JOURNAL OF BACTERIOLOGY, Sept. 1990, p. 4816-4826 Vol. 172, No.

9
0021-9193/90/094816-11$02.00/0
Copyright 1990, American Society for Microbiology

Biosynthesis of Anthraquinones by Interspecies Cloning of

Actinorhodin Biosynthesis Genes in Streptomycetes:
Clarification of Actinorhodin Gene Functions
PAUL L. BARTEL,lt CHUN-BAO ZHU,1t JAY S. LAMPEL,l DONALD C. DOSCH,' NEAL C. CONNORS,'
WILLIAM R. STROHL,1* JOHN M. BEALE, JR.,211 AND HEINZ G. FLOSS2
Department of Microbiology, The Ohio State University, 484 West 12th Avenue, Columbus, Ohio 43210,'
and Department of Chemistry, University of Washington, Seattle, Washington 981952
Received 28 February 1990/Accepted 13 June 1990

Streptomyces galilaeus ATCC 31133 and ATCC 31671, producers of the anthracyclines aclacinomycin A and
2-hydroxyaklavinone, respectively, formed an anthraquinone, aloesaponarin II, when they were transformed

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with DNA from Streptomyces coelicolor containing four genetic loci, actI, actIll, actlV, and actVII, encoding
early reactions in the actinorhodin biosynthesis pathway. Subcloning experiments indicated that a 2.8-kilobase-
pair XhoI fragment containing only the actl and actVII loci was necessary for aloesaponarin II biosynthesis by
S. galilaeus ATCC 31133. Aloesaponarin II was synthesized via the condensation of 8 acetyl coenzyme A
equivalents, followed by a decarboxylation reaction as demonstrated by [1,2-13C2jacetate feeding experiments.
S. coelicolor B22 and B159, actVI blocked mutants, also formed aloesaponarin II as an apparent shunt product.
Mutants of S. coelicolor blocked in several other steps in actinorhodin biosynthesis did not synthesize
aloesaponarin II or other detectable anthraquinones. When S. galilaeus ATCC 31671 was transformed with the
DNA carrying the actI, actIll, and actVII loci, the recombinant strain produced both aloesaponarin H and
aklavinone, suggesting that the actinorhodin biosynthesis DNA encoded a function able to deoxygenate
2-hydroxyaklavinone to aklavinone. When S. galilaeus ATCC 31671 was transformed with a plasmid carrying
only the intact actIII gene (pANT45), aklavinone was formed exclusively. These experiments indicate a function
for the actll gene, which is the reduction of the keto group at C-9 from the carboxy terminus of the assembled
polyketide to the corresponding secondary alcohol. In the presence of the actIll gene, anthraquinones or
anthracyclines formed as a result of dehydration and aromatization lack an oxygen function on the carbon on
which the keto reductase operated. When S. galiaeus ATCC 31671 was transformed with the DNA carrying
the actl, actVII, and actlV loci, the recombinant strain produced two novel anthraquinones, desoxyerythro-
laccin, the 3-hydroxy analog of aloesaponarin II, and 1-0-methyldesoxyerythrolaccin. The results obtained in
these experiments together with earlier data suggest a pathway for the biosynthesis of actinorhodin and related
compounds by S. coelicolor.

The biosynthesis of the benzoisochromanequinone antibi-
otic actinorhodin (4), a polyketide antibiotic produced by
Streptomyces coelicolor, has been studied intensively in
recent years from both the biochemical (7, 13, 14) and
genetic (15, 22-24, 30) viewpoints. Seven classes of blocked
mutants have been described and placed in sequential order
on the basis of cosynthetic and chemical studies (7, 13, 30).
Studies on the molecular genetics of actinorhodin biosynthe-
sis have provided fundamental knowledge on the structure
and organization of antibiotic biosynthesis genes in strepto-
mycetes, including the clustering of antibiotic biosynthesis
structural genes (23, 24), production of the antibiotic by
recombinant strains carrying all of the biosynthesis genes on
a plasmid (23), and the potential homology shared by certain
structural genes within several different pathways (i.e., the
genes encoding polyketide synthases [22]). Actinorhodin
*
Corresponding author.
t Present address: Department of Microbiology, State University
of New York at Stony Brook, Stony Brook, NY 11794.
t Present address: Shanghai Institute for Pharmaceutical Indus-
try, Shanghai, Peoples Republic of China.
Present address: Crop Genetics International, Inc., Hanover,
MD 21076.
|1 Present address: Department of Medicinal and Natural Products
Chemistry, University of Texas at Austin, Austin, TX 78712.
4816
biosynthesis genes also were used in the initial demonstra-
tion of the formation of hybrid antibiotics via interspecies
cloning (17, 29). When the granaticin producer Streptomyces
violaceoruber Tu22 was transformed with actinorhodin bio-
synthesis genes from S. coelicolor, a hybrid compound,
dihydrogranatirhodin, was produced (17). When Streptomy-
ces sp. strain AM-7161 was transformed with certain genes
encoding only part of the actinorhodin pathway, the novel
hybrid antibiotics mederrhodins A and B were formed (17,
29). Here we report the formation of an anthraquinone,
aloesaponarin II, by actinorhodin-nonproducing streptomy-
cetes transformed with the actl, actIII, actVII, and actIV
(actinorhodin) biosynthesis genetic loci from S. coelicolor.
Aloesaponarin II also was produced by the S. coelicolor
actVI mutant strains B22 and B159.
The actIII gene apparently encodes a polyketide reductase
(15, 22), which has been postulated to reduce one of the keto
functions of the actinorhodin polyketide intermediate during
its formation (15). This hypothesis, based on the DNA
sequence of the actIII gene (15) and the inability of the actI
and actIII mutants to cross-feed (30), has been difficult to
prove because no corresponding intermediate has been
isolated. It is probable, however, that the compound which
would theoretically arise from an actIII mutation might be
unstable, as judged from its presumed structure. In this
report, we show that the presence of the actIII gene is
VOL. 172, 1990 HYBRID ANTHRAQUINONE BIOSYNTHESIS 4817

