Vol. 176, No. 22JOURNAL OF BACrERIOLOGY, Nov. 1994, p. 7096-71010021-9193/94/$04.00+ 0Copyright C 1994, American Society for Microbiology

Molecular Cloning and Characterization of the Aklavinone11-Hydroxylase Gene of Streptomyces peucetius

subsp. caesius ATCC 27952tYOUNG-SOO HONG, CHEOL KYU HWANG, SOON-KWANG HONG,

YOUNG HO KIM, AND JUNG JOON LEE*

Genetic Engineering Research Institute, Korea Institute of Scienceand Technology, Yusung, Taejon 305-600, Korea

Received 24 June 1994/Accepted 16 September 1994

The gene encoding aklavinone 11-hydroxylase of Streptomyces peucetius subsp. caesius ATCC 27952 was

cloned and sequenced. The deduced amino acid sequence of the gene contains at least two common motifs ofwell-conserved amino acid sequences of several flavin-type bacterial hydroxylases. The hydroxylase gene isapparently transcribed from a single transcriptional start point. The phenotype of a dnrF mutant generated bygene disruption supports the idea that the dnrF gene encodes aklavinone li-hydroxylase.

Anthracyclines represent a class of antibiotics such as doxo-rubicin, daunorubicin, and aclacinomycin that exhibit extraor-dinary cytotoxic effects. Among them, doxorubicin is the mostuseful in antitumor chemotherapy (2, 18), but its cardiotoxicnature limits its dosage and long-term use (9). Consequently,there is a need to find new anthracyclines and to modifydaunorubicin and doxorubicin to overcome such serious sideeffects.A better understanding of doxorubicin biosynthesis, includ-

ing the organization and function of the biosynthetic genes, willprovide rational means by which to generate hybrid anthracy-clines with improved therapeutic effects (32). From biosyn-thetic studies, many intermediates have been isolated and theirroles in the biosynthesis of anthracyclines have been clarified(16, 31, 35). There is also some understanding of the enzymaticsteps in the biosynthetic pathway (11). In addition, a genecluster encompassing all the doxorubicin biosynthetic geneshas been cloned (25, 33), and the organization and function ofmany genes, including daunorubicin resistance genes (19, 20),regulatory genes (34), and the carminomycin 4-O-methyltrans-ferase gene (22), have been elucidated.We have also reported the cloning of a doxorubicin resis-

tance gene, which proved to be identical to drrAB (19), andpart of a biosynthetic gene cluster linked to the resistance genefrom Streptomyces peucetius subsp. caesius ATCC 27952 (20).Sequence analysis of the upstream region of this doxorubicinresistance gene revealed the presence of another open readingframe which is transcribed divergently from the drrAB genes.The deduced product has a significant resemblance to severalflavin-type bacterial hydroxylases (6, 17, 36, 37). The functionof this gene product has been studied by heterologous expres-sion, bioconversion assay, and gene disruption; the resultssupport our idea that the gene encodes aklavinone 11-hydrox-ylase. The aklavinone 11-hydroxylase activities of Streptomycessp. strain C5 and S. peucetius ATCC 29050 have been reportedby Connors et al. (11) and the location of the gene wassurmised by Guilfoile and Hutchinson (19) and Colombo et al.(10). However, detailed studies of the DNA sequence, gene

* Corresponding author. Electronic mail address: jjlee@geri4680.geri.re.kr.

t This paper is dedicated to Heinz G. Floss on the occasion of his60th birthday.

organization, and enzymatic activity have not been reportedexcept for a small part of the sequence adjacent to the drrAgene (19). Here, we present the complete sequence andtranscriptional organization of the aklavinone 11-hydroxylasegene (dnrF) along with evidence of its function.

