Production of hybrid glycopeptide antibiotics in vitro and in Streptomyces toyocaensis

Solenberg, Patricia J.; Matsushima, Patti; Stack, Douglas R.; Wilkie, Stephen C.; Thompson, Richard C.; Baltz, Richard H.

doi:10.1016/s1074-5521(97)90288-x

Cited by 181 publications

(175 citation statements)

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“…The glycosyltransferase sequence motif When orf2" was first sequenced, its deduced product was suggested to be a glycosyltransferase (Gandecha et al, 1997 ducing Amycolatopsis orientalis, displayed glucosyltransferase activity with a glycopeptide-aglycone as acceptor substrate (Solenberg et al, 1997). Other entries in the protein identification databases were also found in routine searches using the Orf2'" sequence, including DnrS and DauH from daunorubicin (daunomycin)-producing S. peucetius (Otten et al, 1995) and Streptomyces sp.…”

Section: Resultsmentioning

confidence: 99%

Stimulation of polyketide metabolism in Streptomyces fradiae by tylosin and its glycosylated precursors

Fish¹,

Cundliffe

1997

Microbiology

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Three glycosyltransferases are involved in tylosin biosynthesis in Streptomyces fradiae. The first sugar to be added to the polyketide aglycone (tylactone) is mycaminose and the gene encoding mycaminosyltransferase is oH2* (tyhW2). However, targeted disruption of od2* did not lead to the accumulation of tylactone under conditions that normally favour tylosin production; instead, the synthesis of tylactone was virtually abolished. This may, in part, have resulted from a polar effect on the expression of genes downstream of oH2*, particularly od4* (ccr) which encodes crotonyl-CoA reductase, an enzyme that supplies 4-carbon extender units for polyketide metabolism. However, that cannot be the entire explanation, since tylosin production was restored a t about 10% of the wild-type level when oH2* was re-introduced into the disrupted strain. When glycosylated precursors of tylosin were fed to the disrupted strain, they were converted to tylosin, confirming that two of the three glycosyltransferase activities associated with tylosin biosynthesis were still intact. Interestingly, however, tylactone also accumulated under such conditions and, to a much lesser extent, when tylosin was added to similar fermentations. It is concluded that glycosylated macrolides exert a pronounced positive effect on polyketide metabolism in S. fradiae. ;ity OfKeywords : tylosin production, glycosyltransferase, polyketide metabolism, Streptomyces fradiae INTRODUCTIONThe macrolide antibiotic, tylosin (Fig. l ) , is produced by Streptomyces fradiae via a combination of polyketide and 6-deoxyhexose metabolism. Glycosylation of tylactone, the cyclized polyketide product, always begins with the addition of mycaminose followed, in a preferred but not obligatory order, by deoxyallose and then mycarose to generate demethyl-macrocin. Stepwise bis 0-methylation then converts the deoxyallose moiety to mycinose as macrocin is produced and converted to tylosin (Baltz et al., 1983). Characterization of the tylosin biosynthetic pathway (Fig. 2) was facilitated by the availability of non-producing mutants of S. fradiae, generated using NTG (Baltz & Seno, 1981). Collectively, these exhibited nine distinct phenotypes in cross-feeding Abbreviations: OMT, 0-mycaminosyl-tylonolide; DMT, demycinosyltyl osi n. experiments and allowed 13 genetic loci (tylA-M) to be mapped (Fig. 3) following complementation studies using cloned fragments of tyl DNA (Beckmann et al.,

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Section: Resultsmentioning

confidence: 99%

Stimulation of polyketide metabolism in Streptomyces fradiae by tylosin and its glycosylated precursors

Fish¹,

Cundliffe

1997

Microbiology

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“…The abbreviations used are: P450 or CYP, cytochrome P450 monooxygenase; GC/MS, gas chromatography/mass spectrometry. 4 Lamb et al reported that this monoxygenase has sterol 14␣-demethylase activity (12). Although CYP170A1 has sequence similarity to CYP51 and can give Type I binding with lanosterol, the previous exogenous activity in lanosterol demethylation was not reproduced in this work, and as noted previously S. coelicolor does not produce sterols.…”