TABLE 1. Bacterial strains and plasmids useda

Strain or plasmid Relevant characteristics Source or reference
Streptomyces strain
S. galilaeus 31133 Aclacinomycin producer ATCC
S. galilaeus 31671 2-Hydroxyaklavinone producer (actIII-equivalent negative) ATCC
S. peucetius 29050 Daunomycin producer ATCC
S. azureus 14921 Thiostrepton producer ATCC
S. parvulus 2266 Nonactin producer D. Hopwood
S. coelicolor 1190 Actinorhodin producer D. Hopwood
S. coelicolor B17 Blocked mutant (actlV) D. Hopwood (30)
S. coelicolor B18 Blocked mutant (actl) D. Hopwood (30)
S. coelicolor B22 Blocked mutant (actVI) D. Hopwood (30)
S. coelicolor B40 Blocked mutant (actVII) D. Hopwood (30)
S. coelicolor B41 Blocked mutant (actIII) D. Hopwood (30)
S. coelicolor B159 Blocked mutant (actVI) D. Hopwood (30)
Plasmid
pIJ61 Derivative of SLP1.2; LC; Thior Neor D. Hopwood (34)
pIJ350 Derivative of pIJ101; HC; Thior D. Hopwood (19)

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pIJ702 Derivative of pIJ350; HC; Thior Mel' D. Hopwood (16)
pIJ922 Derivative of SCP2*; LC; Thior D. Hopwood (21)
pIJ941 Derivative of SCP2*; LC; Hygr Thior D. Hopwood (21)
pIJ2303 pIJ922 with 32.5-kbp DNA insert from S. coelicolor containing actinorhodin bio- D. Hopwood (23, 24)
synthesis pathway genes
pANT12 pIJ350 with 8.8-kbp PstI subclone from pIJ2303; contains actI, actIlI, actIV, and This work
actVII loci
pANT28 pIJ941 with 8.8-kbp PstI subclone from pANT12; contains actI, actIII, actIV, This work
and actVII loci
pANT31 pIJ61 with 8.8-kbp PstI subclone from pANT12; contains actI, actIII, actIV, and This work
actVII loci
pANT33 pIJ941 with 4.7-kbp PstI-SstI subclone from pANT12; contains actI, actIII, and This work
actVII loci
pANT35 pIJ702 with 5.3-kbp SphI subclone from pANT12; contains actI, actVII, and This work
actIV loci
pANT36 pIJ702 with 5.3-kbp SphI subclone from pANT12; in opposite orientation of This work
pANT35
pANT43 pIJ61 with 2.8-kbp XhoI subclone from pANT12; contains actl and actVII loci This work
pANT44 pIJ61 with 2.2-kbp BamHI subclone from pANT12; contains part of actI locus This work
pANT45 pIJ61 with 1.1-kbp BamHI subclone from pANT12; contains actIII locus This work
pANT46 pIJ61 with 0.8-kbp BamHI subclone from pANT12; contains part of actVII locus This work
pANT56 pIJ941 with 2.8-kbp XhoI subclone from pANT43; contains actI and actVII loci This work
a Abbreviations: ATCC, American Type Culture Collection; FCRC, Frederick Cancer Research Center; LC, low-copy-number plasmid; HC, high-copy-
number plasmid; Thior, thiostrepton resistance; Hygr, hygromycin resistance; Neor, neomycin resistance; Mel', production of melanin; act, actinorhodin genetic
locus.