Cloning and localization of the dnrF gene. We previouslycloned several DNA fragments containing a doxorubicin resis-tance gene from S. peucetius ATCC 27952 (20). Restrictionmapping and Southern analysis identified a 7.5-kb insert DNAin plasmid pMC1 that covers the 4.4-kb insert of pMC4 (20).The resistance gene cloned in pMC1 and pMC4 proved to bethe same as the drrAB genes reported by Guilfoile andHutchinson (19). The function of this cloned DNA, other thanself-resistance, was further analyzed by a complementation andbioconversion assay. The complementation effect of pMC1 wasconfirmed with the yellow mutant F4 (derived from Streptomy-ces sp. strain C5 producing E-rhodomycinone and baumycin),which produces only yellow anthracyclines lacking the C-11hydroxyl group (21). Upon transformation of strain F4 withplasmids, pMC1 caused the production of red color instead ofyellow color, but pMC4 and pMC73 (derived from pMC1 bythe deletion of the 6.0-kb PstI fragment) did not show any colorchange (data not shown). These results strongly suggested thatthe mutation could be complemented by the DNA fragmentlocated upstream of drrAB.To further narrow down the location of the gene that can

complement the yellow mutant, plasmid pMC1 was partiallydigested with BamHI and the 2.2-kb BamHI fragment wasligated into the BglII site of pIJ702 to create pMC213. Strep-tomyces lividans transformants containing various plasmidswere tested for their ability to bioconvert aklavinone intos-rhodomycinone by a feeding experiment in culture broth.After 2 days of growing transformants at 28°C in NDYEmedium (13) containing 25 ,ug of thiostrepton (Sigma) per ml,aklavinone was added to a final concentration of 10 ,ug/ml.After overnight incubation, cultures were analyzed for thepresence of c-rhodomycinone by thin-layer chromatography(TLC) and high-performance liquid chromatography (HPLC).For TLC analysis, plates (silica gel 60 F-254 [Merck & Co.])were developed with a solvent system of chloroform-methanol-formic acid (80:20:2 [vol/vol]) for glycosides and of hexane-chloroform-methanol (50:50:10 [vol/vol]) for aglycones andvisualized by their normal pigmentation and fluorescence

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NOTES 7097

Ps

SuJBg

Ba

2 kb

Ba Ba Ps Ba Ps

dnrF drrA B

Su/Bg

Ba

Aklavinone1 l-hydroxylase

activity

pMC 1

pMC 4

pMC 73 -

pMC 213 +

- pMC 1754 -

FIG. 1. Summary of phenotypes for aklavinone 11-hydroxylaseactivity by a set of derivative plasmids. In plasmid pMC1, the thin lineis used to indicate the DNA fragment from the vector and the thickline represents originally cloned DNA. All constructs were tested fortheir ability to confer the bioconversion of aklavinone to e-rhodomy-cinone on S. lividans 1326. Restriction enzyme abbreviations: Ba,BamHI; Bg, BglII; Ps, PstI; Su, Sau3AI.

under UV irradiation at 365 nm (data not shown). For HPLCanalysis of anthracyclines, a TSK gel ODS-120T column(Tosoh Corp., Tokyo, Japan) was eluted with a linear gradientof 0 to 100% acetonitrile containing 0.1% acetic acid, appliedin 20 min at a flow rate of 1.5 ml/min, and monitored with a

UV detector at 254 nm. As expected, the 2.2-kb BamHIfragment conferred the ability to convert aklavinone tor-rhodomycinone in S. lividans. On the other hand, plasmidpMC1754, which has a shorter BamHI fragment (1.6 kb), didnot endow S. lividans with bioconversion ability (Fig. 1). Theresults of these bioconversion experiments, along with otherreports (10, 19), indicated that the entire gene for aklavinone11-hydroxylase is located in the 2.2-kb BamHI fragment.Sequence analysis of the dnrF region. The nucleotide se-

quence of the 2.2-kb BamHI fragment of pMC213 DNA wasdetermined and analyzed by codon preference analysis (15) toidentify potential coding regions by virtue of a high degree ofbias toward G or C in the third position of Streptomyces codons(4). A complete open reading frame located in the 2.2-kb DNAfragment in the opposite orientation from the drrAB genes wasfound (Fig. 2). The putative open reading frame for aklavinone11-hydroxylase has a GTG translation initiation codon atnucleotides (nt) 352 to 354 and a TGA stop codon at nt 1819to 1821. A possible ribosome binding site, GGAGG, comple-mentary to the 3' end of the 16S rRNA of Streptomycescoelicolor A3(2) (3), is 6 bp upstream of the predicted startcodon. DnrF protein, as deduced from its nucleotide sequence,is composed of 489 amino acids and has a molecular mass of52,289 Da. Two inverted repeat sequences, which may functionas transcription terminators, are found downstream of theTGA stop codon.

