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Jae Kyung Sohng - One of the best experts on this subject based on the ideXlab platform.

  • exploration of cryptic organic photosensitive compound as zincphyrin iv in Streptomyces Venezuelae atcc 15439
    Applied Microbiology and Biotechnology, 2020
    Co-Authors: Hue Thi Nguyen, Anaya Raj Pokhrel, Van Thuy Thi Pham, Chung Thanh Nguyen, Taesu Kim, Dahye Kim, Tokutaro Yamaguchi, Jae Kyung Sohng
    Abstract:

    Zincphyrin IV is a potential organic photosensitizer which is of significant interest for applications in biomedicine, materials science, agriculture (as insecticide), and chemistry. Most studies on Zincphyrin are focused on Zincphyrin III while biosynthesis and application of Zincphyrin IV is comparatively less explored. In this study, we explored Zincphyrin IV production in Streptomyces Venezuelae ATCC 15439 through combination of morphology engineering and “One strain many compounds” approach. The morphology engineering followed by change in culture medium led to activation of cryptic Zincphyrin IV biosynthetic pathway in S. Venezuelae with subsequent detection of Zincphyrin IV. Morphology engineering applied in S. Venezuelae increased the biomass from 7.17 to 10.5 mg/mL after 48 h of culture. Moreover, morphology of engineered strain examined by SEM showed reduced branching and fragmentation of mycelia. The distinct change in color of culture broth visually demonstrated the activation of the cryptic biosynthetic pathway in S. Venezuelae. The production of Zincphyrin IV was found to be initiated after overexpression ssgA, resulting in the increase in titer from 4.21 to 7.54 μg/mL. Furthermore, Zincphyrin IV demonstrated photodynamic antibacterial activity against Bacillus subtilis and photodynamic anticancer activity against human ovarian carcinoma cell lines.

  • Heterologous production of clavulanic acid intermediates in Streptomyces Venezuelae
    Biotechnology and Bioprocess Engineering, 2017
    Co-Authors: Biplav Shrestha, Hum Nath Jnawali, Dipesh Dhakal, Sumangala Darsandhari, Ramesh Prasad Pandey, Anaya Raj Pokhrel, Jae Kyung Sohng
    Abstract:

    Heterologous expression can enhance production of diverse secondary metabolites by redirecting precursor pools towards compound of interest. In this study, Streptomyces Venezuelae YJ028 was utilized as the heterologous host for the expression of four structural clavulanic acid biosynthesis genes, which encode carboxyethylarginine synthase (ceas2), β-lactam synthetase (bls2), clavaminate synthase (cas2), and proclavaminate amidinohydrolase (pah2). These genes were cloned into pIBR25 expression vector containing ermE* promoter to generate pBS4. The cas2 gene was also cloned into pSET152 to generate pCas2. It was then integrated into the genome of S. Venezuelae YJ028. Upon metabolite profiling of recombinant strains by ultra-pressure liquid chromatography-photodiode array (UPLC-PDA) and high resolution liquid chromatography quadruple time-offlight electrospray ionization mass spectrometry (HR-LC-QTOF-ESI/MS), the production of following clavulanic acid intermediates in S. Venezuelae recombinant were confirmed: deoxygaunidinoproclavaminic acid, guanidinoproclavaminic acid, and dihydroclavaminic acid. This work demonstrates the production of β-lactam intermediates of the clavulanic acid pathway by heterologous expression in S. Venezuelae YJ028.

  • metabolic engineering of Streptomyces Venezuelae for malonyl coa biosynthesis to enhance heterologous production of polyketides
    Biotechnology Letters, 2010
    Co-Authors: Sushila Maharjan, Yeo Joon Yoon, Je Won Park, Hei Chan Lee, Jae Kyung Sohng
    Abstract:

    Using metabolic engineering, we developed Streptomyces Venezuelae YJ028 as an efficient heterologous host to increase the malonyl-CoA pool to be directed towards enhanced production of various polyketides. To probe the applicability of newly developed hosts in the heterologous production of polyketides, we expressed type III polyketide synthase, 1,3,6,8-tetrahydroxynaphthalene synthase, in these hosts. Flaviolin production was doubled by expression of acetyl-CoA carboxylase (ACCase) and 4-fold by combined expression of ACCase, metK1-sp and afsR-sp. Thus, the newly developed Streptomyces Venezuelae YJ028 hosts produce heterologous polyketides more efficiently than the parent strain.

