[Show abstract][Hide abstract] ABSTRACT: Expression plasmids carrying different deoxysugar biosynthetic gene cassettes and the gene encoding a substrate-flexible glycosyltransferase DesVII were constructed and introduced into Streptomyces venezuelae YJ003 mutant strain bearing a deletion of a desosamine biosynthetic (des) gene cluster. The resulting recombinants produced macrolide antibiotic YC-17 analogues possessing unnatural sugars replacing native D-desosamine. These metabolites were isolated and further purified using chromatographic techniques and their structures were determined as D-quinovosyl-10-deoxymethynolide, L-rhamnosyl-10-deoxymethynolide, L-olivosyl-10-deoxymethynolide, and D-boivinosyl-10-deoxymethynolide on the basis of 1D and 2D NMR and MS analyses and the stereochemistry of sugars was confirmed using coupling constant values and NOE correlations. Their antibacterial activities were evaluated in vitro against erythromycin-susceptible and -resistant Enterococcus faecium and Staphylococcus aureus. Substitution with L-rhamnose displayed better antibacterial activity than parent compound YC-17 containing native sugar D-desosamine. The present study on relationships between chemical structures and antibacterial activities could be useful in generation of novel advanced antibiotics utilizing combinatorial biosynthesis approach.
Journal of Biotechnology 06/2013; · 3.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Methionine sulfoxide reductase (Msr) is a repair enzyme that reduces oxidized methionine to methionine. The Msr enzyme is divided into MsrA and MsrB, which reduce the S and R configurations of the substrate, respectively. In some pathogenic bacteria MsrA and MsrB exist in a fusion-protein form, MsrAB. In this study, the recombinant MsrA part of MsrAB from Haemophilus influenzae (HIMsrA) was overexpressed, purified and crystallized using the hanging-drop vapour-diffusion method. A diffraction data set was collected to 1.6 Å resolution. The crystal of HIMsrA was found to belong to space group P4(1)2(1)2, with unit-cell parameters a = b = 57.29, c = 186.28 Å, a calculated Matthews coefficient of 1.82 Å(3) Da(-1) and two molecules per asymmetric unit. A preliminary solution was determined by molecular replacement. Refinement of the structure is currently in progress.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 05/2012; 68(Pt 5):557-9. · 0.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Three new flavonoids, 5,6,7,8,3′,4′-hexamethoxyhomoflavone (1), 5,6,7,8,4′-pentamethoxyhomoflavone (2) and 3,6,7,8,2′,5′-hexamethoxyflavone (3) were isolated from the peels of mature fruits of Citrus unshiuMarcow (Rutaceae), together with the three known compounds 6,7,8,4′-tetramethoxyflavone (=6,7,8-trimethoxy-2-(4-methoxyphenyl)-4H-1-benzopyran-4-one), 3,5,7,8,2′,5′-hexamethoxyflavone (=2-(2,5-dimethoxyphenyl)-3,5,7,8-tetramethoxy-4H-1-benzopyran-4-one), and scopoletin (=7-hydroxy-6-methoxy-2H-1-benzopyran-2-one) of which the former two have never been isolated from natural resources, although they have been reported as synthetic compounds. The structures of 1–3 were elucidated on the basis of spectroscopic evidence, including 1D- and 2D-NMR analysis.
[Show abstract][Hide abstract] ABSTRACT: Metabolic engineering of plant-specific phenylpropanoid biosynthesis has attracted an increasing amount of attention recently, owing to the vast potential of flavonoids as nutraceuticals and pharmaceuticals. Recently, we have developed a recombinant Streptomyces venezuelae as a heterologous host for the production of flavonoids. In this study, we successfully improved flavonoid production by expressing two sets of genes predicted to be involved in malonate assimilation. The introduction of matB and matC encoding for malonyl-CoA synthetase and the putative dicarboxylate carrier protein, respectively, from Streptomyces coelicolor into the recombinant S. venezuelae strains expressing flavanone and flavone biosynthetic genes resulted in enhanced production of both flavonoids.
Journal of Microbiology and Biotechnology 11/2011; 21(11):1143-6. · 1.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The adult and the early stages of Cydia piceicola, sp. nov., are described and illustrated from Korea. Larvae of the new species feed on Picea jezoensis (Siebold et Zucc.) Carrière, The life history and larval feeding habits are briefly discussed.