Section: * This Work Was Supported By National Institutes Of Health Gmentioning

confidence: 99%

“…The endogenous biological function of CYP170A1, belonging to the 18-member P450 CYPome of S. coelicolor, has not previously been identified (12). 4 The sco5223 gene encoding CYP170A1 in S. coelicolor is part of a two-gene cluster with a sesquiterpene cyclase gene (sco5222) with which it shares a four-nucleotide ATGA transcriptional overlap at its 5Ј-end. The sco5222 gene product has been shown to catalyze the synthesis of a novel sesquiterpene, epi-isozizaene (1, Fig.…”

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confidence: 99%

Biosynthesis of the Sesquiterpene Antibiotic Albaflavenone in Streptomyces coelicolor A3(2)

Zhao

Lin

Lei

et al. 2008

Journal of Biological Chemistry

165

141

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The presence of CYP170A1 (sco5223) suggested that epiisozizaene might be further oxidized by the transcriptionally coupled P450. We have now established that purified CYP170A1 carries out two sequential allylic oxidations to convert epi-isozizaene to an epimeric mixture of albaflavenols and thence to the sesquiterpene antibiotic albaflavenone. Gas chromatography/mass spectrometry analysis of S. coelicolor culture extracts established the presence of albaflavenone in the wildtype strain, along with its precursors epi-isozizaene and the albaflavenols. Disruption of the CYP170A1 gene abolished biosynthesis of both albaflavenone and the albaflavenols, but not epi-isozizaene. The combined results establish for the first time the presence of albaflavenone in S. coelicolor and clearly demonstrate that the biosynthesis of this antibiotic involves the coupled action of epi-isozizaene synthase and CYP170A1.Biosynthetic pathways for many antibiotics in bacteria and plants are often associated with the presence of cytochromes P450 (P450, CYP).3 The CYPs perform late-stage stereo-and regiospecific metabolic reactions, such as hydroxylation, epoxidation, dehydrogenation, and C-C bond coupling, to modify the parent structural skeletons to give biosynthetic intermediates and/or bioactive products (1-4). Members of the genus Streptomyces produce a wide variety of bioactive secondary metabolites used in human and veterinary therapeutics, including a majority of the medically useful antibiotics (5, 6). Although terpenoids are one of the largest classes of naturally occurring, biologically active substances, with more than 50,000 known compounds, including anti-cancer drugs, antiparasitic, and antibacterial agents, only a relative handful of such terpene metabolites have been isolated from Streptomyces spp (7,8).P450 monooxygenases are a superfamily of heme proteins utilizing atmospheric dioxygen and electrons supplied by NAD(P)H almost always through redox partner proteins to generate, most commonly, oxidized organic products and a molecule of water (9). P450s play an important role in drug metabolism and in the biosynthesis of steroids, lipids, vitamins, antibiotics, and other natural secondary metabolites.Streptomyces coelicolor A3 (2) is genetically one of the most studied actinomycetes and has been a model for genetic analysis of antibiotic production. The complete genome sequence of S. coelicolor has revealed the presence of 18 CYP, 6 ferredoxin, and 4 ferredoxin reductase genes (10, 11). The endogenous biological function of CYP170A1, belonging to the 18-member P450 CYPome of S. coelicolor, has not previously been identified (12). 4 The sco5223 gene encoding CYP170A1 in S. coelicolor is part of a two-gene cluster with a sesquiterpene cyclase gene (sco5222) with which it shares a four-nucleotide ATGA transcriptional overlap at its 5Ј-end. The sco5222 gene product has been shown to catalyze the synthesis of a novel sesquiterpene, epi-isozizaene (1, Fig. 1), by cyclization of the universal sesquiterpene synthase substrate farne...