  • identification and functional characterization of an afsr homolog regulatory gene from Streptomyces Venezuelae atcc 15439
    Journal of Microbiology and Biotechnology, 2009
    Co-Authors: Sushila Maharjan, Hei Chan Lee, Jae Kyung Sohng
    Abstract:

    Sequencing analysis of a 5-kb DNA fragment from Streptomyces Venezuelae ATCC 15439 revealed the presence of one 3.1-kb open reading frame (ORF), designated as afsR-sv. The deduced product of afsR-sv (1,056 aa) was found to have high homology with the global regulatory protein AfsR. Homology-based analysis showed that afsR-sv represents a transcriptional activator belonging to the Streptomyces antibiotic regulatory protein (SARP) family that includes an N-terminal SARP domain containing a bacterial transcriptional activation domain (BTAD), an NB-ARC domain, and a C-terminal tetratricopeptide repeat domain. Gene expression analysis by reverse transcriptase PCR (RT-PCR) demonstrated the activation of transcription of genes belonging to pikromycin production, when afsR-sv was overexpressed in S. Venezuelae. Heterologous expression of the afsR-sv in different Streptomyces strains resulted in increased production of the respective antibiotics, suggesting that afsR-sv is a positive regulator of antibiotics biosynthesis.

  • Heterologous production of epothilones B and D in Streptomyces Venezuelae
    Applied Microbiology and Biotechnology, 2008
    Co-Authors: Sung Ryeol Park, Jae Kyung Sohng, Je Won Park, Won Seok Jung, Yeon-hee Ban, Eunji Kim, Ah Reum Han, Sang Jun Sim, Yeo Joon Yoon
    Abstract:

    Epothilones, produced from the myxobacterium Sorangium cellulosum , are potential anticancer agents that stabilize microtubules in a similar manner to paclitaxel. The entire epothilone biosynthetic gene cluster was heterologously expressed in an engineered strain of Streptomyces Venezuelae bearing a deletion of pikromycin polyketide synthase gene cluster. The resulting strains produced approximately 0.1 μg/l of epothilone B as a sole product after 4 days cultivation. Deletion of an epoF encoding the cytochrome P450 epoxidase gave rise to a mutant that selectively produces 0.4 μg/l of epothilone D. To increase the production level of epothilones B and D, an additional copy of the positive regulatory gene pikD was introduced into the chromosome of both S. venezuleae mutant strains. The resulting strains showed enhanced production of corresponding compounds (approximately 2-fold). However, deletion of putative transport genes, orf3 and orf14 in the epothilone D producing S. Venezuelae mutant strain, led to an approximately 3-fold reduction in epothilone D production. These results introduce S. Venezuelae as an alternative heterologous host for the production of these valuable anticancer agents and demonstrate the possibility of engineering this strain as a generic heterologous host for the production of polyketides and hybrid polyketide-nonribosomal peptides.

Yeo Joon Yoon - One of the best experts on this subject based on the ideXlab platform.

  • complete genome sequence of Streptomyces Venezuelae atcc 15439 a promising cell factory for production of secondary metabolites
    Journal of Biotechnology, 2016
    Co-Authors: Ju Yeon Song, Young Ji Yoo, Si Kyu Lim, Sun Ho Cha, Jieun Kim, Junghye Roe, Jihyun F Kim, Yeo Joon Yoon
    Abstract:

    Streptomyces Venezuelae ATCC 15439, which produces 12- and 14-membered ring macrolide antibiotics, is a platform strain for heterologous expression of secondary metabolites. Its 9.05-Mb genome sequence revealed an abundance of genes involved in the biosynthesis of secondary metabolites and their precursors, which should be useful for the production of bioactive compounds.

  • Developing Streptomyces Venezuelae as a cell factory for the production of small molecules used in drug discovery
    Archives of Pharmacal Research, 2015
    Co-Authors: Eunji Kim, Inho Yang, Yeo Joon Yoon
    Abstract:

    The heterologous expression of biosynthetic pathways is an indispensable tool for drug discovery and development from natural products. Streptomyces Venezuelae is a promising heterologous host as it offers several attractive advantages, such as rapid growth rate, convenient genetic manipulation, and an abundant supply of common biosynthetic building blocks. In recent decades, several S . Venezuelae mutant strains have been constructed and used to facilitate the synthesis and derivatization of diverse natural products. In this review article, we have provided a schematic look at these host strains, which were used to synthesize natural products from genetically engineered biosynthetic gene clusters.

