JOURNAL OF BACTERIOLOGY, June 1990, p. 3427-3434 Vol. 172, No.

6
0021-9193/90/063427-08$02.00/0
Copyright © 1990, American Society for Microbiology

Cloning and Expression of Daunorubicin Biosynthesis Genes from

Streptomyces peucetius and S. peucetius subsp. caesius
SHAREE L. OTTEN, KIM J. STUTZMAN-ENGWALL, AND C. RICHARD HUTCHINSON*
Department of Bacteriology and School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53706
Received 21 December 1989/Accepted 6 April 1990

Genes for the biosynthesis of daunorubicin (daunomycin) and doxorubicin (adriamycin), important
antitumor drugs, were cloned from Streptomycespeucetius (the daunorubicin producer) and S. peucetius subsp.
caesius (the doxorubicin producer) by use of the actIltemla and actII polyketide synthase gene probes.

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Restriction mapping and Southern analysis of the DNA cloned in a cosmid vector established that the DNA
represented three nonoverlapping regions of the S. peucetius subsp. caesius genome. These three regions plus
an additional one that hybridized to the same probes are present in the S. peucetius genome, as reported
previously (K. J. Stutzman-Engwall and C. R. Hutchinson, Proc. Natl. Acad. Sci. USA 86:3135-3139, 1989).
Functional analysis of representative clones from some of these regions in S. lividans, S. peucetius ATCC 29050,
S. peucetius subsp. caesius ATCC 27952, and two of its blocked mutants (strains H6101 and H6125) showed
that many of the antibiotic production genes reside in the region of DNA represented by the group IV clones.
This conclusion is based on the production of r-rhodomycinone, a key intermediate of the daunorubicin
pathway, in certain S. lividans transformants and on the apparent complementation of mutations that block
daunorubicin biosynthesis in strains H6101 and H6125. Some of the transformants of strains 29050, 27952, and
H6125 exhibited substantial overproduction of r-rhodomycinone and daunorubicin.