112

232

352

472

592

712

832

952

1072

1192

1312

1432

1552

1672

1792

1912

2032

2152

BamHIGGATOCCG;TAGAc6AGACCGGCCGGGACGTMTGGTCCAGGCCO cTCCACCGC=CCrCGTMTBCCrGTACAGAaCMGACGMATGGCCCUTCGTcGAG

TGTTCACCTACCMAGTAGTCAClT,GGAGCOGACAAAGCGTGCACrGTAAGTTATTTCGGTCAMTACGTGAGTAC03ACGTTGAGG;ACACrrCATGGCGdrrAB RNA L. P2

I

A P1 dnrF IRNAATGGCTTCGTTCGCOCAGCCA= ACMGGCrAcCCGT=GAcTGCCTTCGAGTGGGcocG=ASCGTGGGTMCGAGCAcGACDCCAALTCAGGAGTGAGprimerl rbs

primer2

QMGCCTTGACGQGATGTCGATGAGTGGGGCGGCGGTCrCGWGGGGCrGTCCAC TCTrZ¢GGGGGGCG xTcGCrGGTAGCGGCATGCCV A L T K P D V D V L V V G GG L G 6 L S T A L F L A R R G A R V L L V E R H A

ASCACCTcLGTCCrGccAAGGCGGcAGQMAPCRCACCATGGLACrG GL ADICGAGALCCGGACACAT GAQGGDGACS T S V L P X A A G Q N P R T H E L F R F G G v A D E I L A T D D I R G A a G D

TTCACCUTCMGGTCGrGAaGCSOTCDCGTl:ACGCrTCGCGAGAGCTTCGCGAACGGrCUOACGGAACAGMC LF T I X V V E R V G G R v P A Q L R E S F E E L V G A T E 0 C T P H P N A L A P

CAGGACCGRGGTGGAGYG tG_ A QLRSGGATC ELGATLCTPH9LALGACO D R V E P V L V A H A A K H G A E I R F A T E L T S F Q A G D D G V T A R L R

GACCGG6CrACOGGAGTAGAGCACCGTGcGAAcGcGGOACGAAGCCGTCIGCIGGTCGGCGBATGCCACAGAGGGCACCD L G TD A E S T V S A R Y L V A A D G P R S A I R E S L G I T R HC G G L A

CACTTATGGGCCCTCGICICTII= A_ _A a G CCGH F T A V I F E A D L T A V V P P G S T G E YA L LH P D F V G T F G P T D R P

AACCGGCACrIATCTACGCaCA0CAGXGACTQ3ACDCFACGACCGCGCCAGCGTTO32GAA9CGGACN R H T F Y V R Y D P E R G E R P E D Y T P O R C T E L I R L A Y D A PVG P

LYYDESLAIVAQRAPIIILLGSY E_G _YLG R_S

D I L D I Q A N D H A A Y I A D R V R E 6 P Y L L Y G D A k X V T P P r a G m 6

GGCAACACMCCArCGGGCACGGCATCOACOTGOOCrggr, r,ITVCaG N T k I G H 6 F D V A N X L A A V L R G E k 6 E I L L D S Y 6 A D G S L V S R

LCOLCO GC 1rLLACTCATAPEC6 AGGACAAARAAR IIIASASALSLACL V V D E S L A I Y A Q R H A P H L L G S V P E E R C T A Q V V L 6 F R Y R S T

BamHIGICCGCSCACXLA _clnlo L CCAOC'GrUCGAWAACTaCMICA V k k E D D D P E P T E D P R R P S G R P G F R A P H V %1 I E Q D 0 T R R S T