required for the formation of antibiotics which are lacking a
hydroxyl group at the ninth carbon from the carboxy termi-
nus of the assembled (intermediate) polyketide chain. Our
data are consistent with the theory that the actIII gene, and
similar genes of other polyketide-producing strains, encodes
a polyketide ketoreductase (15). These concepts were used
to predict and demonstrate the formation of the 3-hydroxy
analog of aloesaponarin II, i.e., desoxyerythrolaccin, by a
polyketide reductase-negative strain, S. galilaeus ATCC
31671, transformed with the actl, actVII, and actIV genetic
loci. The biosynthesis of aloesaponarin II (this work) and
mutactin (38) by blocked mutants of S. coelicolor, in con-
junction with the compounds produced by the recombinant
strains reported in this work, suggests a pathway for the
biosynthesis of actinorhodin.
MATERIALS AND METHODS
Organisms and plasmids. The organisms used in this study
and the sources from which they were obtained are listed in
Table 1. Plasmids obtained for use or developed in this study
also are listed in Table 1. Plasmid pIJ2303 (23, 24), which
contains all of the actinorhodin (act) biosynthesis genes, was
obtained from D. A. Hopwood (John Innes Institute, Nor-
wich, United Kingdom).
Culture maintenance and growth conditions. The strepto-
mycetes were maintained on yeast malt agar as described
previously (9). Strains carrying streptomycete plasmids
pIJ61 (34), pIJ350 (19), pIJ922 (21), and pIJ941 (21) or
derivatives of these plasmids were grown and stored on
plates containing 40 ,ug of thiostrepton per ml. Plates were
incubated at 30C until sporulation occurred and were stored
at 4C. Nitrate-defined medium (NDM), used in 14C- and
13C-labeling experiments, and GPS fermentation medium
have been described previously (8).
Genetic manipulations. Procedures for protoplast forma-
tion, transformation, and regeneration of protoplasts for the
anthracycline-producing Streptomyces strains and for prep-
aration of chromosomal DNA have been described in detail
elsewhere (20). Streptomyces plasmids were isolated by the
methods described by Kieser (18). Standard procedures used
for manipulating anthracycline-nonproducing streptomyce-
tes are detailed by Hopwood et al. (16).
For the construction of pANT12, pIJ2303 was completely
digested with PstI, treated with calf intestinal alkaline phos-
phatase (Boehringer Mannheim Biochemicals, Indianapolis,
Ind.), ligated with PstI-digested pIJ350 by using T4 DNA
ligase (20), and introduced by transformation into Strepto-
myces galilaeus ATCC 31133 and Streptomyces peucetius
4818 BARTEL ET AL.
ATCC 29050 by methods described previously (20). Other
subclones were constructed from pANT12 by similar proce-
dures.
DNA-DNA hybridizations. For hybridization analyses,
samples of chromosomal DNA, purified from S. galilaeus
31133, S. galilaeus 31671, and S. coelicolor A3(2) as de-
scribed previously (20), were digested with BamHI for 12 h,
using 2 U of enzyme per ,ug of DNA. Plasmid pANT45, used
as a probe for the actIII gene, was labeled by the 32p_
oligolabeling procedure described by Feinberg and Vo-
gelstein (12), using 50 jxCi of [c_-32P]dCTP per ,ug of DNA.
Southern blots and hybridizations were performed as de-
scribed by Maniatis et al. (25).
Detection of polyketides. Strains to be tested for the
production of new compounds were grown in 5 ml of GPS
medium in test tubes (16 by 150 mm) for 7 days with rotary
shaking (ca. 250 rpm; 28C) while in a slanted position.
Plasmid-bearing strains were grown in the presence of 10 ,ug
of thiostrepton per ml. Alternatively, mycelia from a plate
culture were inoculated by loop into 50 ml of either GPS
medium or NDM supplemented with thiostrepton (10 ,ug/ml)
in a 250-ml Erlenmeyer flask containing a coiled spring (9).
These cultures were grown at 28C on a rotary shaker (250
rpm) for 3 to 7 days. After incubation, the broth was
extracted with an equal volume of chloroform-methanol
(9:1). The extract was dried under a stream of air and
dissolved in either chloroform or acetone. Concentrated
extracts were spotted onto 0.25-mm silica gel thin-layer
chromatography (TLC) plates (Whatman, Inc.), which were
developed with either solvent system 1 (SS1), containing
benzene, acetone, and methanol (100:10:1), solvent system 2
(SS2), which consisted of chloroform, heptane, and metha-
nol (5:5:1), or solvent system 3 (cyclohexane-ethyl acetate
[1:1]). The compounds on the TLC plates were visualized by
their normal pigmentation and by their fluorescence under
long-wave UV irradiation at 365 nm. Anthraquinones and
anthracyclinones also were separated and identified by high-
performance liquid chromatography (HPLC), using a solvent
system of 65% methanol, 30% water, and 5% glacial acetic
acid (final pH, 2.5). The compounds were detected at 254 nm
with an on-line Hitachi 100-40 spectrophotometer equipped
with an Altex flow cell and quantitated by using a Hewlett-
Packard HP3396A integrator.
Purification of anthraquinones and anthracyclinones. For
purification of large quantities of polyketide metabolites, the
streptomycetes were grown in GPS complex fermentation
medium (8) in a 14-liter New Brunswick Microferm fermen-
tor. The following conditions were used: working volume, 10
liters; temperature, 28C; agitation, 400 rpm; aeration, 8
liters of air per min; pH, initially 7.5 but not controlled. The
fermentors were inoculated with 500 ml of 2-day-old cultures
grown in yeast malt broth. After 5 days of growth, the entire
fermentation culture broths were extracted twice with equal
volumes of ethyl acetate (or, alternatively, with chloroform-
methanol [9:1]). For each purification, the organic layer was
collected and concentrated in vacuo. The concentrated
sample was defatted with 100 ml of hexane, after which the
upper (hexane-lipid) layer was discarded. The lower layer,
which was dried and redissolved in a small volume of
methanol, was loaded onto a Sephadex LH-20 (Pharmacia,
Inc., Piscataway, N.J.) column (2.5 by 45 cm) and eluted
with either methanol or methanol-methylene chloride (80:
20). Fractions containing the compounds of interest (as
determined by TLC) were pooled and dried in vacuo. The
dried samples were dissolved in acetone, and concentrated
samples were applied to a Silica Gel G column (2.5 by 30
J. BACTERIOL.
cm), which was developed with chloroform-heptane-meth-
anol (5:5:2) as the solvent. Fractions containing the com-
pounds of interest (as determined by TLC) were pooled and
dried in vacuo, and the dried samples were dissolved in
acetone and streaked onto preparative TLC plates (silica gel,
2 mm thick; E. Merck AG, Darmstadt, Federal Republic of
Germany). The plates were developed by using SS1, the
appropriate bands were scraped off, and the compounds
were eluted with acetone. These samples were dried in
vacuo and dissolved in a small volume of acetone; if re-
quired, the samples were further purified by preparative
TLC, using SS1 or SS2 as required.
[1,2-13C2]acetate feeding experiments. Cultures of S.
galilaeus(pANT12) were grown in 50 ml of NDM in a 250-ml
flask containing a stainless-steel spring to disperse the myce-
lia (9). After 2 days of growth, 20 ml of the culture was used
to inoculate each of two 2-liter flasks containing 500 ml of
NDM. The flasks (containing stainless-steel springs) were
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incubated with shaking (ca. 250 rpm) at 30C. Sodium
acetate (total of 0.5 g), at a ratio of [1,2-13C2]acetate (99
atoms% 13C)/nonlabeled sodium acetate of 1:2, was added to
the culture in two equal portions after 72 and 96 h of
incubation. After 120 h of incubation, the mycelia were
harvested by centrifugation at 10,000 x g for 10 min, and the
culture broth supernatant was decanted and extracted with
an equal volume of chloroform-methanol (9:1). The mycelial
pellet was extracted once with 100 ml of hexane to remove
neutral lipids. The organic layer was discarded, and the
pellet was extracted twice with 100 ml of acetone. The two
acetone extracts of the mycelial pellet were combined and
concentrated in vacuo, followed by extraction twice with
equal volumes of chloroform-methanol (9:1). These mycelial
extracts were combined with the culture broth extract, and
the entire extract was concentrated in vacuo. Aloesaponarin
II was purified from these extracts as described above.
Analysis by NMR spectroscopy. Samples for nuclear mag-
netic resonance (NMR) spectroscopy were dissolved in 0.4
ml of DMSO-d6 (99+ atoms% 2H). NMR experiments were
performed at a field of 7.1 T on an IBM AF-300 spectrometer
employing an ASPECT-3000 data system. 13C NMR spectra
were acquired with continuous composite pulse proton de-
coupling. Nuclear Overhauser effect (NOE) studies were
conducted on samples of aloesaponarin II by a one-dimen-
sional steady-state difference method.
Chemicals. Sodium [1,2-13C2]acetate (99 atoms% 13C) was
obtained from Cambridge Isotope Laboratories, Woburn,
Mass. Sodium [1,2-14C]acetate (55 mCi/mmol) was obtained
from Dupont, NEN Research Products, Boston, Mass.
Authentic standards for anthracyclines were obtained from
Adria, Inc., the Frederick Cancer Research Center, and
Rhone-Poulenc. Restriction endonucleases were purchased
from Bethesda Research Laboratories, Inc., Gaithersburg,
Md., and deuterated solvents for NMR analysis were ob-
tained from Sigma Chemical Co., St. Louis, Mo.
RESULTS
Production of aloesaponarin II by streptomycetes trans-
formed with actinorhodin biosynthesis genes. Subclones of
pIJ2303, a plasmid containing all of the actinorhodin biosyn-
thesis genes (23), were generated to transform anthracycline-
producing strains for the purpose of searching for new
hybrid polyketide structures. An 8.8-kilobase-pair (kbp) PstI
fragment of DNA from pIJ2303, containing the actI, actIII,
actlV, and actVII loci (Fig. 1), was subcloned into the
high-copy-number vector pIJ350 to yield plasmid pANT12.
VOL. 172, 1990 HYBRID ANTHRAQUINONE BIOSYNTHESIS 4819

0.

