESI-Orbitrap-mass spectrometry and NMR spectroscopy. Based on in vivo measurements of maximum photosystem II efficiency of Norway spruce seedlings, 5-FSA did not influence plant vitality. However, when challenged with H. abietinum 331, ergosterol amounts in infected roots increased significantly for 5-FSA pre-treated seedlings. The severity of the infection was comparable to that observed in the presence of Streptomyces AcH 505. 5-FSA is a structural analogue of salicylic acid, an important signalling molecule active in plant defence. Thus, the expression of two defence-response related marker genes (PR1, Hel) was analysed in 5-FSA treated Arabidopsis thaliana seedlings by Northern blot analysis. The transcription of both marker genes was altered, indicating that 5-FSA is perceived by Arabidopsis in a similar manner to salicylic acid and is able to interfere with Arabidopsis defence signalling. The role of 5-FSA as a potential virulence factor of H. abietinum 331 in the presence of Streptomyces AcH 505 is discussed.

Lanthipeptides are ribosomally synthesized and post-translationally modified microbial secondary metabolites. Here, we report the identification and isolation of streptocollin from Streptomyces collinus Tü 365, a new member of class IV lanthipeptides. Insertion of the constitutive ermE* promoter upstream of the lanthipeptide synthetase gene stcL resulted in peptide production. The streptocollin gene cluster was heterologously expressed in S. coelicolor M1146 and M1152 with 3.5- and 5.5-fold increased yields, respectively. The structure and ring topology of streptocollin were determined by high resolution MS/MS analysis. Streptocollin contains four macrocyclic rings, with one lanthionine and three methyllanthionine residues. To the best of our knowledge, this is the first report on the isolation of a class IV lanthipeptide in preparative amounts, and on the successful heterologous expression of a class IV lanthipeptide gene cluster., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The antibiotic strepturidin (1) was isolated from the microorganism Streptomyces albus DSM 40763, and its structure elucidated by spectroscopic methods and chemical degradation studies. The determination of the relative and absolute stereocenters was partially achieved using chiral GC/EI-MS analysis and microderivatization by acetal ring formation and subsequent 2D-NMR analysis of key (1)H,(1)H-NOESY NMR correlations and extraction of (1)H,(13)C coupling constants from (1)H,(13)C-HMBC NMR spectra. Based on these results, a biosynthesis model was proposed.

Skyllamycin is a non-ribosomally synthesized cyclic depsipeptide from Streptomyces sp. Acta 2897 that inhibits PDGF-signaling. The peptide scaffold contains an N-terminal cinnamoyl moiety, a β-methylation of aspartic acid, three β-hydroxylated amino acids and one rarely occurring α-hydroxy glycine. With the exception of α-hydroxy glycine, the stereochemistry of the amino acids was assigned by comparison to synthetic reference amino acids applying chiral GC-MS and Marfey-HPLC analysis. The stereochemistry of α-hydroxy glycine, which is unstable under basic and acidic conditions, was determined by conformational analysis, employing a combination of data from NOESY-NMR spectroscopy, simulated annealing and free MD simulations. The simulation procedures were applied for both R- and S-configured α-hydroxy glycine of the skyllamycin structure and compared to the NOESY data. Both methods, simulated annealing and free MD simulations independently support S-configured α-hydroxy glycine thus enabling the assignment of all stereocenters in the structure of skyllamycin and devising the role of two-component flavin dependent monooxygenase (Sky39) as S-selective., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