Daunorubicin (daunomycin) and doxorubicin (adriamycin)
are commercially important antibiotics with potent antitu-
mor activity. Daunorubicin, first isolated in 1963 from Strep-
tomyces peucetius (6, 7), was subsequently found in a
number of other Streptomyces spp. (27). Doxorubicin was
isolated in 1969 from S. peucetius subsp. caesius, a mutant
of the wild-type strain (2), and has important clinical appli-
cations in cancer chemotherapy (1), even though both dox-
orubicin and daunorubicin cause cardiotoxic side effects that
are dose limiting and irreversible. The production costs of
these antibiotics are high because of low titers and formation
of a complex mixture of products by the producing bacteria.
Therefore, a genetic study of the biosynthesis of daunorubi-
cin and doxorubicin was undertaken in our laboratory to
elucidate the organization and regulation of the biosynthetic
genes, with the hope that it may also lead to overproducing
strains or strains with a simpler spectrum of secondary
metabolites. This work has been facilitated by the recogni-
tion that the early genes involved in polyketide biosynthesis
by other streptomycetes, such as the S. coelicolor actI and
actIlI genes (9, 17) and the S. glaucescens tcmIa genes (20),
hybridize to the analogous genes in other polyketide produc-
ers (16) and by the fact that antibiotic genes have been found
to be clustered in all of the examples studied (14).
A previous report (24) from this laboratory demonstrated
that the daunorubicin producer S. peucetius (ATCC 29050)
contains five nonoverlapping regions of DNA that hybrid-
ized to the actIl/tcmIa or actIII probe. The properties of S.
peucetius and S. lividans strains transformed with clones
from four of these regions supported the belief that many of
the daunorubicin production (dnr) genes resided in region IV
but also raised the possibility that bona fide dnr genes or
genes that influence daunorubicin production and self-resis-
tance were present in the three other regions (24). The latter
idea is best tested by examining the effects of deletions in
*
Corresponding author.
3427
each of these four regions on daunorubicin production and
resistance.
Since our preliminary evidence (24) indicated that some
portion of region II had been deleted from S. peucetius
subsp. caesius (ATCC 27952), we made a detailed study of
the properties of cosmid clones from three nonoverlapping
regions of DNA in this strain. The effects of these clones and
additional clones from the wild-type 29050 strain in homol-
ogous and heterologous hosts confirm that the genes re-
quired for formation of e-rhodomycinone, a key intermediate
of daunorubicin biosynthesis, are present in region IV. In
addition, we demonstrated that this region contains two
daunorubicin resistance genes and at least some of the genes
for synthesis of the aglycone portion of daunorubicin. We
also found that a substantial increase in the production of
daunorubicin and one of the intermediates of its biosynthesis
occurred upon transformation of the 29050 and 27952 strains
with certain cosmid clones. The absence of a large portion of
DNA in region II in the 27952 strain did not appear to have
a deleterious effect on daunorubicin production or resistance
(a possible effect on doxorubicin production was not exam-
ined); however, the relationship between genes in regions I
and III and daunorubicin production requires further inves-
tigation.
MATERIALS AND M:ETHODS
Biochemicals and chemicals. Apramycin (Apm) was ob-
tained from Eli Lilly & Co., Indianapolis, Ind. Doxorubicin
and daunorubicin were obtained from Sigma Chemical Co.,
St. Louis, Mo.; and E-rhodomycinone was obtained from
Ramesh C. Pandey (Xechem, Inc., Rosemont, Ill.). All other
chemicals and biochemicals were obtained from Sigma or
United States Biochemicals, Cleveland, Ohio. Restriction
enzymes and other recombinant DNA materials were pur-
chased from United States Biochemicals; Promega Biotec
(Madison, Wis.); Bethesda Research Laboratories, Inc.
(Gaithersburg, Md.); or New England BioLabs, Inc. (Bos-
ton, Mass.).
3428 OTTEN ET AL.
Bacterial strains and plasmids. S. lividans 1326 and TK24
(13) were obtained from David Hopwood (John Innes Insti-
tute and AFRC Institute of Plant Science, Norwich, United
Kingdom), and S. lividans JT46 (26) was obtained from
Carton Chen (Institute of Microbiology and Immunology,
National Yang Ming Medical College, Taipei, Taiwan). S.
peucetius ATCC 29050 and S. peucetius subsp. caesius
ATCC 27952 were obtained from the American Type Culture
Collection, Rockville, Md. S. peucetius subsp. caesius
H6101 and H6125 (10, 11) were obtained from Coy-Choke
Ho (University of Malaya, Kuala Lumpur, Malaysia).
Cosmid vector pKC505 (21) was obtained from Richard
Baltz (Eli Lilly & Co.).
Media and growth conditions. Cultures for preparation of
Streptomyces spore stocks were grown on ISP medium 4
(Difco Laboratories, Detroit, Mich.) or YS agar (28) at 30°C
for 7 to 10 days. Streptomyces strains were grown in R2YE
medium (13) at 30°C for preparation of protoplasts and
isolation of chromosomal and plasmid DNAs. Escherichia
coli was grown in LB medium (18) at 37°C. Strains contain-
ing pKC505 were selected with 100 ,ug of Apm per ml for E.
coli and 20 to 25 ,ug of Apm per ml for Streptomyces sp.
strains. Anthracycline production by S. peucetius strains
was determined by one of two methods. (i) Spores or
colonies of the transformants were streaked on ISP medium
4 agar containing 25 ,ug of Apm per ml and incubated for 4 to
6 days at 30°C. A piece of the resulting growth was cut from
the plate and used to inoculate 25 ml of production medium
(8) containing 25 p.g of Apm per ml in a 250-ml baffled flask
with a foam plug closure. The cultures were incubated for 6
to 7 days at 30°C with shaking at 250 rpm on a G25 Gyrotory