GI L LU ILI nAr------- C LC=COiCCACTgV E L F 6 D C 11 V L L A A P E 6 0 Ai 1 G Q A A A RA RI 1{ A S A S T S I S S A

GCGATOCGs=7 7 C sU.rgGGMAA12&KWACrCGGATOCGGGa;GGa63 _ _) B T A GGIw*gGLGA tG S P P P P A N a

AGC=ACCAGC1 6_=.-X'GAO -CGCCG

CrCt1ACACCCrGTATGTAATGAATAIICAGGCCrCGGCACMACGCGAL=AGATCGATGMTGCCAGCGUACrCCA GjUACCGGCCG

GcATGTIGGTGGTCGACOMCGTGAGGATITCCG7COCONCR&5FIG. 2. Nucleotide sequence of the dnrF region and the deduced amino acid sequences of DnrF. The bent arrow at nt 317 indicates the

transcriptional start point determined by primer extension. Another transcriptional start point, at nt 211 and obtained only with S. lividans 1326harboring pMC213, is depicted by a small bent arrow. Sequences complementary to primers are indicated by thin arrows. Putative ribosome bindingsites are underlined. Two inverted repeat sequences are indicated by opposite thick arrows.

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A)

DnrF 3 LTKPD VDVLWGGGL GGLSTALFLA RRGARVLLVE RHASTSVLPK 47Tc G 12 LSTEE VPVLIVGGGL TGLSAALFS QHGVSCRLVE KRCllVLTR 56SchC 10 DTVHR VPVLWGGSL VGISISVFLG RLGVRHMLVE RHAGTSVHPR 54TfdB 5 ... IE TDVLWGTGP AGASAGALLA RYGVR1LIN KYNWTAPTPR 46Consensus DVLIVGAGP TGL A LLA GVRT E

B)** *** ******* * * * * * ***** ********** * ** ***** **

DnrF 300 IREGPVLLVGDAAKVTPPGMGGNTAIGHGFDVAWKLAAVLRGEAGERLLDSYGADGS 358Tc.G 326 YRSGRVFLAGDAAHVHPPAGAFGANGGIQDAHNLAWKLAAVLKGTAGDALLUrYEQERL 384SchC 303 YRAGRVFLAGISAHEnPTGAFGSNTGI_DANLAWKLAVLGGIAGDGLLDTYDAERR 361TfdB 301 LQQGRVFCAGDAVHRHPPTNGLGSNTSIQDSFNLAWKIAMVLNGTADESLLUTYTIERA 359

FIG. 3. Alignment of amino acid sequences from DnrF and otherhydroxylases. This alignment shows the similarity between the dnrFgene product and different hydroxylases around the two motifs in manyFAD- and NAD(P)-dependent enzymes. The two well-conservedmotifs are a possible ADP-binding domain in the N-terminal half (A)and a putative ribityl-binding domain in the C-terminal half (B); theyare compared. Highly conserved residues are in boldface type or areindicated by an asterisk with a putative active site of aspartic acid.TcmG, tetracenomycin hydroxylase from S. glaucescens (12); SchC, apolyketide spore pigment biosynthetic enzyme from S. halstedii (7);TfdB, 2,4-dichlorophenol hydroxylase from A. eutrophus (26).

Comparison of the dnrF gene and DnrF protein. Thesequences of dnrF and its translated product (DnrF) werecompared with those of known genes and proteins by theFASTA program (15). DnrF has a significant resemblance toTcmG protein from Streptomyces glaucescens (12) as well as toother flavin-type bacterial hydroxylases, such as TfdB fromAlcaligenes eutrophus (26) and SchC protein from Streptomyceshalstedii (7). Alignment of the N- and C-terminal regions ofseveral flavin adenine dinucleotide (FAD)- or NAD(P)-depen-dent enzymes has revealed two motifs of well-conserved aminoacid sequences (17, 27, 36, 37), the first motif for the binding ofthe ADP moiety (Fig. 3A) and the second one for the bindingof the ribityl chain of FAD (Fig. 3B). In the second motif, theregion around an aspartic acid is thought to be important forthe binding of the ribityl chain (17). The putative DnrF proteinalso contains these two well-conserved amino acid sequences,which suggests that aklavinone 11-hydroxylase is a FAD- orNAD(P)-dependent enzyme.The high degree of similarity between DnrF and TcmG is

