Vol. 176, No. 20
JOURNAL OF BACTERIOLOGY, Oct. 1994, p. 6270-6280
0021-9193/94/$04.00+0 Copyright © 1994, American Society for Microbiology
Isolation and Sequence Analysis of Polyketide Synthase Genes from the Daunomycin-Producing Streptomyces sp. Strain C5 JINGSONG YE, MICHAEL L. DICKENS, RICHARD PLATER, YUN LI, JESSICA LAWRENCE, AND WILLIAM R. STROHL* Department of Microbiology, Ohio State University, Columbus, Ohio 43210-1292 Received 27 April 1994/Accepted 15 August 1994 A contiguous region of about 30 kbp of DNA putatively encoding reactions in daunomycin biosynthesis was isolated from Streptomyces sp. strain C5 DNA. The DNA sequence of an 8.1-kbp EcoRI fragment, which hybridized with actI polyketide synthase (PKS) and actIII polyketide reductase (PKR) gene probes, was determined, revealing seven complete open reading frames (ORFs), two in one cluster and five in a divergently transcribed cluster. The former two genes are likely to encode PKR and a bifunctional cyclase/dehydrase. The five latter genes encode: (i) a homolog of TcmH, an oxygenase of the tetracenomycin biosynthesis pathway; (ii) a PKS Orfl homolog; (iii) a PKS Orf2 homolog (chain length factor); (iv) a product having moderate sequence identity with Escherichia coli 0-ketoacyl acyl carrier protein synthase III but lacking the conserved active site; and (v) a protein highly similar to several acyltransferases. The DNA within the 8.1-kbp EcoRI fragment restored daunomycin production to two dauA non-daunomycin-producing mutants of Streptomyces sp. strain C5 and restored wild-type antibiotic production to Streptomyces coelicolor B40 (actVII; nonfunctional cyclase/ dehydrase), and to S. coelicolor B41 (actill) and Streptomyces galaeus ATCC 31671, strains defective in PKR activity.
Daunomycin (daunorubicin; Fig. 1), doxorubicin (formerly called adriamycin), and aclacinomycin A, commercially important anthracycline anticancer agents, are polyketides produced from a propionyl starter unit and nine malonyl extender units (43). Previous data from our laboratory on the interfunctionality of anthracycline polyketide synthase (PKS) and actinorhodin PKS components suggest that daunomycin PKS is a type II enzyme complex (5, 44). Type II PKS systems are composed of small, unifunctional or bifunctional enzymes (23, 25). The products of type II PKSs are typically aromatic polyketides which are synthesized by using acetyl or propionyl starter units and only a malonyl moiety for extension (23). The sequences of at least eight Streptomyces type II PKS gene clusters have been published previously (23, 25). Only in the cases of the tetracenomycin (40) and actinorhodin (5, 19, 34, 55) PKSs, however, have biochemical characterizations also been carried out. Thus, most of the information on type II PKSs is derived from the strong conservation of gene structure among the PKS genes (23, 25) and cross-functionality of the components (5, 26, 34, 41). We describe here the structure of a gene region from Streptomyces sp. strain C5 that putatively encodes daunomycin biosynthesis and show that it has a significantly different overall structure from other type II PKS gene regions.
For liquid cultures, S. lividans and S. galilaeus were grown in YEME medium (22) supplemented with 20% (wt/vol) sucrose, and Streptomyces sp. strain C5-derived strains were grown in NDYE medium as described previously (10). Streptomycete strains carrying pIJ486 (51) or derivatives of it were grown on OOH
A 00 0
H
0
OH
OCH,
B HO 1
12
1
OH 1
0
2 H
CH
MATERUILS AND METHODS Bacterial strains and media used. Streptomyces sp. strain C5 and mutants derived from it have been described previously (3, 4). Streptomyces lividans TK24 (22), used as a recombinant host strain, was obtained from D. A. Hopwood. Streptomyces coelicolor mutants, described by Rudd and Hopwood (37), were obtained from H. G. Floss. Streptomyces galilaeus ATCC 31671, which lacks a functional polyketide reductase (PKR), has been described previously (5, 50).
14
4$~o
NHNH2 FIG. 1. The aglycone component of daunomycin is synthesized through a theoretical polyketide intermediate (A) from a propionyl starter molecule and nine C2 units derived from malonyl moieties (43). The intermediate, E-rhodomycinone (B), is also accumulated by cultures of Streptomyces sp. strain CS. The final product, daunomycin (C) (R, =CH3), contains the 2,3,6-trideoxy-4-aminohexose, daunosamine, derived from TDP-D-glucose (48), attached at C-7 of the aglycone. Doxorubicin (R, = CH20H), produced by S. peucetius subsp. caesius, is a related anthracycline. References 4, 43, and 44 detail the proposed pathway for daunomycin biosynthesis in Streptomyces sp. strain CS.