shaker (New Brunswick Scientific Co., Edison, N.J.). (ii)
Spores or colonies of the transformants were streaked on
R2YE agar containing 25 jig of Apm per ml and incubated for
4 to 6 days at 30°C. A single colony was used to inoculate 5
ml of R2YE broth containing 20 to 25 pug of Apm per ml, and
the culture was grown for 48 to 72 h with a toothpick until
turbid. One milliliter of this seed culture was used to
inoculate 25 ml of the production medium containing 20 to 25
,ug of Apm per ml and the production cultures were grown as
described above.
Isolation of chromosomal and plasmid DNAs. Chromo-
somal DNA was isolated from S. peucetius subsp. caesius
by a compilation of the methods of R. Hinterman (Ph.D.
dissertation, Eidgenossische Technische Hochschule, Zur-
ich, Switzerland, 1983) and Hopwood et al. (13). Cells were
grown in 50 ml of R2YE medium to the late-exponential
phase, and approximately 2 g (wet weight) of cells was
treated with 5 ml of TSE buffer (Hinterman, Ph.D. disserta-
tion) containing 2 mg of lysozyme per ml at 37°C with gentle
shaking. One drop of the mixture was tested for lysis by
addition of a drop of 10% sodium dodecyl sulfate at 15-min
intervals until lysis was complete (30 to 60 min.). The lysed
cells were incubated with 3.6 ml of a solution containing 0.25
M EDTA, 2.4% sodium dodecyl sulfate, and 0.5 mg of
pronase per ml (prepared immediately before use) at 37°C for
1 h. The mixture was extracted with 8 ml of phenol-
chloroform (Hinterman, Ph.D. dissertation), and the upper
layer was extracted with 8 ml of chloroform and then treated
with RNase A at a final concentration of 40 ,ug/ml at 37°C for
30 min. The mixture was extracted with equal volumes of
phenol-chloroform and chloroform, and the DNA was pre-
cipitated first with isopropanol and then with ethanol by
standard procedures (13, 18).
The alkaline lysis method described by Maniatis et al. (18)
was used for large-scale plasmid DNA preparations from E.
J. BACTERIOL.
coli, and the boiling method of Holmes and Quigley (12) or
the alkaline extraction procedure of Morelle (19) was used
for small-scale preparations. Plasmid DNA was isolated
from Streptomyces sp. strains by the minilysate procedure of
Kieser (15) or by the large-scale method described by
Stutzman-Engwall and Hutchinson (24).
Transformation procedure. Protoplasts of S. peucetius
strains and S. lividans 1326 were prepared as described by
Hopwood et al. (13), with the following modifications. Cells
were grown in 5 ml of R2YE medium in test tubes (18 by 150
mm) containing a sterile toothpick (to facilitate dispersed
growth) at 30°C with shaking for 2 to 3 days. The culture was
transferred to 50 ml of R2YE containing 0.5% glycine in a
250-ml baffled flask and incubated for 18 to 24 h. The cells
obtained were treated with 20 ml of P buffer containing 2 mg
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of lysozyme per ml, and the resulting protoplasts were
suspended in P buffer at a concentration of -109/ml and
frozen on ice at -80°C. Streptomyces protoplasts were
transformed as described by Hopwood et al. (13), by using
100 ,ul of protoplasts (Q108), 1 ,ug of plasmid DNA in 10 ,ul of
TE buffer, and 500 ,ul of 25% Koch-Light polyethylene
glycol 1000 in P buffer or T buffer (13). Samples (100 ILI) were
plated in 4 ml of soft R2 agar (3) on R2YE regeneration
plates. After incubation at 30°C for 18 h for S. lividans and 30
h for S. peucetius strains, the plates were overlaid with 3 ml
of soft nutrient agar (Difco) containing sufficient Apm to give
final concentrations of 50 to 70 pig/ml for S. lividans and 20
to 25 ,ug/ml for S. peucetius strains. Transformants were
visible after incubation for an additional 3 to 5 days at 30°C.
Competent E. coli DH5 cells were prepared and trans-
formed by the calcium chloride procedure as described by
Maniatis et al. (18), except that glycerol was added to the
resuspended cells to give a final concentration of 20%, and
the cells were stored at -80°C.
Preparation of a cosmid library of S. peucetius subsp.
caesius chromosomal DNA in E. coil. Cosmid vector pKC505
was digested with HpaI, dephosphorylated, and digested
with BamHI as described by Richardson et al. (21). Chro-
mosomal DNA isolated from S. peucetius subsp. caesius
was partially digested with MboI and size fractionated on a
10 to 40% sucrose gradient (13). The fractions containing
approximately 20- to 25-kilobase (kb) fragments were
pooled, precipitated with ethanol, and dissolved in TE. The
vector (4 pug) and chromosomal (700 ng) DNAs were ligated
with 2.5 Weiss units of T4 DNA ligase (United States
Biochemicals) in a reaction volume of 20 pu1 at 12°C over-
night. The ligated DNA was packaged in vitro by using the
Packagene lambda packaging system (Promega Biotec) as
recommended by the manufacturer. The packaged phage
was transduced into E. coli DH1, and Apm-resistant colo-
nies were selected. Plasmid DNA was isolated by the
minilysate method from 10 transductants selected at ran-
dom, and restriction endonuclease digestion indicated that
all contained inserts with an average size of approximately
22 kb. A total of 1,920 transductants were picked to micro-
titer plates containing 100 ,ul of LB medium with Apm (100
p.g/ml) and grown at 37°C overnight. Glycerol was added to
give a final concentration of 20%, and the library was stored
at -800C.
Colony hybridization of the cosmid library. The S. peuce-
tius subsp. caesius cosmid library was replicated to What-
man 541 paper, lysed, and hybridized as described by N. K.
Davis (Abstracts, Fifth International Symposium on the
Genetics of Industrial Microorganisms, Split, Yugoslavia,
1986, p. 92). The 1.8-kb BamHI fragment representing the
tcmIa probe (20) was labeled with [y-32P]dCTP by nick
VOL. 172, 1990 DAUNORUBICIN BIOSYNTHESIS GENES 3429