[Show abstract][Hide abstract] ABSTRACT: Kanamycin is one of the most widely used antibiotics, yet its biosynthetic pathway remains unclear. Current proposals suggest that the kanamycin biosynthetic products are linearly related via single enzymatic transformations. To explore this system, we have reconstructed the entire biosynthetic pathway through the heterologous expression of combinations of putative biosynthetic genes from Streptomyces kanamyceticus in the non-aminoglycoside-producing Streptomyces venezuelae. Unexpectedly, we discovered that the biosynthetic pathway contains an early branch point, governed by the substrate promiscuity of a glycosyltransferase, that leads to the formation of two parallel pathways in which early intermediates are further modified. Glycosyltransferase exchange can alter flux through these two parallel pathways, and the addition of other biosynthetic enzymes can be used to synthesize known and new highly active antibiotics. These results complete our understanding of kanamycin biosynthesis and demonstrate the potential of pathway engineering for direct in vivo production of clinically useful antibiotics and more robust aminoglycosides.
Nature Chemical Biology 11/2011; 7(11):843-52. · 12.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A 14-membered macrolide antibiotic narbomycin produced from Streptomyces venezuelae ATCC 15439 is composed of polyketide macrolactone ring and D-desosamine as a deoxysugar moiety, which acts as an important determinant of its antibacterial activity. In order to generate diverse glycosylated derivatives of narbomycin, expression plasmids carrying different deoxysugar biosynthetic gene cassettes and the gene encoding a substrate-flexible glycosyltransferase DesVII were constructed and introduced into S. venezuelae YJ003 mutant strain bearing a deletion of thymidine-5'-diphospho-D-desosamine biosynthetic gene cluster. The resulting recombinants of S. venezuelae produced a range of new analogs of narbomycin, which possess unnatural sugar moieties instead of native deoxysugar D-desosamine. The structures of narbomycin derivatives were determined through nuclear magnetic resonance spectroscopy and mass spectrometry analyses and their antibacterial activities were evaluated in vitro against erythromycin-susceptible and -resistant Enterococcus faecium and Staphylococcus aureus. Substitution with L-rhamnose or 3-O-demethyl-D-chalcose was demonstrated to exhibit greater antibacterial activity than narbomycin and the clinically relevant erythromycin. This work provides new insight into the functions of deoxysugar biosynthetic enzymes and structure-activity relationships of the sugar moieties attached to the macrolides and demonstrate the potential of combinatorial biosynthesis for the generation of new macrolides carrying diverse sugars with increased antibacterial activities.
Applied Microbiology and Biotechnology 09/2011; 93(3):1147-56. · 3.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rapamycin is a macrocyclic polyketide with immunosuppressive, antifungal, and anticancer activity produced by Streptomyces hygroscopicus ATCC 29253. Rapamycin production by a mutant strain (UV2-2) induced by ultraviolet mutagenesis was improved by approximately 3.2-fold (23.6 mg/l) compared to that of the wild-type strain. The comparative analyses of gene expression and intracellular acyl-CoA pools between wild-type and the UV2-2 strains revealed that the increased production of rapamycin in UV2-2 was due to the prolonged expression of rapamycin biosynthetic genes, but a depletion of intracellular methylmalonyl-CoA limited the rapamycin biosynthesis of the UV2-2 strain. Therefore, three different metabolic pathways involved in the biosynthesis of methylmalonyl-CoA were evaluated to identify the effective precursor supply pathway that can support the high production of rapamycin: propionyl-CoA carboxylase (PCC), methylmalonyl-CoA mutase, and methylmalonyl-CoA ligase. Among them, only the PCC pathway along with supplementation of propionate was found to be effective for an increase in intracellular pool of methylmalonyl-CoA and rapamycin titers in UV2-2 strain (42.8 mg/l), indicating that the PCC pathway is a major methylmalonyl-CoA supply pathway in the rapamycin producer. These results demonstrated that the combined approach involving traditional mutagenesis and metabolic engineering could be successfully applied to the diagnosis of yield-limiting factors and the enhanced production of industrially and clinically important polyketide compounds.
Applied Microbiology and Biotechnology 06/2011; 91(5):1389-97. · 3.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Streptomyces venezuelae YJ028, bearing a deletion of the entire biosynthetic gene cluster encoding the pikromycin polyketide synthases and desosamine biosynthetic enzymes, was used as a bioconversion system for combinatorial biosynthesis of glycosylated derivatives of tylosin. Two engineered deoxysugar biosynthetic pathways for the biosynthesis of TDP-3-O-demethyl-D-chalcose or TDP-Lrhamnose in conjunction with the glycosyltransferaseauxiliary protein pair DesVII/DesVIII were expressed in a S. venezuelae YJ028 mutant strain. Supplementation of each mutant strain capable of producing TDP-3-O-demethyl- D-chalcose or TDP-L-rhamnose with tylosin aglycone tylactone resulted in the production of the 3-O-demethyl- D-chalcose, D-quinovose, or L-rhamnose-glycosylated tylactone.