* Corresponding author. Mailing address: Department of Microbiology, Ohio State University, 484 West 12th Ave., Columbus, OH 43210-1292. Phone: (614) 292-1919. Fax: (614) 292-8120. Electronic mail address:
[email protected].
6270
DAUNOMYCIN PKS GENES
VOL. 176, 1994
6271
B 5.0 kbp
C
A
Bg,2800 S .
K,12300
K,6800 Bg,7300 S
K,3950
S,12800 X
B,13900
S,9000
E /E,25100 X D
Bg
B .B E,27800 BX B S Kg30600
K,20000
kr2 dauA- iAB4dai- iA
ikrl acpA
iaJ &X1
E,17000 B S S
A=A daA mt dak
oJfA oft of' odD
otyf
_A
_ _
worsts~Od pANT121
pANT235
pANT1I3
pANT152
pANT128
pANTISI
pANT164
pANT114
Phage P7 Phage D8
Phage P3
Phage D8-2 FIG. 2. Restriction map of part of the daunomycin biosynthesis gene cluster from Streptomyces sp. strain C5. The DNA sequence of the 8.1-kbp EcoRi fragment spanning the region from E (nucleotide 17000) to E (nucleotide 25100) is described in detail in this paper. The hatched boxes represent fragments to which probes hybridized as follows: a 6.6-kbp BamHI-BglII fragment of phage D8 to which the S. peucetius dnrI-dnrJ probe hybridized (A); a 4.3-kbp SstI fragment of phage P7 to which the actIII (PKR) and actI (PKS) probes hybridized (B); 2.9- and 2.6-kbp BamHI fragments (C and D), respectively, of phage P7 to which the actI probe hybridized (actIll hybridized to only fragment C); and a 4.1-kbp SstI fragment of phage P3 to which the methyltransferase oligonucleotide probe (MT probe) hybridized (E). The sequences of dauA-orfA (PKS Orfl),
dauA-orfB (PKS Orf2), dauA-orfC (putative daunomycin PKS Orf3), dauA-orfD (putative propionyl-CoA:acyl-carrier-protein acyltransferacylase), dau4-orfE (tcmH homolog; putative aklanonic acid-anthraquinol oxygenase), dauA-orfF (actVII homolog; cyclase/dehydrase), and dauB (actIII homolog; PKR) are described in Results and Discussion. The sequences and analyses of the genes encoding acyl carrier protein (acpA), carminomycin methyltransferase (dauK), and dauI and dauJ (dnrI and dnrJ homologs) will be described elsewhere (14, 15, 53). The ORFs marked krl and kr2 represent genes encoding ketoreductases other than dauB found in this region as mentioned in the text, and mt represents the ORF encoding a methyltransferase-like gene downstream of dauA-orfD. The inserts of plasmids pANT113, pANT114, pANT121, pANT128, pANT151, pANT152, pANT164, and pANT235 and phages D8 (14.4-kbp insert), D8-2 (9.5-kbp insert), P7 (13.5-kbp insert), and P3 (12.0-kbp insert) are indicated below the map. Abbreviations for restriction endonuclease sites: B, BamHI; Bg, BglII; E, EcoRI; K, KpnI; S, SstI; X, XhoI.
plates of solid R2YE medium (22) containing 40 jig of thiostrepton per ml. Escherichia coli JM83, used to propagate plasmids for sequencing and restriction analyses, was grown in Luria-Bertani medium (33). Plasmids were introduced into E. coli by standard transformation procedures (33). Other E. coli strains used in this study are described with their particular applications. Ampicillin was added at a concentration of 100 jig/ml to cultures of E. coli harboring pUC19 or derivatives made from it. General genetic manipulations. Procedures for protoplast formation, transformation, and regeneration of protoplasts for Streptomyces sp. strain C5 and mutants derived from it have been described elsewhere (30). S. lividans was transformed with plasmid DNA as described by Hopwood et al. (22). Procedures used for the preparation of Streptomyces plasmid and chromosomal DNAs were described by Hopwood et al. (22). Digestion of DNA with restriction endonucleases was carried out according to the manufacturer's directions. Restriction mapping and other routine molecular methods used in this work were performed as described by Maniatis et al. (33). Library construction, phage isolation, and screening. A complete genomic library of Streptomyces sp. strain C5 DNA was constructed in lambda EMBL3 (20). The streptomycete DNA was partially digested with BamHI, size selected to a range of 9 to 20 kbp on agarose gels, eluted from the gels, and ligated into the phage vector with T4 DNA ligase. The recombinant phages were packaged in vitro and used to infect E. coli LE392 (Pharmacia). Phage DNA was prepared from E. coli by procedures described by Maniatis et al. (33).