intriguing because TcmG introduces three atoms of oxygeninto TcmA2, whose structure is quite different from that ofaklavinone (12). A partially purified protein fraction from thecrude extract of S. lividans harboring pMC213 showed aprotein band with the expected molecular weight for DnrF andthe enzymatic activity to convert aklavinone into e-rhodomy-cinone only in the presence of NADPH (unpublished data),consistent with the report of Connors et al. (11). Similar resultshave been reported for the bioconversion reaction of TcmA2to TcmC when crude extracts of S. lividans carrying pWHM1019 were used as the enzyme source (12). Therefore, the highdegree of amino acid sequence similarity and the same cofac-

tor requirement imply a close evolutionary relationship be-tween TcmG and DnrF.

Disruption of the dnrF gene in the chromosome. To verifythe function of dnrF, a gene disruption experiment was carriedout. An internal 1.3-kb BamHI-SphI fragment of the dnrFcoding region was subcloned into pUC19 to give pUBs. The1-kb SalI fragment containing the neomycin resistance genefrom the pFDNeo-S plasmid (14) was subcloned into the PstIsite of the dnrF fragment in plasmid pUBs by blunt-endligation, which resulted in pUEH-neo. Insertion of the 2.3-kbEcoRI-HindIII fragment from pUEH-neo into pKC1139 (5)resulted in pKN23 (Fig. 4A). pKN23 was introduced into S.peucetius ATCC 27952 by transformation, and transformantswere incubated at 39°C to eliminate autonomously replicatingplasmids (8, 23). A neomycin-resistant transformant whichproduced yellow pigments instead of red ones was isolated andnamed PKN8. The yellow phenotype of the mutant was stableat the permissive temperature in the absence of neomycin.The integration of pKN23 by homologous recombination

was verified by a Southern blot hybridization experiment witha digoxigenin-labeled EcoRI-HindIII fragment (2.3 kb) ofpKN23 as the probe. PstI-digested chromosomal DNA ofPKN8 yielded 8- and 5-kb hybridizing bands in place of the6-kb band of S. peucetius ATCC 27952 (Fig. 4B). This resultindicated that the dnrF gene in strain PKN8 was disrupted bythe integration of plasmid pKN23 through single-crossoverhomologous DNA recombination.The metabolites from strain PKN8 and those from S.

peucetius ATCC 27952 were analyzed in parallel by TLC andHPLC as described above. By TLC, only red metabolites weredetected in S. peucetius ATCC 27952 and a strain harboringautonomously replicating pKN23 and only yellow metaboliteswere detected in the mutant strain PKN8 (data not shown). ByHPLC, the peaks of 11-deoxycarminomycin and 11-deoxy-daunorubicin were determined by comparison with the reten-tion times of standards and by cochromatography of sampleswith standards (Fig. 4C). The peaks corresponding to dauno-rubicin, aklavinone, and e-rhodomycinone are also indicated inFig. 4C. It is known that in the biosynthetic pathway ofdoxorubicin, C-11 hydroxylation of aklavinone can be bypassedto yield a series of yellow 11-deoxy metabolites (10). Thus,these data clearly indicate that the dnrF gene is indeedencoding aklavinone 11-hydroxylase.

Transcriptional analysis of the dnrF gene. To determine theexact genomic size and the direction of dnrF transcription, wecarried out a transcriptional analysis of total RNA preparedfrom 2- and 3-day cultures of S. peucetius ATCC 27952. Forlow-resolution S1 mapping (1, 24), single-stranded DNAs wereprepared from M13mpl8 carrying the 2.2-kb BamHI fragmentof pMC213 in both orientations. DNA protected against S1nuclease treatment was analyzed by Southern blot hybridiza-tion (28), and an approximately 1.5-kb protected fragmentproduced in the opposite direction of the transcript of drrABwas detected by using the digoxigenin-labeled 1.3-kb BamHI-SphI fragment of pMC1 as a probe (data not shown). Primerextension analysis, following the methods described by Stein et