New isolates of Streptomyces champavatii were isolated from marine sediments of the Gotland Deep (Baltic Sea), from the Urania Basin (Eastern Mediterranean), and from the Kiel Bight (Baltic Sea). The isolates produced several oligopeptidic secondary metabolites, including the new octapeptide champacyclin (1a) present in all three strains. Herein, we report on the isolation, structure elucidation and determination of the absolute stereochemistry of this isoleucine/leucine (Ile/Leu = Xle) rich cyclic octapeptide champacyclin (1a). As 2D nuclear magnetic resonance (NMR) spectroscopy could not fully resolve the structure of (1a), additional information on sequence and configuration of stereocenters were obtained by a combination of multi stage mass spectrometry (MSn) studies, amino acid analysis, partial hydrolysis and subsequent enantiomer analytics with gas chromatography positive chmical ionization/electron impact mass spectrometry (GC-PCI/EI-MS) supported by comparison to reference dipeptides. Proof of the head-to-tail cyclization of (1a) was accomplished by solid phase peptide synthesis (SPPS) compared to an alternatively side chain cyclized derivative (2). Champacyclin (1a) is likely synthesized by a non-ribosomal peptide synthetase (NRPS), because of its high content of (D)-amino acids. The compound (1a) showed antimicrobial activity against the phytopathogen Erwinia amylovora causing the fire blight disease of certain plants.

A novel angucycline-type antibiotic, warkmycin, was isolated from the culture filtrate of Streptomyces strain Acta 2930. Its chemical structure was elucidated by HR-MS, one-dimensional and 2D NMR experiments. The compound inhibits the growth of Gram-positive bacteria and shows a strong antiproliferative activity against mouse fibroblast cell line NIH-3T3 and human cancer cell lines HepG2 and HT29.

Langkocyclines A1-A3 and B1 and B2, five new angucycline antibiotics produced by Streptomyces sp. Acta 3034, were detected in the course of our HPLC-diode array screening. The producing strain was isolated from the rhizospheric soil of a Clitorea sp. collected from Burau Bay, Langkawi, Malaysia, and was characterized by morphological, physiological and chemotaxonomic features in addition to 16S ribosomal RNA gene sequence information. Strain Acta 3034 is closely related to Streptomyces psammoticus NBRC 13971(T) and Streptomyces lanatus NBRC 12787(T). Langkocyclines consist of an angular tetracyclic benz[a]anthracene skeleton and hydrolyzable O-glycosidic sugar moieties. The yellow-colored A-type langkocyclines differ in their aglycon from the blue-lilac-colored B-type langkocyclines. The A-type langkocycline aglycon is identical to that of aquayamycin and urdamycin A. The chemical structures of the langkocyclines were elucidated by HR-MS, 1D and 2D NMR experiments. They are biologically active against Gram-positive bacteria and exhibit a moderate antiproliferative activity against various human tumor cell lines.

Rhizosphere-associated Streptomyces sp. AcH 505 (AcH 505) promotes infection of Norway spruce (Picea abies) with the pathogenic fungus Heterobasidion abietinum 331, while Streptomyces sp. GB 4-2 (GB 4-2) enhances spruce defense against the fungus. To identify whether these bacteria influence the availability of the fungal phytotoxin fomannoxin and hence spruce infection, we analyzed the fomannoxin yield in H. abietinum 331-AcH 505 dual cultures. Further, the fate of fomannoxin was studied by adding the compound to cultures of AcH 505, GB 4-2 and nine other soil streptomycetes. Culture filtrates were extracted with ethyl acetate and analyzed by HPLC. Structures of novel compounds were elucidated by HPLC-HR-ESI-Orbitrap-MS and NMR spectroscopy. Phytotoxicity of the compounds was determined by in vivo measurement of maximum photosystem II efficiency of Arabidopsis thaliana seedlings. The amount of fomannoxin in H. abietinum 331-AcH 505 dual cultures was reduced compared to axenic fungus cultures and fungus-plant dual cultures. Following addition of fomannoxin to AcH 505 cultures, the compound disappeared and three novel fomannoxin derivatives without phytotoxic activity were detected. Another novel compound, fomannoxin amide, was discovered following fomannoxin addition to GB 4-2 cultures. Nine other streptomycetes converted fomannoxin into fomannoxin acid or fomannoxin amide. Both compounds exhibit the same phytotoxicity as fomannoxin. We, thus, conclude that the streptomycete-mediated modulation of spruce infection with H. abietinum 331 does not depend on the availability of fomannoxin. We further add evidence to the observation that the lipophilic side chain of fomannoxin is an important structural element for its phytotoxicity.