The phage library of Streptomyces sp. strain C5 DNA was screened for hybridization with either linear DNA fragments containing genes or with degenerate, high-G+C-biased oligonucleotides constructed for DNA sequences expected to be clustered with the daunomycin PKS biosynthesis genes. For probes derived from plasmids, the DNA inserts were removed from the plasmid DNA by using the appropriate restriction endonucleases and isolated by the phenol-freeze-fracture method (24). The inserts were then labelled by the 32P random primer procedure (18) with 50 p.Ci of [cz-32P]dCTP per tig of DNA and random primers (Stratagene, La Jolla, Calif.). Alternatively, degenerate, consensus G+C-biased oligonucleotides were end labelled with 50 jiCi of [y-32P]ATP (>7,000 Ci/mmol; ICN Biochemicals) and DNA kinase (33). Colony hybridizations were carried out by transferring wellseparated E. coli colonies (approximately 300 per plate) containing the phage library to BA-85 nitrocellulose filters (Schleicher & Schuell, Inc., Keene, N.H.) by the Southern blot method (33). Hybridizations were carried out as described by Hopwood et al. (22), with SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate) buffer for DNA fragments and SSPE (0.15 M NaCl, 0.01 M NaH2PO4, 1 mM EDTA [pH 7.4]) buffer for oligonucleotides. DNA sequencing. Plasmids containing inserts to be sequenced were isolated by the methods of Kraft et al. (28). The DNA was sequenced in both directions by the dideoxynucleoside chain termination method (38) with Sequenase version 2.0 (United States Biochemical Corp., Cleveland, Ohio), doublestranded templates, and labelling with [ot-thio-35S]dCTP (1,000 to 1,500 Ci/mmol; Dupont-New England Nuclear, Boston,
6272
YE ET AL.
J. BACTIERIOL.
TABLE 1. Bacterial phages and plasmids used and constructed in this study' Phage or
Relevant
plasmid Phages EMBL3 D8 D8-2 P3 P7
Plasmids pUC19 pIJ486 pANT12 pANT14 pANT15 pANT113 pANT114 pANT121 pANT122 pANT128 pANT151 pANT152 pANT164 pANT233
pANT235
characteristt's
Source or
crcncs1reference Derivative of lambda EMBL3 containing ca. EMBL3 containing ca. EMBL3 containing ca. EMBL3 containing ca.
14.4-kbp insert of Streptomyces sp. strain C5 DNA containing daunomycin biosynthesis genes 9.5-kbp insert of Streptomyces sp. strain C5 DNA containing daunomycin biosynthesis genes 12.0-kbp insert of Streptomyces sp. strain C5 DNA containing daunomycin biosynthesis genes 13.5-kbp insert of Streptomyces sp. strain C5 DNA containing daunomycin biosynthesis genes
2.686 kbp; Ampr, E. coli plasmid 6.2 kbp; derivative of pIJ101; HC, Thior 12.8 kbp; pIJ350 with 8.8-kbp PstI insert from S. coelicolor containing actl, actIII, actVII, actIV, and act~b loci 3.79 kbp; pUC19 containing 1.1-kbp BamHI fragment with the actIII gene from S. coelicolor 4.9 kbp; pUC19 with 2.2-kbp BamHI fragment containing parts of actI-orfl and actI-orJ2 genes of S. coelicolor 6.8 kbp; pUC19 containing a 4.1-kbp SstI fragment hybridizing to methyltransferase gene probe 5.34 kbp; pUC19 with 2.65-kbp EcoRI fragment subcloned from phage P3 containing dauK 10.9 kbp; pUC19 containing 8.2-kbp EcoRI DNA insert from Streptomyces sp. strain C5 DNA in phage P7 14.3 kbp; pIJ486 containing 8.2-kbp EcoRI insert from pANT121 3.8 kbp; 1.2-kbp SstI subclone of pANT235 in pUC19 containing partial sequences of Streptomyces sp. strain C5 homologs of dnrI and dnrJ 7.8 kbp; 5.1-kbp SstI fragment from phage P7 in pUC19 10.5 kbp; 7.8-kbp KpnI fragment from phage P7 in pUC19 9.2 kbp; pIJ486 containing 3.0-kbp EcoRI-KpnI insert from pANT121 6.5 kbp; pUC19 containing 3.8-kbp BamHI fragment from pWHM333 containing dnrI and dnrJ genes of S. peucetius
Pharmacia This work This work This work This work J. N. Reeve 51 5 This work This work This work This work This work This work This work
This work This work This work This work
(45)
9.3 kbp; pUC19 containing a 6.6-kbp BamHI-BglII DNA fragment from Streptomyces sp. strain C5 carrying the dnrRlhybridizing region
a Abbreviations: HC, high-copy-number plasmid;
This work
Thior, thiostrepton resistance; Amlpr, ampicillin resistance; act, actinorhodin genetic loci.