Journal of Microbiology and Biotechnology 06/2011; 21(6):613-6. · 1.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A new reduced hydroxamate, 2,3-dihydrotrichostatin A, was created from trichostatin A by employing a recombinant strain of Streptomyces venezuelae as a microbial catalyst. Compared with trichostatin A, 2,3-dihydrotrichostatin A showed similar antifungal activity against Saccharomyces cerevisiae, but, interestingly, approximately twice the cytostatic activity against human small-cell lung cancer cells. The production of 2,3-dihydrotrichostatin A via microbial biotransformation demonstrates that the regiospecific and substrate-flexible hydrogenation by S. venezuelae provides a new approach for creating natural product analogues with improved bioactive properties.
Journal of Natural Products 05/2011; 74(5):1272-4. · 3.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Doxorubicin, one of the most widely used anticancer drugs, is composed of a tetracyclic polyketide aglycone and l-daunosamine as a deoxysugar moiety, which acts as an important determinant of its biological activity. This is exemplified by the fewer side effects of semisynthetic epirubicin (4'-epi-doxorubicin). An efficient combinatorial biosynthetic system that can convert the exogenous aglycone ε-rhodomycinone into diverse glycosylated derivatives of doxorubicin or its biosynthetic intermediates, rhodomycin D and daunorubicin, was developed through the use of Streptomyces venezuelae mutants carrying plasmids that direct the biosynthesis of different nucleotide deoxysugars and their transfer onto aglycone, as well as the postglycosylation modifications. This system improved epirubicin production from ε-rhodomycinone by selecting a substrate flexible glycosyltransferase, AknS, which was able to transfer the unnatural sugar donors and a TDP-4-ketohexose reductase, AvrE, which efficiently supported the biosynthesis of TDP-4-epi-l-daunosamine. Furthermore, a range of doxorubicin analogs containing diverse deoxysugar moieties, seven of which are novel rhodomycin D derivatives, were generated. This provides new insights into the functions of deoxysugar biosynthetic enzymes and demonstrates the potential of the S. venezuelae-based combinatorial biosynthetic system as a simple biological tool for modifying structurally complex sugar moieties attached to anthracyclines as an alternative to chemical syntheses for improving anticancer agents.
Applied and Environmental Microbiology 05/2011; 77(14):4912-23. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Manganese oxide nanocrystals are combined with aluminum oxide nanocrystals to improve their crystallinity via calcination without a significant increase of crystal size. A nanocomposite, consisting of two metal oxides, can be synthesized by the reaction between permanganate anions and aluminum oxyhydroxide keggin cations. The as-prepared manganese oxide–aluminum oxide nanocomposite is X-ray amorphous whereas heat-treatment gives rise to the crystallization of an α-MnO2 phase at 600 °C and Mn3O4/Mn2O3 and γ-Al2O3 phases at 800 °C. Electron microscopy and N2 adsorption-desorption-isotherm analysis clearly demonstrate that the as-prepared nanocomposite is composed of a porous assembly of monodisperse primary particles with a size of ∼20 nm and a surface area of >410 m2 g−1. Of particular interest is that the small particle size of the as-prepared nanocomposite is well-maintained up to 600 °C, a result of the prevention of the growth of manganate grains through nanoscale mixing with alumina grains. The calcined nanocomposite shows very-high catalytic activity for the oxidation of cyclohexene with an extremely high conversion efficiency of >95% within 15 min. The present results show that the improvement of the crystallinity without significant crystal growth is very crucial for optimizing the catalytic activity of manganese oxide nanocrystals.
[Show abstract][Hide abstract] ABSTRACT: Microbial cultures produce complex and potentially interesting mixtures of biosynthetic intermediates and derivatives of metabolites. These mixtures' reliable identification is important and so too is the development of techniques for their analysis. Here, a simple and highly selective method of detecting the biosynthetic congeners involved in the pentangular polyphenol pradimicin (PR) pathway from Actinomadura hibisca fermentation was developed. Solid-phase extraction (SPE) cleanup using an OASIS HLB cartridge was a simple and reliable tool for the extraction of PRs from a fermentation broth. The separation of each natural PR analog--eluted with a gradient system of aqueous acetonitrile through a reversed-phase C(18) column containing ammonium acetate and acetic acid as additives--allowed their simultaneous profiling. The combined use of SPE cleanup and chromatographic separation, coupled with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) detection was demonstrated to be sufficiently accurate and reliable to analyze the natural PR analogs produced from A. hibisca. Ten natural PRs were identified: four alanine-containing (PRA, PRC, PRL, and PRB), two glycine-substituted (PRD and PRE), and four serine-substituted (PRFA-1, PRFA-2, PRFL, and PRFB). This report demonstrates the first use of both SPE cleanup and HPLC-ESI-MS/MS to profile a wide range of structurally closely related PRs in a bacterial fermentation broth.