Elaiomycins K and L, two new azoxy-type antibiotics, were detected by HPLC-diode array screening in the culture filtrate extract of Streptomyces sp. Tü 6399. The structures were determined by high-resolution MS and 2-dimensional (1)H and (13)C correlated NMR spectroscopy including (15)N-NMR experiments and established these compounds as new members of the elaiomycin family. Both metabolites show a weak antibacterial activity against Bacillus subtilis and Staphylococcus lentus as well as against the phytophathogenic strain Xanthomonas campestris.

Streptomycetes were isolated out of a soil sample taken from the rhizosphere of a spruce stand and screened by HPLC-diode array analysis for the production of secondary metabolites. This led to the detection of silvalactam, a novel 24-membered macrolactam antibiotic in extracts of Streptomyces strain Tü 6392. The structure was determined by MS and NMR spectroscopy experiments. Silvalactam shows a potent antiproliferative activity against various cancerous and non-cancerous cell lines.

A new 32-membered macrolactone antibiotic, named langkolide, was isolated from the mycelium of Streptomyces sp. Acta 3062. The langkolide structure was determined by HR-MS and 1D and 2D NMR as a 32-membered macrolactone connected from an overhanging polyketide tail to a naphthoquinone unit mediated by two carbohydrate moieties. The producing strain was isolated from a rhizosphere soil of Clitorea sp. collected at Burau Bay, Langkawi, Malaysia, and was characterized by its morphological and chemotaxonomic features in addition to its 16S rRNA gene sequence. It was identified as a member of the Streptomyces galbus clade. Langkolide exhibited various bioactivities including antimicrobial and antiproliferative activities. Furthermore, langkolide inhibited human recombinant phosphodiesterase 4 with an IC(50) value of 0.48 μM.

Lantibiotics are a large group of ribosomally synthesized peptides post-translationally modified to incorporate the amino acid lanthionine. They are classified, according to their biosynthetic pathway and bioactivity, into three major subtypes. Of Actinomycetes type III lantibiotics, only four peptides (SapB, SapT, LabA1, and LabA2) have been described and structurally characterized, although homologous gene clusters are abundant in other Actinomycetes. All these gene clusters share a similar architecture with a characteristic Ser/Ser/Cys motif in precursor peptides, which has previously been suggested to act as a precursor for lanthionine (SapB) and labionin (LabA2) rings. Mass spectrometry screening led to the discovery and characterization of three new representatives of type III lantibiotics: Avermipeptin (Avi), Erythreapeptin (Ery), and Griseopeptin (Gri) from Streptomyces avermitilis DSM 46492, Saccharopolyspora erythraea NRRL 2338, and Streptomyces griseus DSM 40236, respectively. Apart from the assignment of these peptides to their corresponding gene clusters, additional investigations on Avi, Ery and Gri peptides indicate stepwise leader processing by putative aminopeptidase-like protease(s), thus yielding mixtures of differently N-terminal-processed lantibiotic peptides. Similar peptide processing was observed for a heterologously expressed eryth biosynthetic gene cluster expressed in a Streptomyces host system. Remarkably, all isolates of the new type III lantibiotics contain both the amino acids lanthionine and labionin, thus implying dual-mode cyclase activity of the processing lyase-kinase-cyclase enzymes. These findings have implications for the structures and maturation of other type III lantibiotics from Actinomycetes., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Three new 22-membered macrolactone antibiotics, atacamycins A-C, were produced by Streptomyces sp. C38, a strain isolated from a hyper-arid soil collected from the Atacama Desert in the north of Chile. The metabolites were discovered in our HPLC-diode array screening and isolated from the mycelium by extraction and chromatographic purification steps. The structures were determined by mass spectrometry and NMR experiments. Atacamycins A, B and C exhibited moderate inhibitory activities against the enzyme phosphodiesterase (PDE-4B2), whereas atacamycin A showed a moderate antiproliferative activity against adeno carcinoma and breast carcinoma cells.

Two novel alkyhydrazides, elaiomycins B and C, together with the azoxy antibiotic elaiomycin were isolated from Streptomyces sp. BK 190. The structures were established by 1D- and 2D-NMR spectroscopy including (15)N NMR studies and high-resolution orbitrap-ESI-mass spectrometry.