translation with the kit from Bethesda Research Laborato- 'Group

ries as recommended by the manufacturer. Hybridization II 111II 11 III III
was carried out in a solution containing 5 x SSC (13), 20 mM
sodium phosphate (pH 7.0), 50% formamide, and 0.1 mg of 301
denatured salmon sperm DNA per ml at 42°C overnight. The 501
filters were washed twice with 5x SSC-20 mM sodium 502
phosphate (pH 7.0) at 42°C for 30 min. Autoradiography was
carried out at -80°C overnight by using Kodak X-Omat AR
film and intensifying screens. Group II
Plasmid DNA was isolated from the colonies and sub-
jected to Southern blot hybridization under appropriate III 1 111 1
stringency conditions (24) to confirm that it hybridized to the 313
temIa probe DNA. 315
Additional clones were isolated from the S. peucetius Group III
cosmid library (24) by hybridization with a 1.9-kb BamHI

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II I II
fragment from pWHM337 and a 3.3-kb BamHI fragment (15, (3.8/2.3, _ (3.0/2.5,
0.5) 0.7/05) 511 23)
from pWHM333.
Analysis of anthracycline metabolite production. Cultures 510
were grown in production medium (8) as described above, 507
and anthracycline metabolites were hydrolyzed with oxalic 316
acid and extracted with chloroform as previously described
(24). Extracts were subjected to high-pressure liquid chro-
matography with a Waters 501 or 6000 solvent delivery Group IV
system, a 484 or 441 UV detector operated at 254 nm, a II I
Waters Baseline 810 chromatography workstation or 740 (6/3.9/05) (0.01/7.2 1I
10 kb
data module, and a Rheodyne 7125 or Waters U6K injector. 0.5)
Filtered samples of the chloroform solution (10 pAl) were 340
analyzed on a Waters Nova-Pak C18 cartridge (8 mm by 10 337
cm or 5 mm by 10 cm) with a mobile phase of methanol- 517
water, adjusted to pH 2.5 with phosphoric acid in a ratio 335
ranging from 75:25 to 85:15 at a flow rate of 1.5 to 2.5 ml/min.
Daunorubicin, doxorubicin, and e-rhodomycinone (5 and 10
,ug/ml in methanol) were used as external standards. -333
The extracts were also subjected to thin-layer chromatog- 516
raphy analysis on silica gel 60 F-254 (Merck & Co., Inc.,
Rahway, N.J.) by using a solvent system of chloroform- 5i5
methanol (95:5) for aglycones and chloroform-methanol-
514
acetic acid-water (80:20:16:6) for glycosides (5). Plates were
visualized under UV light (310 nm) with a TM-36 Chromato 339
Vue Transilluminator (Ultra-Violet Products, Inc., Milwau-
kee, Wis.). FIG. 1. pWHM clones from S. peucetius (300 series) and S.
Cosynthesis of blocked mutants. Blocked mutants of S. peucetius subsp. caesius (500 series). The individual cosmid clones
are indicated by the thin lines beneath BamHI restriction maps of
peucetius subsp. caesius were tested for the ability to the four regions of the genomes represented by the four groups of
cosynthesize e-rhodomycinone and daunorubicin by growth clones. BamHI sites are indicated by short vertical lines, and in
in mixed liquid culture. The mutants were grown separately groups III and IV, the sizes (kilobases) of unmapped BamHI
in 25 ml of production medium in 250-ml baffled flasks for 3 fragments are indicated in parentheses below three different regions
days. A 5-ml sample of each culture was transferred to 25 ml of the genomic maps. The location of tcmIa-hybridizing DNA is
of fresh production medium, and the culture was incubated indicated by short, thick lines below the restriction map of each
for 3 days. Feeding experiments were also conducted by region.
adding a solution of e-rhodomycinone in dimethyl sulfoxide
(10 mg/ml) to 3-day-old cultures to give a final concentration Materials and Methods and screened with tcmIa, the poly-
of 30 ,ug/ml, followed by incubation of the cultures for an ketide synthase gene from S. glaucescens (4, 20), to identify
additional 3 days. 20 colonies that hybridized to the probe. Restriction endo-
Analysis of daunorubicin resistance. S. lividans strains nuclease mapping of plasmid DNAs isolated from these
were tested by the gradient plate method (25) using square colonies revealed that 18 of the clones could be ordered into
plates (100 by 15 mm) and 25-ml layers of R2YE agar. three groups with no apparent overlap among the groups
Approximately 105 spores were applied in parallel lanes (Fig. 1).
across a gradient of 0 to 50 ,ug of daunorubicin per ml. The Seven new clones were identified from the S. peucetius
resistance of S. peucetius strains was determined by patch- library, and these were ordered into the five groups previ-
ing stationary-phase mycelia grown on R2YE agar onto ously described (24). Chromosome walking experiments
R2YE plates containing 0 to 100 ,g of daunorubicin per ml in with 32P-labeled BamHI fragments from the ends of clones
5-,ug/ml increments up to 50 ptg/ml. pWHM333 and pWHM337 (24) expanded the region of DNA
covered by the group IV clones to -94 kb. The absence of
RESULTS overlap between groups I to IV was verified by Southern blot
Isolation of cosmid clones. A pKC505 cosmid library of S. hybridization of 32P-labeled fragments from the ends of
peucetius subsp. caesius DNA was prepared as described in clones pWHM313, pWHM317, pWHM333, and pWHM337
3430 OTTEN ET AL.
to representative cosmid clones from the three other groups
(data not shown).
Comparison of the clones from the 29050 and 27952 strains
showed that the restriction maps of the regions of DNA
represented by the group I, III, and IV clones were appar-
ently identical and that clones analogous to the group II
clones from the 29050 strain were absent in the DNA library
from the 27952 strain. No clones were isolated from the
library of 27952 DNA that corresponds to group V from S.
peucetius 29050 because the latter clones were obtained
primarily by hybridization with the S. coelicolor actIII probe
(24).
Expression of representative cosmid clones in S. lividans. By
