Cloning, sequencing, and analysis of aklaviketone reductase from Streptomyces sp. strain C5

Dickens, M L; Ye, J; Strohl, William R.

doi:10.1128/jb.178.11.3384-3388.1996

Cited by 45 publications

(51 citation statements)

References 27 publications

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“…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. strain C5 (Dickens et al, 1996), respectively, but the closest match (50 % identity) was found with the product of eryCZIl (orf8) from the erythromycin producer, Saccharopolyspora erythraea (Haydock, 1992). These proteins all contain a consensus sequence motif (Fig.…”

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 acyltransferase encoded by dpsD has been speculated to function as propionyl-SCoA:acyl carrier protein (ACP)-SH acyltransferase (41). The gene encoding ACP, dpsG, is located about 6.8 kbp upstream of the genes encoding the daunorubicin KS ␣ and KS ␤ (8,12,41). Because the daunorubicin/doxorubicin PKS gene cluster of Streptomyces sp.…”

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Minimal Streptomyces sp. strain C5 daunorubicin polyketide biosynthesis genes required for aklanonic acid biosynthesis

Rajgarhia

Strohl

1997

J Bacteriol

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The structure of the Streptomyces sp. strain C5 daunorubicin type II polyketide synthase (PKS) gene region is different from that of other known type II PKS gene clusters. Directly downstream of the genes encoding ketoacylsynthase ␣ and ␤ (KS ␣ , KS ␤ ) are two genes (dpsC, dpsD) encoding proteins of unproven function, both absent from other type II PKS gene clusters. Also in contrast to other type II PKS clusters, the gene encoding the acyl carrier protein (ACP), dpsG, is located about 6.8 kbp upstream of the genes encoding the daunorubicin KS ␣ and KS ␤ . In this work, we demonstrate that the minimal genes required to produce aklanonic acid in heterologous hosts are dpsG (ACP), dauI (regulatory activator), dpsA (KS ␣ ), dpsB (KS ␤ ), dpsF (aromatase), dpsE (polyketide reductase), and dauG (putative deoxyaklanonic acid oxygenase). The two unusual open reading frames, dpsC (KASIII homolog lacking a known active site) and dpsD (acyltransferase homolog), are not required to synthesize aklanonic acid. Additionally, replacement of dpsD or dpsCD in Streptomyces sp. strain C5 with a neomycin resistance gene (aphI) results in mutant strains that still produced anthracyclines.Daunorubicin (daunomycin) and doxorubicin (adriamycin), clinically important anthracycline chemotherapeutic agents produced by Streptomyces sp. strain C5 and Streptomyces peucetius ATCC 29050, are synthesized by condensation of nine extender units derived from malonyl coenzyme A (CoA) onto a propionyl moiety to make a theoretical C 21 polyketide intermediate (16,(33)(34)(35) which is reduced at C-9 (34), cyclized, and aromatized to form aklanonic acid (Fig.

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“…The aglycone is formed by condensing nine extender units derived from malonyl coenzyme A onto a propionyl moiety to make a C 21 polyketide intermediate (21,23,(42)(43)(44)(45)50), which is reduced at C-9 from the carboxy terminus (43,44), cyclized, and aromatized to form aklanonic acid (18,19,23,40,(42)(43)(44). Aklanonic acid is converted in four steps to ε-rhodomycinone (3,7,15,16,30,40,42,44), an intermediate that is accumulated in large quantities by most daunorubicin-producing strains (27,42,44,46). It is postulated that ε-rhodomycinone is glycosylated by condensation with TDP-daunosamine (3,8,23,38,42,44) to form the first glycoside intermediate, here named rhodomycin D, which is then converted by a series of reactions to daunorubicin and doxorubicin.…”

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In vivo and in vitro bioconversion of epsilon-rhodomycinone glycoside to doxorubicin: functions of DauP, DauK, and DoxA

1997

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We recently determined the function of the gene product of Streptomyces sp. strain C5 doxA, a cytochrome P-450-like protein, to be daunorubicin C-14 hydroxylase (M. L. Dickens and W. R. Strohl, J. Bacteriol. 178: [3389][3390][3391][3392][3393][3394][3395] 1996). In the present study, we show that DoxA also catalyzes the hydroxylation of 13-deoxycarminomycin and 13-deoxydaunorubicin to 13-dihydrocarminomycin and 13-dihydrodaunorubicin, respectively, as well as oxidizing the 13-dihydro-anthracyclines to their respective 13-keto forms. The Streptomyces sp. strain C5 dauP gene product also was shown unequivocally to remove the carbomethoxy group of the -rhodomycinone-glycoside (rhodomycin D) to form 10-carboxy-13-deoxycarminomycin. Additionally, Streptomyces sp. strain C5 DauK was found to methylate the anthracyclines rhodomycin D, 10-carboxy-13-deoxycarminomycin, and 13-deoxy-carminomycin, at the 4-hydroxyl position, indicating a broader substrate specificity than was previously known. The products of Streptomyces sp. strain C5 doxA, dauK, and dauP were sufficient and necessary to confer on Streptomyces lividans TK24 the ability to convert rhodomycin D, the first glycoside in daunorubicin and doxorubicin biosynthesis, to doxorubicin. Daunorubicin (daunomycin [11,17]) and doxorubicin (14-hydroxydaunorubicin; adriamycin [2]) ( Fig. 1) are clinically important anthracycline chemotherapeutic agents (2, 11, 17), which are synthesized by a type II polyketide synthase (23,(42)(43)(44). The aglycone is formed by condensing nine extender units derived from malonyl coenzyme A onto a propionyl moiety to make a C 21 polyketide intermediate (21,23,(42)(43)(44)(45)50), which is reduced at C-9 from the carboxy terminus (43, 44), cyclized, and aromatized to form aklanonic acid (18,19,23,40,(42)(43)(44). Aklanonic acid is converted in four steps to ε-rhodomycinone (3,7,15,16,30,40,42,44), an intermediate that is accumulated in large quantities by most daunorubicin-producing strains (27,42,44,46). It is postulated that ε-rhodomycinone is glycosylated by condensation with TDP-daunosamine (3,8,23,38,42,44) to form the first glycoside intermediate, here named rhodomycin D, which is then converted by a series of reactions to daunorubicin and doxorubicin. Previously, however, the order of the reactions and the enzymes catalyzing some of the steps in the conversion of rhodomycin D to doxorubicin were not known. We recently showed that the doxA gene of Streptomyces sp. strain C5 encoded a cytochrome P-450 enzyme that was capable of conferring on Streptomyces lividans TK24 the ability to convert daunorubicin to doxorubicin (14). In the present work, we describe the enzymatic activities from Streptomyces sp. strain C5 that are responsible for the conversion of rhodomycin D to doxorubicin both in vivo and in vitro (Fig. 1). MATERIALS AND METHODSBacterial strains, plasmids, media, and genetic manipulations. Streptomyces sp. strain C5, originally obtained from the Frederick Cancer Research Center (33) and previously described in detail (3), was...

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Cloning, sequencing, and analysis of aklaviketone reductase from Streptomyces sp. strain C5

Cited by 45 publications

References 27 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

Minimal Streptomyces sp. strain C5 daunorubicin polyketide biosynthesis genes required for aklanonic acid biosynthesis

In vivo and in vitro bioconversion of epsilon-rhodomycinone glycoside to doxorubicin: functions of DauP, DauK, and DoxA

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