Piceamycin, a new macrolactam polyketide antibiotic, was detected by HPLC-diode array screening in extracts of Streptomyces sp. GB 4-2, which was isolated from the mycorrhizosphere of Norway spruce. The structure of piceamycin was determined by mass spectrometry and NMR experiments. It showed inhibitory activity against Gram-positive bacteria, selected human tumor cell lines and protein tyrosine phosphatase 1B.

Gombapyrones A-D, new members of the alpha-pyrone family of secondary metabolites, were produced by Streptomyces griseoruber Acta 3662, which was isolated from bamboo tree rhizosphere. The strain was characterized by its morphological and chemotaxonomical features and by 16S rDNA sequencing as S. griseobuber. The gombapyrone structures were determined by mass spectrometry and by NMR experiments, and were found to have an inhibitory activity against protein tyrosine phosphatase 1B and glycogen synthase kinase 3beta.

Caboxamycin, a new benzoxazole antibiotic, was detected by HPLC-diode array screening in extracts of the marine strain Streptomyces sp. NTK 937, which was isolated from deep-sea sediment collected in the Canary Basin. The structure of caboxamycin was determined by mass spectrometry, NMR experiments and X-ray analysis. It showed inhibitory activity against Gram-positive bacteria, selected human tumor cell lines and the enzyme phosphodiesterase.

Albidopyrone, a new alpha-pyrone-containing secondary metabolite, was produced by Streptomyces sp. NTK 227, a strain isolated from Atlantic Ocean sediment and found to be a member of the Streptomyces albidoflavus 16S rRNA gene clade. The structure of the compound was determined by MS and NMR spectroscopy, and found to have a moderate inhibitory activity against protein-tyrosin phosphatase B.

The new benzoxazole derivative nataxazole was isolated from Streptomyces sp. (strain Tü 6176). Nataxazole is related in structure to the potent antitumor compounds UK-1 and AJI9561 and showed similar strong growth inhibitory activity against various human tumor cell lines.

Mycorrhiza helper bacterium Streptomyces strain AcH 505 stimulates ectomycorrhiza formation between spruce and fly agaric by supporting fungal growth whereas growth of pathogenic fungi is suppressed. A fungal growth promoting substance was isolated and the chemical structure elucidated by mass spectrometry and NMR spectroscopy. The absolute configuration of the novel fungal growth promoting compound auxofuran (1) was deduced from NMR data with the help of Mosher esters.

Three new members of the fluostatin family, fluostatins C-E, were discovered in a culture filtrate extract of strain Acta 1383 during an HPLC screening program. The producing strain belongs to the genus Streptomyces and is closely related to type strains classified in the Streptomyces lavendulae 16S rRNA subclade. Fluostatins are named by their characteristic fluorenone chromophore. Fluostatin C shows moderate activity against selected human tumor cell lines.

The antibiotic compound pyrroindomycin B contains an indole ring chlorinated in the 5 position. The indole ring is probably derived from tryptophan, and thus primers derived from conserved regions of tryptophan halogenases were used to amplify and clone a DNA fragment that was then used to isolate a tryptophan 5-halogenase gene (pyrH) from a cosmid library of the pyrroindomycin producer Streptomyces rugosporus LL-42D005. A gene disruption mutant in the tryptophan 5-halogenase gene no longer produced pyrroindomycin B, but still produced pyrroindomycin A, the nonhalogenated derivative. The halogenase gene could be overexpressed in Pseudomonas fluorescens BL915 DeltaORF1 and was purified to homogeneity by immobilized metal chelate ion affinity chromatography. Chlorinating and brominating activities with tryptophan as a substrate were detected in cell-free extracts and for the purified enzyme.

New lipopeptide antibiotics, colourless arylomycins A series and yellow arylomycins B series were detected in the culture filtrate and mycelium extracts of Streptomyces sp. Tü 6075 by HPLC-diode-array and HPLC-electrospray-mass-spectrometry screening. Arylomycins are a family of lipohexapeptide antibiotics, which represent the first examples of biaryl-bridged lipopeptides. They show antibiotic activities against Gram-positive bacteria.