  • enhanced flavonoid production in Streptomyces Venezuelae via metabolic engineering
    Journal of Microbiology and Biotechnology, 2011
    Co-Authors: Sung Ryeol Park, Je Won Park, Ah Reum Han, Mi Sun Ahn, Yeo Joon Yoon
    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.

  • Biosynthesis of glycosylated derivatives of tylosin in Streptomyces Venezuelae.
    Journal of microbiology and biotechnology, 2011
    Co-Authors: Ah Reum Han, Sung Ryeol Park, Je Won Park, Eun Yeol Lee, Dong-myung Kim, Byung-gee Kim, Yeo Joon Yoon
    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.

  • metabolic engineering of Streptomyces Venezuelae for malonyl coa biosynthesis to enhance heterologous production of polyketides
    Biotechnology Letters, 2010
    Co-Authors: Sushila Maharjan, Yeo Joon Yoon, Je Won Park, Hei Chan Lee, Jae Kyung Sohng
    Abstract:

    Using metabolic engineering, we developed Streptomyces Venezuelae YJ028 as an efficient heterologous host to increase the malonyl-CoA pool to be directed towards enhanced production of various polyketides. To probe the applicability of newly developed hosts in the heterologous production of polyketides, we expressed type III polyketide synthase, 1,3,6,8-tetrahydroxynaphthalene synthase, in these hosts. Flaviolin production was doubled by expression of acetyl-CoA carboxylase (ACCase) and 4-fold by combined expression of ACCase, metK1-sp and afsR-sp. Thus, the newly developed Streptomyces Venezuelae YJ028 hosts produce heterologous polyketides more efficiently than the parent strain.

David L. Jakeman - One of the best experts on this subject based on the ideXlab platform.

  • on the catalytic activity of a gt1 family glycosyltransferase from Streptomyces Venezuelae isp5230
    Journal of Organic Chemistry, 2019
    Co-Authors: Stephanie M Forget, Sydney B Shepard, Ebrahim Soleimani, David L. Jakeman
    Abstract:

    GT1 family glycosyltansferase, Sv0189, from Streptomyces Venezuelae ISP5230 (ATCC 10721) was characterized. The recombinantly produced protein Sv0189 possessed UDP-glycosyltransferase activity. Screening, using an assay employing unnatural nitrophenyl glycosides as activated donors, resulted in the discovery of a broad substrate scope with respect to both acceptor molecules and donor sugars. In addition to polyphenols, including anthraquinones, simple aromatics containing primary or secondary alcohols, a variety of complex natural products and synthetic drugs were glucosylated or xylosylated by Sv0189. Regioselectivity was established through the isolation and characterization of glucosylated products. Sv0189 and homologous proteins are widely distributed among Streptomyces species, and their apparent substrate promiscuity reveals potential for their development as biocatalysts for glycodiversification.

  • furan and lactam jadomycin biosynthetic congeners isolated from Streptomyces Venezuelae isp5230 cultured with ne trifluoroacetyl l lysine
    Journal of Natural Products, 2017
    Co-Authors: Stephanie M Forget, Andrew W. Robertson, David P. Overy, Russell G. Kerr, David L. Jakeman
    Abstract:

    Angucycline antibiotics are composed of a classical four-ring angularly linked polyaromatic backbone. Differential cyclization chemistry of the A- and B-rings in jadomycin biosynthesis led to the discovery of two new furan analogues, while oxidation led to a ring-opened form of the jadomycin Ne-trifluoroacetyl-l-lysine (TFAL) congener. The compounds were isolated from Streptomyces Venezuelae ISP5230 cultures grown with TFAL. Biosynthetic incorporation using d-[1-13C]-glucose in cultures enabled the unambiguous assignment of the aldehyde, alcohol, and amide functionalities present in these new congeners through NMR spectroscopy. Tandem mass spectrometry analysis of cultures grown with 15Nα- or 15Ne-lysine demonstrated the incorporation of Nα exclusively into the angucycline backbone, contrasting results with ornithine [J. Am. Chem. Soc. 2015, 137, 3271]. Compounds were evaluated against antimicrobial and cancer cell panels and found to possess good activity against Gram-positive bacteria.