Journal of Chromatography A 02/2011; 1218(16):2284-91. · 4.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The development of an efficient analytical protocol for the reliable identification of the biosynthetic intermediates found in microbial cultures, which usually produce complex intermediates of the metabolites of interest, is both challenging and essential for further studies into gene-to-metabolite networks. A simple and highly selective method for detecting the biosynthetic intermediates involved in the aminoglycosidic nebramycin pathway of Streptomyces tenebrarius was developed and validated. Cleanup utilizing a solid-phase extraction (OASIS MCX SPE) technique provides a simple and reproducible method for extracting the nebramycin factors from a fermentation broth. The separation of each factor through a reversed-phase C(18) column using an ion-pairing reagent allowed the simultaneous profiling of the aminoglycosides. By employing the authentic tobramycin spiked into a blank fermentation medium, the combined use of acid extraction, OASIS MCX SPE cleanup, and HFBA (heptafluorobutyric acid)-mediated chromatographic separation coupled with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) detection was proven to be sufficiently accurate and reliable to analyze the nebramycin factors produced in a culture broth. The detection limit of tobramycin spiked in the culture broth was approximately 1.8 ng mL(-1). The mean recovery ranged from 89 to 92%, the intra- and inter-day precision (RSD) was <6% and their accuracy ranged from 88 to 93%. A total of nine nebramycin factors including apramycin, 6''-O-carbamoylkanamycin B, 6''-O-carbamoyltobramycin, 3'-hydoxyapramycin, tobramycin, kanamycin B, NK-1012-1, nebramine, and neamine were identified. This is the first report on the integrated LC-ESI-MS/MS characterization of a wide range of nebramycin factors from a bacterial fermentation broth.
[Show abstract][Hide abstract] ABSTRACT: Polyketides comprise one of the major families of natural products. They are found in a wide variety of bacteria, fungi, and plants and include a large number of medically important compounds. Polyketides are biosynthesized by polyketide synthases (PKSs). One of the major groups of polyketides are the macrolides, the activities of which are derived from the presence of a macrolactone ring to which one or more 6-deoxysugars are attached. The core macrocyclic ring is biosynthesized from acyl-CoA precursors by PKS. Genetic manipulation of PKS-encoding genes can result in predictable changes in the structure of the macrolactone component, many of which are not easily achieved through standard chemical derivatization or total synthesis. Furthermore, many of the changes, including post-PKS modifications such as glycosylation and oxidation, can be combined for further structural diversification. This review highlights the current state of novel macrolide production with a focus on the genetic engineering of PKS and post-PKS tailoring genes. Such engineering of the metabolic pathways for macrolide biosynthesis provides attractive alternatives for the production of diverse non-natural compounds. Other issues of importance, including the engineering of precursor pathways and heterologous expression of macrolide biosynthetic genes, are also considered.
Applied Microbiology and Biotechnology 11/2009; 85(5):1227-39. · 3.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Phenylpropanoids, including flavonoids and stilbenes, are plant secondary metabolites with potential pharmacological and nutraceutical properties. To expand the applicability of Streptomyces venezuelae as a heterologous host to plant polyketide production, flavonoid and stilbene biosynthetic genes were expressed in an engineered strain of S. venezuelae DHS2001 bearing a deletion of native pikromycin polyketide synthase gene. A plasmid expressing the 4-coumarate/cinnamate:coenzyme A ligase from Streptomyces coelicolor (ScCCL) and the chalcone synthase from Arabidopsis thaliana (atCHS) under the control of a single ermE* promoter was constructed and introduced into S. venezuelae DHS2001. The resulting strain produced racemic naringenin and pinocembrin from 4-coumaric acid and cinnamic acid, respectively. Placement of an additional ermE* promoter upstream of the codon-optimized atCHS (atCHS(op)) gene significantly increased the yield of both flavanones. Expression of codon-optimized chalcone isomerase gene from Medicago sativa, together with ScCCL and atCHS(op) genes led to production of (2S)-flavanones, but the yield was reduced. On the other hand, a recombinant strain harboring the ScCCL and codon-optimized stilbene synthase gene from Arachis hypogaea generated stilbenes such as resveratrol and pinosylvin. This is the first report on the heterologous expression of plant phenylpropanoid biosynthetic pathways in Streptomyces genus.
Journal of Biotechnology 06/2009; 141(3-4):181-8. · 3.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An unusual set of reduced macrolide antibiotics was discovered by combination of organic synthesis and a biosynthetic approach using the unique metabolic diversity of Streptomyces venezuelae; two unnatural 16-membered ring macrolides are also created by employing this bio-catalyst.
Chemical Communications 12/2008; · 6.38 Impact Factor