Actinobacteria are among the most prolific sources of medically and agriculturally important compounds, derived from their biosynthetic gene clusters (BGCs) for specialized (secondary) pathways of metabolism. Genomics witnesses that the majority of actinobacterial BGCs are silent, most likely due to their low or zero transcription. Much effort is put into the search for approaches towards activation of silent BGCs, as this is believed to revitalize the discovery of novel natural products. We hypothesized that the global transcriptional factor AdpA, due to its highly degenerate operator sequence, could be used to upregulate the expression of silent BGCs. Using Streptomyces cyanogenus S136 as a test case, we showed that plasmids expressing either full-length adpA or its DNA-binding domain led to significant changes in the metabolome. These were evident as changes in the accumulation of colored compounds, bioactivity, as well as the emergence of a new pattern of secondary metabolites as revealed by HPLC-ESI-mass spectrometry. We further focused on the most abundant secondary metabolite and identified it as the polyene antibiotic lucensomycin. Finally, we uncovered the entire gene cluster for lucensomycin biosynthesis (lcm), that remained elusive for five decades until now, and outlined an evidence-based scenario for its adpA-mediated activation. [ABSTRACT FROM AUTHOR]

Summary: Members of actinobacterial genus Streptomyces possess a sophisticated life cycle and are the deepest source of bioactive secondary metabolites. Although morphogenesis and secondary metabolism are subject to transcriptional co‐regulation, streptomycetes employ an additional mechanism to initiate the aforementioned processes. This mechanism is based on delayed translation of rare leucyl codon UUA by the only cognate tRNALeuUAA (encoded by bldA). The bldA–based genetic switch is an extensively documented example of translational regulation in Streptomyces. Yet, after five decades since the discovery of bldA, factors that shape its function and peculiar conditionality remained elusive. Here we address the hypothesis that post‐transcriptional tRNA modifications play a role in tRNA‐based mechanisms of translational control in Streptomyces. Particularly, we studied two Streptomyces albus J1074 genes, XNR_1074 (miaA) and XNR_1078 (miaB), encoding tRNA (adenosine(37)‐N6)‐dimethylallyltransferase and tRNA (N6‐isopentenyl adenosine(37)‐C2)‐methylthiotransferase respectively. These enzymes produce, in a sequential manner, a hypermodified ms2i6A37 residue in most of the A36‐A37‐containing tRNAs. We show that miaB and especially miaA null mutant of S. albus possess altered morphogenesis and secondary metabolism. We provide genetic evidence that miaA deficiency impacts translational level of gene expression, most likely through impaired decoding of codons UXX and UUA in particular. [ABSTRACT FROM AUTHOR]

Nonribosomal peptides (NRPs), a large family of natural products, possess numerous pharmaceutically significant bioactivities. However, many native microbial producers of NRPs are not cultivable or have low production yields making mass production infeasible. The recombinant production of natural products in a surrogate host has emerged as a strategy to overcome these limitations. De novo recombinant production of the NRP antibiotic valinomycin in an engineered Escherichia coli host strain was established with the necessary biosynthetic pathway constituents from Streptomyces tsusimaensis. In the present study, the initially modest valinomycin yields could be significantly increased from 0.3 up to 2.4 mg L by switching from a batch to an enzyme-based fed-batch mode in shake flasks. A subsequent design of experiment-driven optimization of parallel fed-batch cultivations in 24-well plates with online monitoring of dissolved oxygen and pH led to valinomycin yields up to 6.4 mg L. Finally, repeated glucose polymer feeding to enzyme-based high cell density cultivations in shake flasks resulted in cell densities of OD >50 and a valinomycin titer of appr. 10 mg L. This represents a 33-fold improvement compared to the initial batch cultivations and is the highest concentration of a nonribosomal peptide which has been produced in E. coli without feeding of specific precursors so far to our knowledge. Also, such a small-scale optimization under fed-batch conditions may be generally applicable for the development and scale-up of natural product production processes in E. coli. [ABSTRACT FROM AUTHOR]