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“…Additional examples are found in the biosynthesis of the vancomycin-type antibiotics: balhimycin (10), chloroeremomycin (11), and vancomycin, produced by different strains of Amycolatopsis ( Fig. 1) (12,13). In the biosyntheses of these compounds, the P450s OxyA, OxyB, and OxyC have been shown in vivo to be responsible for the oxidative phenolic coupling of the aromatic side chains found in the vancomycin aglycone (14 -18).…”

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Structural Characterization of OxyD, a Cytochrome P450 Involved in β-Hydroxytyrosine Formation in Vancomycin Biosynthesis

Cryle

Meinhart

Schlichting

2010

Journal of Biological Chemistry

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The cytochrome P450 OxyD from the balhimycin glycopeptide antibiotic biosynthetic operon of Amycolatopsis mediterranei is involved in the biosynthesis of the modified amino acid ␤-R-hydroxytyrosine, an essential precursor for biosynthesis of the vancomycin-type aglycone. OxyD binds the substrate tyrosine not free in solution, but rather covalently linked to the carrier protein (CP) domain of the non-ribosomal peptide synthase BpsD, exhibiting micromolar binding affinity to a tyrosineloaded carrier protein construct. The crystal structure of OxyD was determined to 2.1-Å resolution, revealing a potential binding site for the carrier protein-bound substrate in a different orientation to that seen with the acyl carrier protein-bound P450 BioI Hydroxylated amino acid residues are widely utilized in nature as precursors for complex natural products (see Fig. 1). The oxidative modification of amino acids at the ␤-position is a process that occurs in many different classes of medicinally important biomolecules, including the vancomycin-type antibiotics (reviewed in Refs. 2, 3). These glycopeptide antibiotics have had great clinical success in treating Gram-positive bacterial infections that are resistant to other classes of antibiotics and function through blocking cell wall biosynthesis via complex formation with the cell wall precursor peptidoglycan peptidyl units terminating in -Lys-D-Ala-D-Ala (2, 3). The biosynthesis of the vancomycin peptide requires the incorporation of two amino acid residues derived from ␤-R-hydroxytyrosine. Another group of medicinally important molecules that contain hydroxylated amino acid residues is the aminocoumarincontaining angucycline family of antibiotic compounds, which include coumermycin A, clorobiocin, and simocyclinone and act upon bacterial DNA gyrase. ␤-R-Hydroxytyrosine is an essential precursor in the biosynthesis of these types of antibiotics, where it is needed for the formation of both the A (Fig. 1, blue) and B (Fig. 1, red) rings in the aminocoumarin core. Further examples of the incorporation of hydroxylated amino acids into the biosyntheses of medicinally important compounds can be found in the biosyntheses of the nikkomycin-type antibiotics, zorbamycin and echinomycin ( Fig. 1) (4 -7).Despite the large structural difference in the compounds highlighted above and the difference in the biosyntheses of the compounds overall, the formation of the ␤-hydroxyamino acid precursors follows a conserved route. This involves the oxidation of the amino acid by a member of the cytochrome P450 (P450) 2 superfamily. P450s form a superfamily of heme containing monoxygenases that catalyze a vast array of biologically important transformations (8). The archetypical P450 reaction is the hydroxylation of unactivated C-H bonds, a difficult oxidation made more impressive by the ability of P450s to utilize their oxidative power both regio-and stereoselectively. Many P450s play important roles in natural product biosyntheses, where such selectivity is channeled toward the synthesis of comp...

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Production of hybrid glycopeptide antibiotics in vitro and in Streptomyces toyocaensis

Cited by 181 publications

References 20 publications

Stimulation of polyketide metabolism in Streptomyces fradiae by tylosin and its glycosylated precursors

Stimulation of polyketide metabolism in Streptomyces fradiae by tylosin and its glycosylated precursors

Biosynthesis of the Sesquiterpene Antibiotic Albaflavenone in Streptomyces coelicolor A3(2)

Structural Characterization of OxyD, a Cytochrome P450 Involved in β-Hydroxytyrosine Formation in Vancomycin Biosynthesis

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