I- a a ao0
C.) 4
.I rc co,
a
0
U- U- g- cm

actill Abesaponarinhl
'' actl actvU actlV
p p
pANT12 +
sp mm

pANT35 +
p s
pANT33 +
x x
PANT43 +
a B
pANT44 -

B B
pANT45 -

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B B
pANT46
FIG. 1. Structure of part of the act genetic loci and restriction map of the actinorhodin biosynthesis genes. The restriction site numbers
12, 13, 14, 17, 18, and 20 correspond to the numbers given those sites by Malpartida and Hopwood (24). Sites 12a, 13a, 13b, 14a, 17a, 18a,
and 18b were determined in this work. The restriction fragments which conferred aloesaponarin II production on S. galilaeus ATCC 31133
are identified. Abbreviations for restriction endonuclease sites: P, PstI; Sp, SphI; S, SstI; X, XhoI; B, BamHI.

This plasmid was introduced by transformation into S.
galilaeus ATCC 31133, a strain that normally produces
aclacinomycin A (28). When transformed with pANT12,
however, this strain formed significant quantities of a new
yellow compound, which was distinct from anthracyclines
produced by S. galilaeus by its migration on TLC, elution by
HPLC, and unusual orange fluorescence at 365 nm, a prop-
erty not shared by the anthracyclines (Table 2). The yellow
compound was purified from cultures of S. galilaeus
31133(pANT12) and identified by its '3C and 'H NMR
spectra (Table 3) and by comparison with an authentic
reference sample (35) to be 1,7-dihydroxy-9-methylan-
thraquinone, also known as aloesaponarin 11 (35; Fig. 2A).
The positioning of the methyl group on the anthraquinone
ring system was confirmed by proton NOE experiments.
Control experiments and subcloning of antinorhodin genes.
When the 8.8-kbp PstI DNA fragment from pANT12 was
subcloned in the low-copy-number vectors pIJ941 and pIJ61
to construct pANT28 and pANT31, respectively, and intro-
duced by transformation into S. galilaeus 31133, aloesapon-
arin II was the major product formed by the recombinant
cultures (Table 3). The amount of aloesaponarin II produced
by S. galilaeus cultures grown in GPS medium was inversely
related to the copy number of the plasmids carrying the act
genes; S. galilaeus 31133 carrying pANT12 (plJ101 replicon,
Sti-; reported copy number, 50 to 100 [10]), pANT31 (SLP1
replicon; reported copy number, 4 to 5 [2]), and pANT28
(SCP2* replicon; reported copy number, 1 to 2 [21]) yielded
1.1, 5.7, and 10.6 jig of aloesaponarin II per ml of whole
culture broth (mycelia plus filtrate), respectively. Most of
the aloesaponarin II produced by the recombinant S. gali-
laeus 31133 cultures was in the mycelial fraction; very little
aloesaponarin II was found extracellularly.
S. galilaeus 31133 transformed with pIJ702, pIJ941, or
pIJ61 did not form aloesaponarin II, indicating that its
production was due to the presence of the antinorhodin
biosynthesis-encoding DNA and not to the presence of the
plasmids. S. peucetius, another anthracycline-producing
streptomycete (9, 33), and the anthracycline-nonproducing
streptomycetes Streptomyces azureus and Streptomyces
parvulus also produced aloesaponarin II when transformed
with plasmids containing the actI, actIII, actVII, and actIV
loci (Table 4).
Several subclones of the act DNA fragment in pANT12
were prepared to determine which of the antinorhodin
biosynthesis genes were needed to confer aloesaponarin II
biosynthesis on S. galilaeus 31133. To construct subclones
of the actinorhodin DNA, we determined a more detail
restriction map of the act DNA on pANT12. The restriction
sites 12a, 13a, 13b, 14a, 17a, 18a, and 18b are sites which we

TABLE 2. Chromatographic analysis of anthraquinones and anthracyclinones produced by nonrecombinant and recombinant cultures
Analysis by TLC
HPLC
Compound Rf inb: retention time
Pigment Fluorescence' (min)a
SS1 SS2 SS3
Aklavinone 0.30 0.35 0.42 Yellow Yellow 8.9
2-Hydroxyaklavinone 0.09 0.25 0.11 Yellow Light yellow 14.8
Aloesaponarin II 0.37 0.33 0.52 Yellow Bright orange 14.9
Desoxyerythrolaccin 0.11 0.19 0.32 Red Purple 11.5
1-O-methyldesoxyerythrolaccin 0.37 0.30 0.52 Red Pink 20.1
a Using a solvent system of 65% methanol-30% water-5% acetic acid (pH 2.5) and a Whatman C18 reverse-phase column.
b Solvent
systems SS1 to SS3 are defined in Materials and Methods.
c Color emitted when viewed under UV light at 365 nm.
4820 BARTEL ET AL. J. BACTERIOL.