  • Streptomyces Venezuelae isp5230 maintains excretion of jadomycin upon disruption of the mfs transporter jadl located within the natural product biosynthetic gene cluster
    Frontiers in Microbiology, 2017
    Co-Authors: Stephanie M Forget, Leo C Vining, Jennifer Mcvey, David L. Jakeman
    Abstract:

    JadL was identified as a Major Facilitator Superfamily (MFS) transporter (T.C. 2.A.1) through sequence homology. The protein is encoded by jadL, situated within the jadomycin biosynthetic gene cluster. JadL has, therefore, been assigned a putative role in host defence by exporting its probable substrates, the jadomycins, a family of secondary metabolites produced by Streptomyces Venezuelae ISP5230. Herein, we evaluate this assumption through the construction and analysis of a jadL disrupted mutant, S. Venezuelae VS678 (delta_jadL::aac(3)IV). Quantitative determination of jadomycin production with the jadL disrupted mutant did not to show a significant decrease in production in comparison to the wildtype strain , as determined by HPLC and by tandem mass spectrometry. These results suggest that efflux of jadomycin occurs upon disruption of jadL, or that jadL is not involved in jadomycin efflux. Potentially, other transporters within S. Venezuelae ISP5230 may adopt this role upon inactivation of jadL to export jadomycins.

  • Furan and Lactam Jadomycin Biosynthetic Congeners Isolated from Streptomyces Venezuelae ISP5230 Cultured with Nε‑Trifluoroacetyl‑l‑lysine
    2017
    Co-Authors: Stephanie M. Forget, Andrew W. Robertson, David P. Overy, Russell G. Kerr, David L. Jakeman
    Abstract:

    Angucycline antibiotics are composed of a classical four-ring angularly linked polyaromatic backbone. Differential cyclization chemistry of the A- and B-rings in jadomycin biosynthesis led to the discovery of two new furan analogues, while oxidation led to a ring-opened form of the jadomycin Nε-trifluoroacetyl-l-lysine (TFAL) congener. The compounds were isolated from Streptomyces Venezuelae ISP5230 cultures grown with TFAL. Biosynthetic incorporation using d-[1-13C]-glucose in cultures enabled the unambiguous assignment of the aldehyde, alcohol, and amide functionalities present in these new congeners through NMR spectroscopy. Tandem mass spectrometry analysis of cultures grown with 15Nα- or 15Nε-lysine demonstrated the incorporation of Nα exclusively into the angucycline backbone, contrasting results with ornithine [J. Am. Chem. Soc. 2015, 137, 3271]. Compounds were evaluated against antimicrobial and cancer cell panels and found to possess good activity against Gram-positive bacteria

  • characterization of l digitoxosyl phenanthroviridin from Streptomyces Venezuelae isp5230
    Journal of Natural Products, 2015
    Co-Authors: Andrew W. Robertson, Camilo F Martinezfarina, Raymond T Syvitski, David L. Jakeman
    Abstract:

    The jadomycin-derived compound l-digitoxosyl-phenanthroviridin was isolated from fermentations of Streptomyces Venezuelae ISP5230 grown in nutrient-deficient media with l-lysine as the sole nitrogen source. Structural elucidation was accomplished using a combination of high-resolution MS, LC-MS/MS, and 1D- and 2D-NMR. The compound was evaluated against the National Cancer Institute (NCI) 60 human tumor cell line screen in both the one-dose and five-dose screens, and cytotoxicity was compared to a small library of jadomycin analogues to probe the structure-activity relationship.

David H. Sherman - One of the best experts on this subject based on the ideXlab platform.

  • neopikromycin and novapikromycin from the pikromycin biosynthetic pathway of Streptomyces Venezuelae
    Journal of Natural Products, 2006
    Co-Authors: Sang Kil Lee, Sung Ryeol Park, Je Won Park, Won Seok Jung, Ji Won Kim, Cha Yong Choi, Eungsoo Kim, Beomseok Kim, Jong Seog Ahn, David H. Sherman
    Abstract:

    Two new macrolides from the pikromycin biosynthetic pathway of Streptomyces Venezuelae, neopikromycin (9) and novapikromycin (10), were identified and structurally characterized through mass spectrometry and NMR spectroscopy. The established structures showed that 9 and 10 have hydroxyl groups at C-14 (9) and at both C-12 and C-14 (10), on the basis of a comparison with narbomycin (7). The purified PikC cytochrome P450 monooxygenase catalyzes the in vitro hydroxylation of 7 and pikromycin (8) to yield 9 and 10, respectively, thus expanding the substrate- and regio-flexibility of this enzyme.