using several representative cosmid clones, transformants of
S. lividans 1326, TK24, and JT46 were prepared for analysis
of the expression of the cloned DNA. The presence of the
intact cosmid in representative transformants was verified
by endonuclease digestion and agarose gel electrophoresis of
plasmid DNA reisolated from the transformant and amplified
in E. coli (the latter step was necessary to obtain sufficient
amounts of plasmid DNA). No change in the restriction
pattern of the transforming plasmid was observed in the
cases tested. Nonetheless, the plasmid was not stably main-
tained in these hosts without selection for Apm resistance.
Clones pWHM337 and pWHM339 from the 29050 library
and pWHM517 from the 27952 library imparted significant
resistance to the growth-inhibitory effects of daunorubicin
when S. lividans transformants were tested as described in
Materials and Methods. Their ability to grow on concentra-
tions of antibiotic that were approximately twice the MIC
value for the host strain suggests that a daunorubicin resis-
tance gene is located in the 7.5-kb region of group IV DNA
that is unique to pWHM517 relative to pWHM516. This
assumption was confirmed by analysis of subclones of
pWHM337 and pWHM517 by which we located a daunoru-
bicin resistance determinant in region IV (see Fig. 5) (P. G.
Guilfoile, unpublished data). The fact that clone pWHM339
does not overlap with pWHM337 or pWHM517 indicates
that region IV contains a second daunorubicin resistance
determinant located at least 20 kb from the first one. Fur-
thermore, clone pWHM315 from region II also conferred
significant daunorubicin resistance to S. lividans (24).
Transformants of S. lividans 1326, TK24, and JT46 con-
taining representative clones were grown in a daunorubicin
production medium (8), and 10-fold-concentrated chloro-
form extracts were analyzed by thin-layer chromatography
and high-pressure liquid chromatography for the presence of
anthracyclines or related products. Colored, fluorescent
compounds not present in the controls were clearly visible in
S. lividans strains containing clones from groups I, III, and
IV. The nature of the substances produced by the first two
sets of transformants was not studied further. S. livi-
dans(pWHM333) and S. lividans(pWHM334) appeared, on
the basis of thin-layer chromatography and high-pressure
liquid chromatography analyses, to produce aklavinone and
aklaviketone, known intermediates of daunorubicin biosyn-
thesis (23) (Fig. 2). We have previously reported that S.
lividans(pWHM338) produces e-rhodomycinone (24), the
nature of which has been confirmed by high-resolution mass
spectral analysis (Jim McAlpine, personal communication).
Since none of the clones caused daunorubicin production,
the transformants were tested in mixed liquid cultures in
pairwise combinations within groups and between groups.
Only in combinations of group IV clones were new products
obtained. S. lividans(pWHM337) or S. lividans(pWHM335)
plus transformants containing pWHM333 or pWHM334 pro-
J. BACTERIOL.
COSEnz O0 004
CH36CK2COSCoA 0 0-
+ 0a0i0
-kanni
CH2COSCo0 Decapolyketide
0 0 0 0H40040 0
Akianonic acid
002
0 CO^
OH
[Aklaviketonel
OH 0 HO 0H OH 0 0O OH
Aklavinone e-Rhodomycinone
0 HO0 0
04$
[TDP-L-daunosamine] 0HO CP
CH,O NO
Downloaded from http://jb.asm.org/ on October 9, 2019 by guest
O
NM2
N0H4
'Rhodomycine' Daunorubicin
| C-14 oxidation
TDP-D-glucose Doxorubicin
FIG. 2. Biosynthetic pathway for daunorubicin and doxorubicin.
Thick arrows indicate several steps, and thin arrows indicate single
steps between the biosynthetic intermediates. "Rhodomycine" in-
dicates that this compound is similar to the structures of the known
rhodomycines that are glycosides of rhodosamine (2,3,6,-trideoxy-
3-dimethylamino-L-1yxo-hexose).
duced e-rhodomycinone; similarly, S. lividans(pWHM517)
produced e-rhodomycinone in mixed culture with transfor-
mants containing either pWHM514 or pWHM516. These
results indicate that all of the genes required for e-rhodomy-
cinone biosynthesis by the pathway summarized in Fig. 2 are
contained in the 33.7-kb region of S. peucetius DNA encom-
passed by pWHM338 and the 29-kb region of S. peucetius
subsp. caesius DNA encompassed by pWHM516 and
pWHM517.
Characterization of S. peucetius subsp. caesius blocked
mutants H6101 and H6125. Strains H6101 and H6125 are
blocked mutants of S. peucetius subsp. caesius prepared by
Ho et al. (10, 11). According to those workers, H6125
produces no daunorubicin but accumulates e-rhodomyci-
none. When grown in the production medium (8), strain
H6125 accumulated approximately 45% of the e-rhodomyci-
none produced by strain 27952 (see Fig. 3A). H6101 pro-
duced no daunorubicin, e-rhodomycinone, or other known
intermediates of anthracycline biosynthesis but did produce
a number of yellow-colored, blue-fluorescent compounds
that were not identified. Mycelial cells taken from H6101 and
H6125 cultures at the normal stage of daunorubicin produc-
tion did not grow in 20 to 25 ,ug of daunorubicin per ml,
whereas growth of strain 27952 was unaffected at a concen-
tration of 100 ,ug/ml. Spores of strains H6101 and H6125
were significantly more sensitive, because inhibition of
growth occurred at 10 ,ug of daunorubicin per ml.
H6101 and H6125 were tested for the ability to cosynthe-
size daunorubicin by growth in mixed liquid cultures, but no
daunorubicin was produced. Exogenous e-rhodomycinone
was also added to H6101 cultures at a level of 30 ,ig/ml, but
no conversion to daunorubicin was observed. Lack of anti-
biotic production was also seen in cosynthesis experiments
between H6101 and each of the Streptomyces sp. C5 dauC,
dauD, dauE, and dauH mutants (23) that are known to
secrete intermediates of c-rhodomycinone formation or e-
rhodomycinone itself (Jeff Buboltz, unpublished data). The
VOL. 172, 1990 DAUNORUBICIN BIOSYNTHESIS GENES 3431