TABLE 3. "3C and 'H NMR data on the structure and carbon- TABLE 4. Formation of polyketides by nonrecombinant
carbon coupling patterns for aloesaponarin IIa and recombinant strains
13C 'H Strain Plasmid Major polyketide
Carbon Chemical Chemical compound(s) formed
no. shifb Multi- Jcc Multi- JHH Aclacinomycin A
plicityd (Hz)' shift plicity (Hz) S. galilaeus 31133 None
(ppm)c S. galilaeus 31133 pIJ702 Aclacinomycin A
9' 23.4 q 41.4 2.65 s - S. galilaeus 31133 pIJ61 Aclacinomycin A
9 145.2 s 41.4 - - - S. galilaeus 31133 pIJ941 Aclacinomycin A
12 116.4 s 61.0 - - - S. galilaeus 31133 pANT12 Aloesaponarin II
10 189.2 s 61.0 - - - S. galilaeus 31133 pANT28 Aloesaponarin II
13 122.2 s 60.3 - - - S. galilaeus 31133 pANT31 Aloesaponarin II
1 162.4 s 60.3 - - - S. galilaeus 31133 pANT33 Aloesaponarin II
2 124.1 d 59.0 7.35 dd 8.0, 1.3 S. galilaeus 31133 pANT35 Aloesaponarin II
3 135.8 d 59.0 7.70 t 8.0 S. galilaeus 31133 pANT43 Aloesaponarin II
4 118.1 d 62.1 7.65 dd 8.0, 1.3 S. galilaeus 31133 pANT44 Aclacinomycin A
14 132.5 s 62.2 - - - S. galilaeus 31133 pANT45 Aclacinomycin A
5 182.1 s 52.0 - - - S. galilaeus 31133 pANT46 Aclacinomycin A
11 136.8 s 52.1 - - - S. galilaeus 31133 pANT56 Aloesaponarin II

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6 112.0 d 62.2 7.48 d 2.6 S. galilaeus 31671 None 2-Hydroxyaklavinone
7 161.4 s 62.1 - - - S. galilaeus 31671 pANT12 Aloesaponarin II
8 124.5 d NC 7.06 d 2.6 S. galilaeus 31671 pANT28 Aloesaponarin II
S. galilaeus 31671 pANT31 Aloesaponarin II
a Abbreviations and symbol: s, singlet; d, doublet; q, quartet; dd, doublet of S. galilaeus 31671 pANT33 Aloesaponarin II; aklavinone
doublets; JCC, coupling constant of 13C (determined after incorporation of S. galilaeus 31671 pANT35 Desoxyerythrolaccin; 1-0-
sodium [1,2-13C2]acetate); JHH, coupling constant of 1H; NC, not coupled; -, methyldesoxyerythrolaccin
no signal acquired. S. galilaeus 31671 pANT43 1-O-Methyldesoxyerythrolaccin
b See Fig. 2A.
c Chemical shifts for both 13C and 'H were referenced internally to solvent
S. galilaeus 31671 pANT44 2-Hydroxyaklavinone
resonance.
S. galilaeus 31671 pANT45 Aklavinone
I Determined by edited DEPT (distortion enhancement by polarization S. galilaeus 31671 pANT46 2-Hydroxyaklavinone
transfer) analysis. S. peucetius 29050 None Daunomycin
S. peucetius 29050 pANT12 Aloesaponarin II
S. parvulus 2266 None NKa
S. parvulus 2266 pANT12 Aloesaponarin II
S. azureus 14921 None NK
S. azureus 14921 pANT28 Aloesaponarin II
S. azureus 14921 pANT56 NK
A. B. a
NK, None known to be produced.

4 6
HO OH determined for the purpose of subcloning the act genes (Fig.
2 1). Subclones of the actinorhodin DNA which conferred
R 0
aloesaponarin II formation on S. galilaeus 31133 are shown
CH3 in Fig. 1 and Table 4. The 2.8-kbp XhoI fragment in pANT43
B (Fig. 1) was the smallest DNA fragment which conferred the
ALOESAPONARIN 11 DESOXYERYTHROLACCIN -OH ability to synthesize aloesaponarin II on S. galilaeus 31133
1 -Q-METHYLDESOXY- (Table 4). On the other hand, S. azureus(pANT56) (pIJ941
ERYTHROLACCIN -OCH3
containing the 2.8-kbp Xhol fragment; Table 1) did not
produce aloesaponarin II (Table 4). The fact that the 2.8-kbp
C. D. XhoI insert, containing only the actI and actVII loci, con-
ferred the ability to form aloesaponarin II on the anthracy-
H 0 COOH o OCH3 cline-producing strain, S. galilaeus ATCC 31133, but not on
S. azureus (a strain not known to synthesize polyketides)
R. indicates that the actIII and (or) actIV functions, apparently
required for aloesaponarin II biosynthesis, are supplied by
OH H 0 OH OH
S. galilaeus 31133.
CH3 Production of aloesaponarin II by S. coelicolor mutants.
B The proposed functions for the first four genes of the
ACTINORHODIN -H
AKLAVINONE actinorhodin biosynthesis pathway, actI, actIII, actIV, and
2-HYDROXYAKLAVINONE -OH actVII (which are present in pANT12), suggest that they are
FIG. 2. Structures of compounds described in the text. (A) theoretically capable of converting malonyl coenzyme A
Structure of aloesaponarin II with numbered carbons, and its 13C (malonyl-CoA) and acetyl-CoA to a precursor of actinorho-
enrichment and _3C-'3C coupling pattern after feeding with sodium din (13). To determine whether any of the S. coelicolor
[1,2-l'3C2]acetate. Symbols: 0, enriched carbon showing no 13C-13c mutants blocked in actinorhodin biosyhthesis produced even
coupling; - , pairs of enriched, '3C-'3C coupled carbons. See minor quantities of aloesaponarin II, we analyzed the chlo-
Table 2 for NMR data. (B) Structures of desoxyerythrolaccin (R =
OH) and 1-O-methyldesoxyerythrolaccin (R = OCH3) with num- roform-methanol (9:1) extracts of several cultures of S.
bered carbons. See Table S for NMR data. (C) Structure of acti- coelicolor blocked mutants, grown for 5 days in either GPS
norhodin. (D) Structures of the anthracyclinones aklavinone (R = medium or NDM, for the presence of aloesaponarin II.
H) and 2-hydroxyaklavinone (R = OH). When grown in either medium, S. coelicolor B22 and B159
VOL. 172, 1990 HYBRID ANTHRAQUINONE BIOSYNTHESIS 4821