Mass.). Conditions for DNA sequencing are described in the brochure accompanying the Sequenase enzyme (United States Biochemical Corp.). Sequencing reactions were carried out with 7-deaza-dGTP nucleotide mixes to reduce compressions. Universal and reverse primers were used to obtain the initial sequences in the inserts before the generation of specific primers for the sequences within the inserts. DNA and deduced amino acid sequence analyses and database searches. DNA sequence data were analyzed by means of Clone Manager (Science and Educational Software, Inc., Stateline, Pa.), Genepro (Riverside Scientific, Inc., Seattle, Wash.), and the Sequence Analysis Software package of the Genetics Computer Group (Madison, Wis.) (13). The sequenced DNA was analyzed by FRAME (8) and CODON PREFERENCE (52) algorithms and use of an IBM-PC program (27) to determine the presence and direction of potential open reading frames (ORFs). Amino acid sequences of potential gene products were compared with those in the databases by means of BLAST (1). Detection of anthracyclines. Strains to be tested for antibiotic production were grown for 7 days in 50 ml of NDYE or YEME medium supplemented with thiostrepton (10 ,ug/ml) in a 250-ml Erlenmeyer flask containing a coiled spring (10). Cultures were extracted, and anthracyclines were analyzed by thin-layer chromatography and high-performance liquid chromatography (HPLC) as described previously (10, 11). Nucleotide sequence accession number. The DNA sequence data described in this paper have been deposited at GenBank with the accession number L34880.
RESULTS AND DISCUSSION Construction of probes for Streptomyces sp. strain C5 daunomycin biosynthesis gene cluster. A 6.6-kbp BamHI-BglII DNA fragment from Streptomyces sp. strain C5 hybridized at high stringency to a 3.8-kbp BamHI fragment carrying dnrI and dnrJ from Streptomyces peucetius (45). Thus, a partial library of
BamHI- and BglII-digested Streptomyces sp. strain C5 DNA of around 6.6 kbp was ligated into BamHI-digested pUC19 and the mixture was used to transform E. coli JM83. Plasmid pANT235, containing a 6.6-kbp BamHI-BglII DNA insert that hybridized at high stringency with the S. peucetius dnrI-dnrJ probe (Fig. 2), was isolated by colony hybridization. A probe for the PKR gene was constructed by subcloning the 1.1-kbp BamHI fragment containing S. coelicolor actIII (21) from pANT12 (5) into pUC19 to make pANT14. A PKS probe was constructed by subcloning a 2.2-kbp BamHI DNA fragment (32), containing most of the S. coelicolor actI-orfl and orf2 genes (19), from pANT12 (5) into pUC19 to make pANT15. In order to isolate the Streptomyces sp. strain C5 genes encoding aklanonic acid methyltransferase (10) or carminomycin methyltransferase (12, 31), a consensus, degenerate, high-G+C-biased, 47-mer oligonucleotide probe (5'-GGC[G]-
GAC-TTC-TTC-GAG-CCG[C]-CTG[C]-CCG-CGC[G]AAG-GCC[G]-GAC-GCC-ATC-ATC-CT-3') (MT probe) was synthesized for a highly conserved region (GDFFEPLPRKADAIIL) of several methyltransferases, including S. peucetius DnrK (31), Streptomyces glaucescens TcmO and TcmN
FIG. 3. Nucleotide sequence of the 8.1-kbp EcoRI DNA fragment from Streptomyces sp. strain CS containing the daunomycin PKS region. The deduced amino acid sequences of the proposed translation products are given below the nucleotide sequence. For genes reading from right to left, the second strand reading in the opposite direction has been added. Where double-stranded sequence is shown, the top strand reads from 5' to 3'. The numbers at the right indicate nucleotide positions. Potential ribosome binding sites (rbs) are underlined. Inverted repeats (dashed arrows) and stop codons (*) are indicated.
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