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      (#Issue1)Medium optimization of Streptomyces sp. 17944 for tirandamycin B production and isolation and structural elucidation of tirandamycins H, I, and J

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          Abstract

          We have recently isolated tirandamycin (TAM) B from Streptomyces sp. 17944 as a Brugia malayi AsnRS ( BmAsnRS) inhibitor that efficiently kills the adult B. malayi parasites and does not exhibit general cytotoxicity to human hepatic cells. We now report (i) the comparison of metabolite profiles of S. sp. 17944 in six different media, (ii) identification of a medium enabling the production of TAM B as essentially the sole metabolite, and with improved titer, and (iii) isolation and structural elucidation of three new TAM congeners. These findings shed new insights into the structure-activity relationship of TAM B as a BmAsnRS inhibitor, highlighting the δ-hydroxymethyl-α,β-epoxyketone moiety as the critical pharmacophore, and should greatly facilitate the production and isolation of sufficient quantities of TAM B for further mechanistic and preclinical studies to advance the candidacy of TAM B as an antifilarial drug lead. The current study also serves as an excellent reminder that traditional medium and fermentation optimization should continue to be very effective in improving metabolite flux and titer.

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          Most cited references29

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          Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp.

          We have constructed cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. All vectors contain the 760-bp oriT fragment from the IncP plasmid, RK2. Transfer functions need to be supplied in trans by the E. coli donor strain. We have incorporated into these vectors selectable antibiotic-resistance markers (AmR, ThR, SpR) that function in Streptomyces spp. and other features that should allow for: (i) integration via homologous recombination between cloned DNA and the Streptomyces spp. chromosome, (ii) autonomous replication, or (iii) site-specific integration at the bacteriophage phi C31 attachment site. Shuttle cosmids for constructing genomic libraries and bacteriophage P1 cloning vector capable of accepting approx. 100-kb fragments are also described. A simple mating procedure has been developed for the conjugal transfer of these vectors from E. coli to Streptomyces spp. that involves plating of the donor strain and either germinated spores or mycelial fragments of the recipient strain. We have shown that several of these vectors can be introduced into Streptomyces fradiae, a strain that is notoriously difficult to transform by PEG-mediated protoplast transformation.
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            Carbohydrate structural determination by NMR spectroscopy: modern methods and limitations.

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              Tirandamycin biosynthesis is mediated by co-dependent oxidative enzymes

              Elucidation of natural product biosynthetic pathways provides important insights about the assembly of potent bioactive molecules, and expands access to unique enzymes able to selectively modify complex substrates. Here we show full reconstitution in vitro of an unusual multi-step oxidative cascade for post-assembly line tailoring of tirandamycin antibiotics. This pathway involves a remarkably versatile and iterative cytochrome P450 monooxygenase (TamI) and an FAD-dependent oxidase (TamL), which act co-dependently through repeated exchange of substrates. TamI hydroxylates tirandamycin C (TirC) to generate tirandamycin E (TirE), a heretofore unidentified tirandamycin intermediate. TirE is subsequently oxidized by TamL, giving rise to the ketone of tirandamycin D (TirD), after which a unique exchange back to TamI enables successive epoxidation and hydroxylation to afford, respectively, the final products tirandamycin A (TirA) and tirandamycin B (TirB). Ligand-free, substrate- and product-bound crystal structures of bicovalently flavinylated TamL oxidase reveal a likely mechanism for the C-10 oxidation of TirE.
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                Author and article information

                Journal
                0151115
                4490
                J Antibiot (Tokyo)
                J. Antibiot.
                The Journal of antibiotics
                0021-8820
                28 May 2013
                29 May 2013
                January 2014
                01 July 2014
                : 67
                : 1
                : 127-132
                Affiliations
                [1 ]Department of Chemistry, The Scripps Research Institute, The Scripps Research Institute, Jupiter, Florida 33458, United States
                [2 ]Department of Molecular Therapeutics, The Scripps Research Institute, The Scripps Research Institute, Jupiter, Florida 33458, United States
                [3 ]Natural Products Library Initiative, The Scripps Research Institute, The Scripps Research Institute, Jupiter, Florida 33458, United States
                [4 ]School of Life Science, Northeast Agricultural University, Harbin, Heilongjiang 150030, China
                [5 ]Biotechnology and Bioengineering Center and Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, United States
                Author notes
                [* ]To whom correspondence should be addressed: Ben Shen, The Scripps Research Institute, 130 Scripps Way, #3A1, Jupiter, Florida 33458, United States. Tel: (561) 228-2456, Fax: (561) 228-2472, shenb@ 123456scripps.edu
                Article
                NIHMS468361
                10.1038/ja.2013.50
                3773001
                23715040
                29070b15-1aac-459b-ba98-40d951f5ea09

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                History
                Funding
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: P41 GM086184 || GM
                Categories
                Article

                Infectious disease & Microbiology
                asnrs inhibitor,filariasis,medium optimization,s. sp 17944,tirandamycin

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