fact that H6101 was unable to make E-rhodomycinone or A

convert it to daunorubicin in these experiments suggests that
this strain contains a regulatory mutation or that it has two or 1200
more blocks in the pathway. The defect in daunorubicin
resistance in strains H6101 and H6125 would not be ex-
pected to prevent observable conversion of e-rhodomyci- 1000
none to daunorubicin, since strain 27952 produces only E
about 4 ,ug of daunorubicin per ml and H6101 and H6125 are "
800
resistant at this level. a.!
Expression of cosmid clones in S. peucetius strains. Expres-
o 600
sion of the cosmid clones was studied in strains 29050 and E
27952 and in nonproducing mutant strains H6101 and H6125. 0
U
The results of these experiments are summarized in Fig. 3, 400
which includes some data reported previously (24) for com-
parison. The data were obtained under highly reproducible

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conditions and are comparable for different transformants of 200
the same strain.
(i) Complementation of mutations in strains H6101 and
H6125. Strain H6125 produced daunorubicin when trans- LO
formed with pWHM339, pWHM514, or pWHM515, and the (N
C0 - O - co rn %a U,
amount varied between 25% and -200% of the titer obtained ID Pi, r') v- rI"' I')
F' -
U.) -
U) U rI
with strain 27952 (Fig. 3A). This result suggests that the
B
DNA from group IV contains some (or all) of the genes that
govern the later steps in the daunorubicin biosynthetic 6

pathway (Fig. 2), in addition to the genes for e-rhodomyci- 0m 150

none biosynthesis, as demonstrated above. Interestingly, the C ~~~~~~~~~~~~~~~

growth of the H6125(pWHM339) transformant was unaf- )

fected by 60 ,ug of daunorubicin per ml, which supports the

conclusion stated above that there is a daunorubicin resis-
tance determinant in this region.
Daunorubicin and e-rhodomycinone production was re-
stored in strain H6101 by transformation with pWHM334,
pWHM338, pWHM341, and pWHM517 (Fig. 3B). Since
clones pWHM334 and pWHM341 contain no overlap, these 0 r- ITX
ao
I: In U) In In)
data suggest that there are two regions of DNA within the n

group IV clones capable of restoring production to H6101. In C

addition, the data indicate that the two regions have different
effects on the amounts of e-rhodomycinone and daunorubi- 100 40
cin produced. Region 1, represented by pWHM334 and
E
pWHM338, affected predominantly e-rhodomycinone pro-
duction, giving titers of r-rhodomycinone and daunorubicin C0 75 30 _
-S
that were approximately 350% and 25%, respectively, of the
titers obtained with strain 27952. In contrast, region 2, which Eo 5Q
s 6L26 20 Oc
is represented by pWHM341 and pWHM517, had a greater
impact on daunorubicin production, giving titers that were
approximately 180% (pWHM341) or 650o (pWHM517) of B 25 10 X
the titer of the parent strain. The E-rhodomycinone levels 14

were approximately 160%)o of that of the parent strain. Since

0
the only DNA common to both pWHM341 and pWHM517 is u)
0 IN F- lw %0 v
a 1.9-kb BamHI fragment (this fragment is absent in Ca
N1 F., U) rI Ul) U,)
pWHM334 and pWHM338, which represent region 1), the
gene(s) from region 2 that restores antibiotic production in D
H6101 may be located within this BamHI fragment.
(ii) Overproduction of daunorubicin and e-rhodomycinone. 20(0 40
Clones from group IV caused significant increases in e-
rhodomycinone production, which ranged from 3- to 11-fold E
m
in the producing strains (Fig. 3C and D) and from 11- to C0 15C0 30 =S
5
b
a:E
'a lOCD 20 o
0
FIG. 3. Production of s-rhodomycinone and daunorubicin by r
0
strains of S. peucetius and S. peucetius subsp. caesius. The number 9

of replicate cultures and the standard error are, respectively, s

10
indicated by the number and the short line extending above each
bar, which shows the amount of metabolite produced by cultures
grown as described in Materials and Methods. Strains were trans- (N
formed with the pWHM clones indicated. Panels: A, H6125; B, U,)
' F-- '0 LA v
H6101; C, ATCC 29050; D, ATCC 27952. F--
(N4
-
U)
-
U)
-
U)
-
Ur)
3432 OTTEN ET AL. J. BACTERIOL.

production observed was specific to the group I and III

clones, three cosmids were selected at random from the
20 27952 library and analyzed for their effects on metabolite
15 production. Restriction endonuclease digestion of these
cosmids (pWHM518 to pWHM520) revealed no apparent
c
10 overlap with the regions of DNA represented by the group I,
III, or IV clones. All of these clones caused inhibition of
5 anthracycline production similar to that observed with group
C0
I and III clones when tested in strains 29050, 27952, and
H6125 (Fig. 4). These results indicate that the inhibition of
0