(actVI mutants) each produced a compound with the same TABLE 5. 13C and 'H NMR data on the structure of desoxy-
Rf value, the same pigmentation, and the same fluorescence erythrolaccin isolated from S. galilaeus 31671(pANT35)a
characteristics as aloesaponarin II (Table 2). Strain B22 also 13C 'H
produced trace amounts of two other yellow compounds (Rf Carbon Chemical Chemical
values of 0.60 and 0.70 in SS2; P. L. Bartel, unpublished no. shiftc Multiplicityd shift Multiplicity (Hz)
data). The major pigmented product of B22 culture extracts (ppM) (ppm)
was purified and determined by "C and 'H NMR to be
aloesaponarin II (data not shown). None of the other S. 1 163.9 s - - -
coelicolor blocked mutants tested, including B17, B18, B40, 2 107.0 d 6.52 d 2.90
and B41 (described in Table 1), produced detectable levels of 3 164.3 s - - -
aloesaponarin II. 4 108.1 d 7.19 d 2.90
5 182.5 s - - -
Labeling with [1,2-"CJ]acetate. To determine the pattern 6 112.3 d 7.45 d 2.65
of acetate incorporation into aloesaponarin II, sodium [1,2- 7 161.6 s - - -
"C2]acetate was administered to a culture of S. galilaeus 8 124.7 d 7.15 d 2.65
31133(pANT12), and the product was purified. The carbon- 9 144.8 s - - -
carbon spin-coupling pattern of the labeled aloesaponarin II 9' 23.3 q 2.70 s -
demonstrated that all carbon atoms except C-2 were coupled 10 187.2 s - - -
to one neighboring carbon (Fig. 2A; Table 3). This finding 11 136.7 s - - -