FIG. 4. Generation of a dnrF-deficient mutant strain and its phenotypic analysis. (A) Scheme representing the integration of pKN23 (see thetext for construction) into the homologous region of S. peucetius ATCC 27952 chromosomal DNA and the fragments generated after PstI digestion.Abbreviations: Ba, BamHI; Ps, PstI; Sp, SphI; neo, neomycin. (B) Analysis of chromosomal DNA by Southern blot hybridization. PstI-digestedchromosomal DNA of wild-type S. peucetius ATCC 27952 (lane 1) and the mutant strain PKN8 (lane 2) were analyzed by Southern blothybridization by using the 2.3-kb EcoRI-HindIII fragment from pUEH-neo as a probe. (C) HPLC analysis of the metabolites produced by the wildtype (tracing a), the wild type harboring plasmid pKN23 (tracing b), and the mutant strain PKN8 (tracing c). Abbreviations: C-rho,e-rhodomycinone; dnr, daunorubicin; akn, aklavinone; lld-dnr, 11-deoxydaunorubicin; lid-car, 11-deoxycarminomycin.

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NOTES 7099

APs '

Hd

PS Ba B P SpB1 I I 'I I I

Ec

Ps 6kb

PS B BI I I

PS

s L3kb PS

PSnzSPs Sp Bs

PS

Ps 8kb PS

Ps Stkb

PS 2.3kb PS

b

PS B

6kb-

2.3kb_

-'8kb_S5kb

- 23kb

PS

C0a

C1-0

:I

c

1.- T-

"T- I'

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primer I prinicr2I Ir I I

51

GGT

GC

3'

5

-GG

GT

\c3C~~~~~~~~~~3,FIG. 5. Primer extension analyses of the dnrF gene. RNA (25 ,g)

from S. peucetius ATCC 27952 was hybridized to primers (Fig. 2)complementary to the 5' end of the dnrF gene as described in the text.Lane 1, primer 1 and yeast tRNA as a negative control; lane 2, primer1 and RNA from 2-day culture; lane 3, primer 1 and RNA from 3-dayculture; lane 4, primer 2 and RNA from 3-day culture; lane 5, primer2 and RNA from 2-day culture; lane 6, primer 2 and yeast tRNA as a

negative control; lanes A, C, G, and T, sequencing reactions generatedwith primers 1 (left) and 2 (right). Arrows indicate bands found in bothprimer extension reactions.

al. (29) and Guilfoile and Hutchinson (19), was used to map

mRNA 5' ends more precisely. Superscript Moloney murineleukemia virus reverse transcriptase (Bethesda Research Lab-oratories) was used for extension reactions. Two 30-merprimers, primer 1 (5'-GAGACCGCCGCCCACCACGAGGACATCGAC-3') and primer 2 (5'-ATCGACATCCGGCTITCGTCAAGGCCACCTC-3'), were designed to have complemen-tary sequences to the putative dnrF transcript and to corre-spond to nt 373 to 402 and 349 to 378, respectively. As shownin Fig. 5, the apparent 5' end of dnrF mRNA was the G residue35 bp upstream from the GTG translational start codon. Thispromoter (named Pl in Fig. 2) lacks obvious -10 and -35consensus sequences found in several other Streptomyces pro-moters (30).When RNA of S. lividans harboring pMC213 was analyzed

by primer extension, two transcriptional start points wereobserved. One (P1) was located at the same position (nt 317; a

G residue) as that of S. peucetius ATCC 27952, and another(P2) was the T residue at nt 211 (Fig. 2 and detailed data notshown). It would be interesting to determine whether thisdifference reflects any differential expression of regulatoryfactors in S. peucetius and S. lividans.

Nucleotide sequence accession number. The DNA sequencedata described in this paper have been deposited at GenBankunder the accession number U09844.

We are grateful to Anna L. Colombo of Farmitalia Carlo Erba forsamples of 11-deoxycarminomycin and 11-deoxydaunorubicin and to

Mercian Co. of Japan for aklavinone. We also thank C. R. Hutchinsonfor helpful discussions and critical reading of the manuscript and D. A.Hopwood for Streptomyces strains and plasmids.

This work was supported by a grant from the Ministry of Science andTechnology of Korea.

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