40 e-rhodomycinone and daunorubicin production observed

L- E was due to a nonspecific effect.
o cm30
c
4)
D
C
DISCUSSION
0 20

Downloaded from http://jb.asm.org/ on October 9, 2019 by guest

C

u
The isolation of three groups of apparently nonoverlap-
10 ping clones that hybridized to the tcmIa probe from the
0 cosmid library of S. peucetius subsp. caesius 27952 DNA is
0 consistent with the observation of four groups of nonover-
lapping clones in parental strain 29050 (24) that hybridized to
Iw 20 the same probe. Restriction mapping of the DNA cloned
15 from strain 27952 indicated that the three groups were
apparently identical to group I, III, and IV clones of strain
29050. Clones analogous to group II of the latter strain were
not observed in strain 27952. Colony hybridization experi-
s ; I
ments and Southern analysis of chromosomal DNA have
I
00
shown that the genome of strain 29752 contains a deletion of
at least 4 kb which includes the tcmIa-hybridizing region of
the group II clones isolated from strain 29050 (data not
cn Ln
shown). This deletion did not have a negative effect on
daunorubicin production or resistance, since strain 27952
_ U

Y 0 0
o
0
- 0
0 0 produced significantly more daunorubicin than did strain
o

C 0. I)n n )n L) In In
C'
LO
FIG. 4. Production of a-rhodomycinone and daunorubicin by
strains of S. peucetius subsp. caesius. The bars show the amount of
metabolite produced by cultures grown as described in Materials
and Methods. The standard error was +±15%. Strains were trans-
formed with pKC505 or the pWHM clones indicated.
74-fold in the blocked mutant H6125 (Fig. 3A). Some of
the increased e-rhodomycinone production in H6125 may be
due to the fact that this strain is blocked in its ability to
convert e-rhodomycinone to daunorubicin; however, clone
pWHMSS, which complemented this mutation, still gave
very high levels of e-rhodomycinone (Fig. 3A). (pWHM339
produced much lower levels of both E-rhodomycinone and
daunorubicin when transformed into H6125 [Fig. 3A] and
also caused decreased levels of e-rhodomycinone and
daunorubicin when transformed into strain 29050 [Fig. 3C]).
Transformants of strains 27952 and H6101 containing
pWHMS17 and transformants of strain 29050 containing
pWHM337 or pWHM517 produced significantly higher
amounts of daunorubicin (three- to eightfold) relative to the
respective untransformed strains (Figs. 3B to D). Some of
the other group IV clones gave smaller increases in dauno-
rubicin production in some strains (Fig. 3).
(iii) Effects of group I and Ill clones. Group I and III clones
obtained from the strains 29050 and 27952 did not comple-
ment the mutations in strain H6101 or H6125 and generally
inhibited production of e-rhodomycinone and daunorubicin
in all of the strains tested (Fig. 4). The exception was
pWHM317, which caused a large increase in production of
e-rhodomycinone by H6125(pWHM317) transformants, as
previously described (24).
To determine whether the depression of anthracycline
29050 (Fig. 3D) and was as resistant to daunorubicin as the
latter strain. Whether or not this deletion is somehow
responsible for the doxorubicin-producing ability of strain
27952 (1) remains to be determined, but it is unlikely that this
region of DNA is essential for daunorubicin or doxorubicin
production.
The question of the exact location of the daunorubicin
production genes, in essence, whether they are in more than
one region (24), is not settled. Because of the properties of
the group IV clones studied from both of the S. peucetius
strains, we believe that region IV is directly associated with
daunorubicin biosynthesis and appears to contain all of the
genes required for synthesis of e-rhodomycinone and some
(if not all) of the genes for synthesis of the aglycone portion
of daunorubicin. The results obtained with the other groups
of clones do not provide such a strong link with anthracy-
cline production. Nevertheless, the presence of a daunoru-
bicin resistance gene in region II (the presence or absence of
which in strain 27952 cannot be determined on the basis of
the present data) and the ability of a group III clone to
stimulate r-rhodmycinone production suggest that these two
regions influence anthracycline production. Alternatively,
these regions might code for other types of polyketide
metabolites, such as spore pigments, a function recently
discovered for actl/tcmIa-homologous DNA in S. coelicolor
(K. F. Chater, personal communication) and S. avermitilis
(T. MacNeil, personal communication). Further insight into
this matter must await the construction of strains with
specific deletions in regions I and III and larger deletions of
region II.
On the basis of the expression of the clones from group IV
in S. peucetius and S. lividans strains, one can draw some
preliminary conclusions about the probable locations of
some of the daunorubicin biosynthesis genes in this cluster.
VOL. 172, 1990
B B B1 B1BBB BB B B B B 58
(73.an.5) (10.117.2) D
resstane
e-rhodomnycinone
FIG. 5. Approximate locations of some of the genes for dauno-
rubicin (Dnr) production in region IV of the S. peucetius genome.
PKS indicates the location of tcmIa-hybridizing DNA. The large
bracket and partial bracket below the map indicate the approximate
extents of the regions that govern formation of e-rhodomycinone
and conversion of this intermediate to daunorubicin, as discussed in
the text. Restriction site abbreviations: B, BamHI; Bg, Bglll; E,
EcoRI. The sizes (kilobases) of unmapped BamHI fragments are
indicated in parentheses below three.regions of the genomic map.
The genes for the early part of the pathway are expected to