Downloaded from http://jb.asm.org/ on June 23, 2017 by guest

indicates that aloesaponarin II was formed by condensation
of 8 acetyl-CoA equivalents, followed by a decarboxylation
which abolished coupling at C-2 by scission of the carbon-
carbon bond (Fig. 2A).
Formation of natural and hybrid polyketides by recombi-
nant S. galilaeus 31671. Mutants of S. galilaeus that produce
2-hydroxy derivatives of aklavinone-related anthracyclines
have been isolated and characterized (26). One of these
strains was deposited in the American Type Culture Collec-
tion (Rockville, Md.) as S. galilaeus ATCC 31671, a strain
that produces 2-hydroxyaklavinone (see Fig. 2D for struc-
ture) as its major product (26). S. galilaeus 31671(pANT12)
produced aloesaponarin II as its major product' (Table 4).
However, when S. galilaeus 31671 was transformed with
pANT33, a plasmid that contains the actI, actIII, and actVII
loci on a 4.7-kbp PstI-SstI DNA fragment (Fig. 1), both
aloesaponarin II and aklavinone were formed (Table 4). The
latter was -identified by comparison with authentic aklavi-
none (Table 2), obtained by acid hydrolysis (0.1 N HCl, 60
min) of aclacinomycin A and purified by preparative TLC
using 'SS1. Because 'S. galilaeus 31671, which normally
produces small quantities of 2-hydroxyaklavinone, formed
aklavinone as a result of being transformed with pANT33, a
function encoded by pANT33-was apparently responsible for
the deoxygenation at C-2 (Fig. 2D). When pANT35, a
subclone of pANT12 containing a 4.3-kbp SphI DNA frag-
ment with the actI, actVII, and actIV loci, was introduced
into S. galilaeus 31671, at least three new compounds were
produced. The major compound was identified by "C and
'H NMR (Table 5) and comparison with authentic material
as 1,3,7-trihydroxy-9-methylanthraquinone, also known as
desoxyerythrolaccin (27, 35) (Fig. 2B). A second abundantly
produced compound was identified by 13C and 'H NMR
(data not shown) as 1-O-methyldesoxyerythrolaccin (Fig.
2B; Table 4). The position of the 0-methyl group was
confirmed by proton NOE experiments. After several puri-
fication steps, this'compound was still slightly contaminated
with a second 0-methylated compound, either 3-0-methyl-
or 7-O-methyldesoxyerythrolaccin, which usually comi-
grated with the 1-0-methyl derivative. The position of the
0-methyl group of the contaminant, however, has not been
established unambiguously. When S. galilaeus 31671 was
transformed with pANT43, which contains the actI and
actVII loci (Table 1), only 1-O-methyldesoxyerythrolaccin
(and its associated contaminant) was produced. Wild-type S.
galilaeus 31133(pANT43), on the other hand, produced the
3-desoxy derivative of desoxyerythrolaccin, aloesaponarin
II. These data demonstrate that the actIII gene function, not
12 122.2 s - - -
13 109.7 s - - -
14 134.2 s - - -
a
Abbreviations and symbol: s, singlet; d, doublet; q, quartet; JHH, 1H-1H
coupling constant; -, no signal acquired.
b
See Fig. 2B.
c Chemical shifts for both '3C and 'H were referenced internally to solvent
resonance.
d Determined by edited DEPT analysis.
required for aloesaponarin II formation by S. galilaeus
31133, is required for aloesaponarin II biosynthesis by S.
galilaeus 31671. This finding indicates that S. galilaeus
31133, a strain which synthesizes aklavinone, contains an
actIII-equivalent gene function that is lacking in the 2-
hydroxyaklavinone-producing strain S. galilaeus 31671. To
test this hypothesis, we constructed plasmid pANT45, which
contains the 1,035-bp BamHI fragment carrying the intact
actIII gene (15) in the low-copy-number streptomycete vec-
tor pIJ61 (34). When S. galilaeus 31671 was transformed
with pANT45, aklavinone was the only product formed
(Table 4). The structures of 2-hydroxyaklavinone produced
by S. galilaeus 31671 and aklavinone produced by the same
strain carrying pANT45 were confirmed by purifying the
chloroform-methanol (9:1)-soluble products as'described in
Materials and Methods and analyzing them by TLC, HPLC
(Table 2), and 'H NMR (data not shown). Thus, the actIlI
gene from S. coelicolor, responsible for a discrete biosyn-
thetic step in the actinorhodin biosynthesis pathway (15), is
able to complement a'missing function of S. galilaeus 31671
to confer aklavinone formation.
In control experiments, cultures of S. galilaeus 31671
transformed with pIJ702, pIJ61, pIJ941, pANT44 (containing
a 2.2-kbp BamHI fragment with part of the actl genetic
locus), or pANT46 (containing a 0.8-kbp BamHI fragment
with part of the actVII locus) produced 2-hydroxyaklavinone
as their major product. In none of these cases was aklavi-
none, desoxyerythrolaccin, 1-O-methyldesoxyerythrolaccin,
or aloesaponarin II produced.
Southern hybridizations to identify actIII-homologous se-
quences. Streptomyces sp. strain C5 and S. peucetius ATCC
29050, strains which produce daunomycin and similar an-
thracyclines that lack an oxygen function at C-2, contain an
actIII-hybridizing DNA fragment (22, 33; J. L. Lampel and
W. R. Strohl, unpublished data). On the other hand, Strep-
tomyces glaucescens ETH 22794, a strain which produces
tetracenomycin, an anthracycline that is apparently pro-
duced without such a reduction step (22, 37), does not
4822 BARTEL ET AL.
12 3 4 5 7
6.1-
3.9-
2.5
1.4--
4'
FIG. 3. Southern blots showing hybridization of the actIII gene
(plasmid pANT45) to DNA isolated from S. coelicolor and from S.
galilaeus 31133 and 31671. Lanes: 1, BamHI-digested DNA from S.
coelicolor showing a 1,035-bp fragment (15); 2 to 4, S. galilaeus
31133 DNA digested with BamHI, PstI, and SstI, respectively; 5 to
7, S. galilaeus 31671 DNA digested with BamHI, PstI, and SstI,
respectively. The hybridizations were carried out at 42C using 50%
formamide, with a single wash at 55C for 1 h using 0.2x SSC buffer.
contain DNA fragments that hybridize to the actIII gene
(22). Our data indicate that S. galilaeus 31133 possesses an
active actIII-equivalent function but that S. galilaeus 31671,
a derivative of 31133, does not. We probed BamHI, PstI,
and SstI restriction endonuclease-digested DNA samples
from cultures of each of these strains to determine whether
there were any differences in the presence of potential
actIII-hybridizing sequences. The hybridization data show
that both S. galilaeus 31133 and 31671 contain sequences of
the same size and restriction digestion patterns that are
homologous to actIII from S. coelicolor (Fig. 3). This finding
confirms that S. galilaeus 31133 possesses a gene homolo-
gous to the actIII gene, as suggested by the complementa-
tion data. Furthermore, the hybridizations suggest that the
mutation of the actIII-homologous gene of S. galilaeus
31671, made by using N-nitro-N'-nitrosoguanidine (26), is
probably a point mutation (or a series of point mutations).
DISCUSSION
Production of aloesaponarin II. In our attempts to generate
hybrid antibiotics by transforming discrete restriction frag-
ments of the actinorhodin biosynthesis gene cluster into
various streptomycetes, we found that an anthraquinone,
aloesaponarin II (35), was produced by recombinant strep-
tomycetes that normally do not produce that compound.
Aloesaponarin II was originally isolated from aloe plants
(35), and to our knowledge it has never before been detected
as a bacterial product.
The formation of aloesaponarin II by streptomycetes
transformed with DNA encoding actinorhodin biosynthesis
was correlated with the presence of the actI, actIII, actVII,
and actIV loci in certain strains. Strains B22 and B159, actVI
mutants of S. coelicolor (30), also were found to produce
aloesaponarin II as an apparent shunt product of actinorho-
din biosynthesis. 'This concurs with the order of the act
mutants delineated by cross-feeding and cosynthesis studies
(30), which suggests that the actVI mutants should form
products which are the result of the functions of the actI,
actIII, actIV, and actVII loci, the same genetic loci present
in plasmids pANT12, pANT28, and pANT31 (Table 1).
Thus, our data indicate that aloesaponarin II production is
J. BACTERIOL.
conferred by the activity of the gene products encoded by
the actI, actIff, actIV, and actVII loci in the absence of an
active actVI gene product, whether these are present and
active in S. coelicolor (as in the case of the actVI blocked
mutants B22 and B159) or cloned into a heterologous recip-
ient (e.g., S. galilaeus, S. peucetius, S. azureus, or S.
parvulus).
Minimal genetic requirements for aloesaponarin II biosyn-
thesis. The minimal fragment of DNA encoding actinorhodin
biosynthesis which confers aloesaponarin II production on
S. galilaeus 31133, the 2.8-kbp XhoI fragment (pANT43),
appears to contain the actI and actVII loci (24). The XhoI
site is 50 bp upstream from the BamHI site (15), the latter of
which appears to interrupt actI gene function (24). The actI
locus has been reported to be transcribed on a polycistronic
message that begins somewhere upstream of the BamHI site
14 (Fig. 1) and continues through the actVII and actIV
genetic loci (24). This suggests that the 50-bp segment
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between XhoI site 13b and BamHI site 14 probably contains
the promoter region required for this polycistronic tran-
script.
The data showing that pANT43, a plasmid carrying only
the actI and actVII loci, conferred aloesaponarin II forma-
tion on S. galilaeus 31133 indicate either that the functions
encoded by the actIII and actIV loci are not required for
aloesaponarin II production or that these functions are
carried out by S. galilaeus 31133 gene products. Since
pANT28 (containing actI, actIII, actVII, and actlV loci)
confers aloesaponarin II production on S. azureus (a strain
not known to produce polyketides) but pANT56 (containing
just the actl and actVII loci) does not, it appears that the
actIll and (or) actIV loci, or their equivalents, are required
for aloesaponarin II biosynthesis. Since the actIll and actlV
loci are not present in pANT43 and pANT56, plasmids
which confer aloesaponarin II formation on S. galilaeus
31133, these functions appear to be carried out by S.
galilaeus gene products. When the actl, actVII, and actlV
genes were cloned into an S. galilaeus strain lacking a
functional actIII-like gene (strain ATCC 31671), a different
product was formed, further indicating that the S. galilaeus
31133 actIII-like gene was cross-functional.
Pathway for aloesaponarin II and actinorhodin biosynthe-
sis. Based on the precursors required for formation of
actinorhodin (2x C16; 2x 8 acetyl-CoA equivalents [14]) and
anthracyclines (C21; 9 acetyl-CoA equivalents condensed
onto a propionyl-CoA starter unit [6]) (Fig. 4), two routes are
possible for the formation of aloesaponarin II (C15): (i)
propionyl-CoA might be used as a starter unit with the
condensation of 6 acetyl-CoA equivalents, or (ii) an acetyl-
CoA starter unit might be used, onto which 7 acetyl-CoA
equivalents would be condensed, followed by a decarboxy-
lation. The carbon-carbon coupling patterns determined by
13C NMR showed that aloesaponarin II is formed by the
condensation of 8 acetyl-CoA equivalents, followed by a
decarboxylation at the carboxy-terminal end of the poly-
ketide (Fig. 2A and 5). Thus, the aloesaponarin II precursor
polyketide was not synthesized with use of a propionyl-CoA
starter unit, consistent with formation of the aloesaponarin
II polyketide in S. galilaeus 31133(pANT12) being catalyzed
primarily by the act gene products.
The major differences between aloesaponarin II and acti-
norhodin monomer are the folding pattern in the third ring
and the loss of one carbon by decarboxylation in the
formation of aloesaponarin II (Fig. 2A, 2C, 4, and 5). Since
aloesaponarin II is a shunt product in S. coelicolor B22 and
also is a product of S. azureus(pANT28), it is apparent that
VOL. 172, 1990 HYBRID ANTHRAQUINONE BIOSYNTHESIS 4823