  • heterologous expression of tylosin polyketide synthase and production of a hybrid bioactive macrolide in Streptomyces Venezuelae
    Applied Microbiology and Biotechnology, 2006
    Co-Authors: Won Seok Jung, Jae Kyung Sohng, Sung Ryeol Park, Ah Reum Han, Byung-gee Kim, Sang Kil Lee, Cha Yong Choi, Jay Sung Joong Hong, Soon Jeong Jeong, David H. Sherman
    Abstract:

    Tylosin polyketide synthase (Tyl PKS) was heterologously expressed in an engineered strain of Streptomyces Venezuelae bearing a deletion of pikromycin PKS gene cluster using two compatible low-copy plasmids, each under the control of a pikAI promoter. The mutant strain produced 0.5 mg/l of the 16-membered ring macrolactone, tylactone, after a 4-day culture, which is a considerably reduced culture period to reach the maximum production level compared to other Streptomyces hosts. To improve the production level of tylactone, several precursors for ethylmalonyl-CoA were fed to the growing medium, leading to a 2.8-fold improvement (1.4 mg/ml); however, switching the pikAI promoter to an actI promoter had no observable effect. In addition, a small amount of desosamine-glycosylated tylactone was detected from the extract of the mutant strain, revealing that the native glycosyltransferase DesVII displayed relaxed substrate specificity in accepting the 16-membered ring macrolactone to produce the glycosylated tylactone. These results demonstrate a successful attempt for a heterologous expression of Tyl PKS in S. Venezuelae and introduce S. Venezuelae as a rapid heterologous expression system for the production of secondary metabolites.

  • isolation and structure determination of novamethymycin a new bioactive metabolite of the methymycin biosynthetic pathway in Streptomyces Venezuelae
    Journal of Natural Products, 2001
    Co-Authors: Qibo Zhang, David H. Sherman
    Abstract:

    Novamethymycin (9), a novel macrolide antibiotic, was isolated from Streptomyces Venezuelae, the producer of methymycin (4) and neomethymycin (5). Spectroscopic analysis of 9 indicated that it is highly related to 4 and 5 but contains hydroxy groups at both C-10 and C-12 on the macrolactone ring. Bioconversion studies using the PikC cytochrome P450 hydroxylase demonstrated that 4 is converted to 9, further broadening the remarkable substrate flexibility of this enzyme.

  • characterization and analysis of the pikd regulatory factor in the pikromycin biosynthetic pathway of Streptomyces Venezuelae
    Journal of Bacteriology, 2001
    Co-Authors: Daniel J Wilson, Yongquan Xue, Kevin A Reynolds, David H. Sherman
    Abstract:

    The Streptomyces Venezuelae pikD gene from the pikromycin biosynthetic cluster was analyzed, and its deduced product (PikD) was found to have amino acid sequence homology with a small family of bacterial regulatory proteins. Database comparisons revealed two hypothetical domains, including an N-terminal triphosphate-binding domain and a C-terminal helix-turn-helix DNA-binding motif. Analysis of PikD was initiated by deletion of the corresponding gene (pikD) from the chromosome of S. Venezuelae, resulting in complete loss of antibiotic production. Complementation by a plasmid carrying pikD restored macrolide biosynthesis, demonstrating that PikD is a positive regulator. Mutations were made in the predicted nucleotide triphosphate-binding domain, confirming the active-site amino acid residues of the Walker A and B motifs. Feeding of macrolide intermediates was carried out to gauge the points of operon control by PikD. Although the pikD mutant strain was unable to convert macrolactones (10-deoxymethynolide and narbonolide) to glycosylated products, macrolide intermediates (YC-17 and narbomycin) were hydroxylated with high efficiency. To study further the control of biosynthesis, presumed promoter regions from pik cluster loci were linked to the xylE reporter and placed in S. Venezuelae wild-type and pikD mutant strains. This analysis demonstrated that PikD-mediated transcriptional regulation occurs at promoters controlling expression of pikRII, pikAI, and desI but not those controlling pikRI or pikC.