lie in the middle of the cluster, since the actl and tcmIa
probes, which code for polyketide synthases (4, 17, 20, 22),
hybridized to this region (Fig. 5). The genes involved in
e-rhodomycinone production appear to be contained within
the 29-kb region defined by clones pWHM516 and
pWHM517 and in the 33.7-kb region defined by clone
pWHM338, since S. lividans produced this metabolite upon
transformation with these clones. (This conclusion, of
course, rests on the assumption that S. lividans does not
provide metabolites essential for e-rhodomycinone biosyn-
thesis.) Complementation of the mutation in strain H6125
that causes accumulation of s-rhodomycinone by clones
pWHM339 and pWHM515 but not by clones pWHM333,
pWHM338, and pWHM516 suggests that some of the genes
for the late steps in the pathway are located to the right of the
presumptive polyketide synthase genes (Fig. 5). One dauno-
rubicin resistance gene is located on the left side of the
cluster and may be linked to a gene that encodes aklavinone
li-hydroxylase on the basis of a report that these two genes
are adjacent in a highly productive strain derived from S.
peucetius subsp. caesius (A. L. Colombo, Abstracts of the
Seventh International Symposium on the Biology of Actino-
mycetes, abstr. P5-1, p. 148, 1988). Another daunorubicin
resistance determinant, contained in clone pWHM339, may
reside on the right of the polyketide synthase genes. Fur-
thermore, mutations in Streptomyces sp. strain C5 (23) that
block formation of the methyl ester of aklanonic acid (dauC),
its cyclization to aklaviketone (dauD), and reduction of
aklaviketone to aklavinone (dauE) (Fig. 2) are comple-
mented by DNA that maps near the tcmIa-hybridizing region
of the group IV clones (W. R. Strohl, personal communica-
tion).
The results of the expression of the group IV clones in
strain H6101 were unexpected, since two nonoverlapping
and well-separated regions of DNA within region IV caused
production of e-rhodomycinone and daunorubicin in this
strain; pWHM334 (representative of region 1) had a greater
effect on e-rhodomycinone production, and pWHM341 (rep-
resentative of region 2) had a greater effect on daunorubicin
production. There are several possible explanations of these
effects; for instance, H6101 could contain more than one
mutation that affects daunorubicin biosynthesis and resis-
tance, or metabolite production may have been restored in
some transformants by a mechanism other than strict com-
plementation or repair of a pathway-specific mutation. Ex-
periments are under way to investigate these and other
possibilities.
The increases in E-rhodomycinone and daunorubicin pro-
duction in transformants containing group IV clones were
DAUNORUBICIN BIOSYNTHESIS GENES 3433
B 55 B very substantial, and further study of this matter may lead
to useful applications for the construction of more-produc-
kb I tive strains. At first glance, one would assume that these
effects were due to expression of a number of genes, since
(E-rhOdOfYCy---n
cl >
each cosmid clone probably contains several dnr genes
daunotknbi) which may have had additive or even opposite effects on
metabolite production. Despite these uncertainties, the com-
parative increases among the different clones suggest that
there are at least two regions involved in high e-rhodomy-
cinone and daunorubicin production. These regions have
been narrowed down by subcloning experiments to a 2.0-
kb BglII-BamHI fragment present in clones pWHM334,
pWHM333, pWHM338, pWHM337, pWHM514, pWHM
515, pWHM516, and pWHM517 and to a 1.9-kb BamHI
fragment contained in clones pWHM337, pWHM340,
Downloaded from http://jb.asm.org/ on October 9, 2019 by guest
pWHM341, and pWHM517 (unpublished data). Therefore,
the genes responsible for high e-rhodomycinone and dauno-
rubicin production appear to lie on the left side of the
tcmIa-hybridizing region of the cluster and could include a
regulatory gene, a resistance gene, or a gene for a rate-
limiting step in the pathway. The latter seems to be some
point in the conversion of e-rhodomycinone to daunorubicin,
since e-rhodomycinone normally accumulates in S. peuce-
tius fermentations (23).
In summary, several groups of nonoverlapping clones that
hybridize with genes involved in the early steps of poly-
ketide biosynthesis were cloned from S. peucetius and S.
peucetius subsp. caesius and at least one of these groups is
directly involved in daunorubicin biosynthesis. Preliminary
conclusions on the organization of the gene cluster and the
location of some dnr genes were drawn on the basis of the
expression of the clones in heterologous and homologous
hosts. These conclusions provide a framework that will
facilitate further study of the organization, regulation, and
function of the dnr genes. Moreover, the significant in-
creases in secondary metabolite production obtained by
self-cloning in a wild-type strain (S. peucetius) and a more-
productive mutant (S. peucetius subsp. caesius) indicate that
this approach could be generally useful for identifying DNA
segments cloned from actinomycetes that could then be used
for genetic engineering of overproducing strains.
ACKNOWLEDGMENTS
We thank Pamela Kukuk, Kris Jensen, Ray Mirande, Dave
Helke, and Jeff Bubolz for technical assistance; Pat Guilfoile for
fruitful discussions; and David Hopwood for critical reading of the
manuscript.
This research was supported by grants from Xechem, Inc.,
Chicago, Ill.; Calbiochem-Behring, La Jolla, Calif.; and the Gradu-
ate School of the University of Wisconsin.
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