The actinomycin biosynthetic gene cluster of Streptomyces chrysomallus harbors two paralogous genes. acml and acmL, encoding methyltransferases. To unveil their suspected role in the formation of 3-hydroxy-4-methyl-anthranilic acid (4-MHA), the building block of the actinomycin chromophore, each gene was expressed in Escherichia coli. Testing the resulting ∼40 kDa His6-tagged proteins with compounds of biogenetic relevance as substrates and S-adenosyl-L-methionine revealed that each exclusively methylated 3-hydroxykynurenine (3-HK) with formation of 3-hydroxy-4-methylkynurenine (4-MHK) identified by its in vitro conversion to 4-MHA with hydroxykynureninase. Acml and AcmL methylate also hydroxyphenyl-amino propanoic acids such as p-tyrosine, m-tyrosine, or 3, 4-dihydroxy-L-phenylalanine (DOPA) but at a lower rate than 3-HK. The presence of the α-amino group was necessary for substrate recognition. Phenolic acids with shorter chains such as 4-hydoxyphenyl-c-glycine (HPG), 3-hydroxybenzoic acid (3-HB), or 3-hydroxyanthranilic acid (3-HA) gave no product. Both enzymes were stereospecific for the optical configuration at α-C with unprecedented antipodal selectivity for the D-enantiomer of 3-HK and the L-enantiomer of p-tyrosine or m-tyrosine. Acml and AcmL show sequence similarity to various C-and O-methyltransferases from bacteria. Phylogenetic analysis places them into the clade of C-methyltransferases comprising among others orthologues involved us 4-MHA formation of other biosynthesis systems and methyltransferases putatively involved in the C-methylation of tyrosine. Remarkably, computational remodelling of Acml and AcmL structures revealed significant similarity with the 3-D structures of type 1 O-methyltransferases from plants such as caffeic acid O-methyltransferase (COMT) and other phenylpropanoid methyltransferases. The relevance of 3-HK or 3-HA methylation in the actinomycin biosynthesis pathways of different actinomycetes is discussed. [ABSTRACT FROM AUTHOR]

Background: A bacterial artificial chromosomal library of Planobispora rosea, a genetically intractable actinomycete strain, was constructed using Escherichia coli-Streptomyces artificial chromosome (ESAC) and screened for the presence of genes known to be involved in the biosynthesis of antibiotics. Results: One clone with a 40 kb insert showed antimicrobial activity against Gram positive bacteria. Insert sequence analysis and subcloning experiments revealed that the bioactivity was due to a 3.5 kb DNA fragment containing two open reading frames. These orfs encode two proteins with high similarity to a putative membrane protein of Streptomyces coelicolor and to the nogalamycin resistance protein SnorO of Streptomyces nogalater, respectively. The role of these two Orfs is unknown in Planobispora. Disruption and complementation experiments revealed that both proteins are necessary for the antibacterial activity and chemical analysis demonstrated that the antibiotic activity was due to thiostrepton, antibiotic used as recombinant clone selection marker. Conclusion: Two Planobispora rosea orfs are responsible for increasing intracellular amounts and storage of thiostrepton in Streptomyces lividans. [ABSTRACT FROM AUTHOR]

The cyclic depsipeptide skyllamycin A is a potent inhibitor of the platelet-derived growth factor (PDGF) signaling pathway by inhibiting binding of homodimeric PDGF BB to the PDGF -receptor. Its structure contains a cinnamoyl side chain and shows a high amount of/3-hydroxylated amino acids as well as an unusual a-hydroxyglycine moiety as a rare structural modification. The skyllamycin biosynthetic gene cluster was cloned and sequenced from Streptomyces sp. Acta 2897. Its analysis revealed the presence of open reading frames encoding proteins for fatty acid precursor biosynthesis, non-ribosomal peptide synthetases, regulators, and transporters along with other modifying enzymes. Specific in-frame mutagenesis of these tailoring enzymes resulted in the production of novel skyllamycin derivatives revealing that j3-hydroxy groups in skyllamycin A are introduced by a promiscuous cytochrome P450 monooxygenase, whereas a two-component flavin-dependent monooxygenase is involved in a-hydroxylation. [ABSTRACT FROM AUTHOR]