  • Biosynthesis and combinatorial biosynthesis of pikromycin-related macrolides in Streptomyces Venezuelae.
    Metabolic engineering, 2001
    Co-Authors: Yongquan Xue, David H. Sherman
    Abstract:

    Pikromycin-related macrolides have recently attracted significant research interest because they are structurally related to the semisynthetic ketolide antibiotics that have demonstrated promising potential in combating multi-drug-resistant respiratory pathogens. Cloning and in-depth studies of the pikromycin biosynthetic gene cluster from Streptomyces Venezuelae have led to new avenues in modular polyketide synthases, deoxysugar biosynthesis, cytochrome P450 hydroxylase, secondary metabolite gene regulation, and antibiotic resistance. Moreover, the knowledge and tools used for these studies are proving to be valuable in the development of advanced technologies for combinatorial biosynthesis of new macrolide antibiotics. This review summarizes these new developments and introduces S. Venezuelae as a powerful new system for secondary metabolite pathway engineering from bench-top genetic manipulation to product fermentation.

Leo C Vining - One of the best experts on this subject based on the ideXlab platform.

  • Streptomyces Venezuelae isp5230 maintains excretion of jadomycin upon disruption of the mfs transporter jadl located within the natural product biosynthetic gene cluster
    Frontiers in Microbiology, 2017
    Co-Authors: Stephanie M Forget, Leo C Vining, Jennifer Mcvey, David L. Jakeman
    Abstract:

    JadL was identified as a Major Facilitator Superfamily (MFS) transporter (T.C. 2.A.1) through sequence homology. The protein is encoded by jadL, situated within the jadomycin biosynthetic gene cluster. JadL has, therefore, been assigned a putative role in host defence by exporting its probable substrates, the jadomycins, a family of secondary metabolites produced by Streptomyces Venezuelae ISP5230. Herein, we evaluate this assumption through the construction and analysis of a jadL disrupted mutant, S. Venezuelae VS678 (delta_jadL::aac(3)IV). Quantitative determination of jadomycin production with the jadL disrupted mutant did not to show a significant decrease in production in comparison to the wildtype strain , as determined by HPLC and by tandem mass spectrometry. These results suggest that efflux of jadomycin occurs upon disruption of jadL, or that jadL is not involved in jadomycin efflux. Potentially, other transporters within S. Venezuelae ISP5230 may adopt this role upon inactivation of jadL to export jadomycins.

  • use of degenerate primers and touchdown pcr to amplify a halogenase gene fragment from Streptomyces Venezuelae isp5230
    Journal of Industrial Microbiology & Biotechnology, 2002
    Co-Authors: Mahmood Piraee, Leo C Vining
    Abstract:

    Consensus amino acid sequences of FADH(2)-dependent bacterial halogenases were used to design PCR primers amplifying a halogenase gene fragment from the chloramphenicol producer Streptomyces Venezuelae ISP5230. The sequence-specific degenerate primers (MPF1 and MPR2) were used with a touchdown PCR procedure in the first PCR-assisted cloning of a halogenase gene fragment. In the region of the 290-bp PCR product containing the reverse primer, the deduced amino acid sequence exhibited characteristics of a beta-alpha-beta fold present in FAD-binding sites of certain monooxygenases. When used to probe Southern blots of restriction-enzyme-digested DNA, the [alpha-(32)P]dCTP-labeled PCR product hybridized specifically with DNA fragments from genomic DNA of S. Venezuelae ISP5230. Primers MPF1 and MPR2 also allowed amplification by PCR of approximately 290-bp DNA fragments from several other streptomycetes. The fragments from Streptomyces aureofaciens NRRL2209 and Streptomyces coelicolor A3(2) showed sequence identity with halogenase genes from these species. Thus, the PCR primers are of potential value for amplification and subsequent isolation of actinomycete halogenase genes.

  • biosynthesis of the dideoxysugar component of jadomycin b genes in the jad cluster of Streptomyces Venezuelae isp5230 for l digitoxose assembly and transfer to the angucycline aglycone
    Microbiology, 2002
    Co-Authors: Liru Wang, Robert L White, Leo C Vining
    Abstract:

    Eight additional genes, jadX, O, P, Q, S, T, U and V, in the jad cluster of Streptomyces Venezuelae ISP5230, were located immediately downstream of jadN by chromosome walking. Sequence analyses and comparisons implicated them in biosynthesis of the 2,6-dideoxysugar in jadomycin B. The genes were cloned in Escherichia coli, inactivated by inserting an apramycin resistance cassette with a promoter driving transcription of downstream genes, and transferred into Streptomyces Venezuelae by intergeneric conjugation. Analysis by HPLC and NMR of intermediates accumulated by cultures of the insertionally inactivated Streptomyces Venezuelae mutants indicated that jadO, P, Q, S, T, U and V mediate formation of the dideoxysugar moiety of jadomycin B and its attachment to the aglycone. Based on these results and sequence similarities to genes described in other species producing deoxysugar derivatives, a biosynthetic pathway is proposed in which the jadQ product (glucose-1-phosphate nucleotidyltransferase) activates glucose to its nucleotide diphosphate (NDP) derivative, and the jadT product (a 4,6-dehydratase) converts this to NDP-4-keto-6-deoxy-D-glucose. An NDP-hexose 2,3-dehydratase and an oxidoreductase, encoded by jadO and jadP, respectively, catalyse ensuing reactions that produce an NDP-2,6-dideoxy-D-threo-4-hexulose. The product of jadU (NDP-4-keto-2,6-dideoxy-5-epimerase) converts this intermediate to its L-erythro form and the jadV product (NDP-4-keto-2,6-dideoxyhexose 4-ketoreductase) reduces the keto group of the NDP-4-hexulose to give an activated form of the L-digitoxose moiety in jadomycin B. Finally, a glycosyltransferase encoded by jadS transfers the activated sugar to jadomycin aglycone. The function of jadX is unclear; the gene is not essential for jadomycin B biosynthesis, but its presence ensures complete conversion of the aglycone to the glycoside. The deduced amino acid sequence of a 612 bp ORF (jadR*) downstream of the dideoxysugar biosynthesis genes resembles many TetR-family transcriptional regulator sequences.

  • a repressor response regulator gene pair controlling jadomycin b production in Streptomyces Venezuelae isp5230
    Gene, 2001
    Co-Authors: Keqian Yang, Liru Wang, Lei Han, Leo C Vining
    Abstract:

    A second regulatory gene (jadR(1)) is located immediately upstream of the putative repressor gene (jadR(2)) in the jad cluster for biosynthesis of the antibiotic jadomycin B in Streptomyces Venezuelae ISP5230. It encodes a 234-amino acid polypeptide with a sequence resembling those of response regulator proteins in two-component control systems. Features in the conserved C-terminal domain of JadR(1) place the protein in the OmpR-PhoB subfamily of response regulators. In mutants where jadR(1) was deleted or disrupted, jadomycin B was not produced, implying that the gene has an essential role in biosynthesis of the antibiotic. Cloning jadR(1) from S. Venezuelae in pJV73A, and introducing additional copies of the gene into the wild-type parent by plasmid transformation gave unstable strains with pJV73A integrated into the chromosome. The transformants initially showed increased production of jadomycin B but gave lower titers as excess copies of jadR(1) were lost; mature cultures stabilized with a wild-type level of antibiotic production. The mutant from which jadR(1) had been deleted could not be transformed with pJV73A. Altering the composition of jadR genes in the chromosome by integration of vectors carrying intact and disrupted copies of jadR(1) and jadR(2) provided evidence that the two genes form a regulatory pair different in function from previously reported two-component systems controlling antibiotic biosynthesis in streptomycetes.

  • the pdx genetic marker adjacent to the chloramphenicol biosynthesis gene cluster in Streptomyces Venezuelae isp5230 functional characterization
    Microbiology, 2001
    Co-Authors: Nathan A Magarvey, K A Aidoo, Leo C Vining
    Abstract:

    The pdx-4 mutation in Streptomyces Venezuelae ISP5230 confers a growth requirement for pyridoxal (pdx) and is a marker for the genetically mapped cluster of genes associated with chloramphenicol biosynthesis. A gene regulating salvage synthesis of vitamin B6 cofactors in S. Venezuelae was cloned by transforming a pdx-4 mutant host with the plasmid vector pDQ101 carrying a library of wild-type genomic DNA fragments, and by selecting for complementation of the host’s pdx requirement. However, the corresponding replicative plasmid could not be isolated. Southern hybridizations and transduction analysis indicated that the complementing plasmid had integrated into the chromosome; after excision by a second crossover, the plasmid failed to propagate. To avoid loss of the recombinant vector, a pdxdependent Streptomyces lividans mutant, KAA1, with a phenotype matching that of S. Venezuelae pdx-4, was isolated for use as the cloning host.