Number of
Starter Units Extender Units OOH OOH
8 6 4 2 20 HO 0
0 2319 dauB

A. Propionyl-CoA 9 H 0 0
00 10
O 00 0 O 0 0 0
O OCH3 O OCOCH3
HO NZ
A B. H

OH H OH OH 0 H bH

COOH OOH
HO
05e - 3
act/Ul H

B. Acetyl-CoA 6 101 3

0 0

Downloaded from http://jb.asm.org/ on June 23, 2017 by guest

0COOH
H H 0~~~ COOH

C IC ]D

0
OH
2 H3 -2 OH CH3

COOH
HO
H 19~
C. Acetyl-CoA 6 10t:
0
HO H O
E.9H H

N3 0
OH H3

FIG. 4. Structures and theoretical polyketide precursors of the polyketides aklavinone (A), actinorhodin (B), and aloesaponarin 11 (C),
formed as a result of cloning act genes in S. galilaeus 31133 and 31671. The starter units and number of extending acetyl units (from
malonyl-CoA) are identified for each compound. The proposed activity of the actIfI and actIII-homologous genes is shown in relation to the
products formed. If the actIII function, polyketide reductase, is present, then the keto group at the ninth carbon from the carboxyl end of
the polyketide chain in each case is reduced. Upon ring closure and aromatization, a hydroxyl group is lost from that carbon and the resultant
molecule is deoxygenated in that position. If the actIII function is not present, then the resultant molecules contain hydroxyl groups in the
respective positions (marked with dots). (A) 2-Hydroxyaklavinone (A) and aklavinone (B) biosynthesis; (B) biosynthesis of the postulated
hydroxyactinorhodin monomer molecule (C; which has not been found) and actinorhodin monomer (D); (C) biosynthesis of desoxyerythro-
laccin (E) and its 3-deoxy analog, aloesaponarin 11 (F).

the unusual folding pattern is not solely caused by activities
associated with anthracycline biosynthesis. We have dem-
onstrated that the polyketide precursor of actinorhodin can
undergo an entirely different type of cyclization in S. coeli-
color to produce mutactin, representing a novel carbon
skeleton (38). Mutactin is formed in traces by the wild-type
strain, but its production is enhanced 100-fold in the actVII
mutant B40 (38).
By combining the data obtained on the biosynthesis of
aloesaponarin II, formed by the act!, actIII, actVII, and
actIV loci (Fig. 1; Table 4), and mutactin (38), requiring only
the actI and actIII loci from the actinorhodin pathway, we
have developed a hypothetical route for the formation of
actinorhodin, aloesaponarin II, and mutactin (Fig. 5). We
propose that aloesaponarin II is formed in cultures of S.
galilaeus 31133(pANT12) and S. coelicolor B22 and B159 by
the following sequence of events. (i) A C16 polyketide is
formed by the action of the actinorhodin polyketide synthase
complex (actI gene products [22]), which catalyzes the
sequential condensation of seven acetyl-CoA equivalents
(from malonyl-CoA) onto an initial acetyl-CoA starter unit
(Fig. 4 and 5). (ii) The polyketide is reduced at the ninth
carbon from the carboxyl end by the action of the polyketide
reductase. This enzyme is encoded either by the actIII gene
(15), if present, or by the actIII-equivalent gene of the S.
galilaeus 31133 genome, as would occur when this organism
is transformed with pANT35 or pANT43. The first ring
would be formed by the action of gene products encoded by
the act! locus, perhaps as a stabilization event (11), by bond
formation between C-7 and C-12 (Fig. 5). These steps are
shared with the biosynthesis of actinorhodin as well as
mutactin (38). (iii) The closure of the second ring, by bond
formation between C-5 and C-14, and aromatization of the
first ring would be carried out by a function encoded by
actVII. This step is shared with the biosynthesis of acti-
norhodin, but in the actVII mutant S. coelicolor B40, this
step cannot occur, allowing formation of the mutactin pre-
cursor. This occurs by activation of C-6 toward aldol con-
densation with C-15 (Fig. 5). (iv) The postulated bicyclic
intermediate is dehydrated by the gene product(s) encoded
by actIV to yield a compound which is the precursor to
either actinorhodin or aloesaponarin II (Fig. 5). In the
presence of actVI, this precursor would be cyclized by
attack of the hydroxyl oxygen on the carbonyl group,
4824 BARTEL ET AL.
171 HOOCCH2COSCoA ct
CH,COSCoA
7C02
FIG. 5. Hypothetical pathway for the biosynthesis of mutactin (38), aloesaponarin II (this work), and actinorhodin (13, 14), based on
results obtained in this paper and data from other sources (7, 13-15, 38). The probable functions of the gene products of actI (polyketide
synthase), actIII (polyketide reductase), actVII (cyclase and/or dehydratase), and actIV (dehydratase) are given. In the presence of the actVI
gene product (cyclase and/or reductase), the pathway would follow reaction A to form actinorhodin; in the absence of the actVI gene product,
the pathway would follow reaction B to form aloesaponarin II.
followed by dehydration and reduction to give the heterocy-
clic actinorhodin precursor, which has been isolated from an
actVa mutant (7) (Fig. 5, reaction A). In the absence of
actVI, however, aldol condensation of C-2 with the carbonyl
group at C-15 followed by dehydrative decarboxylation
would form the precursor to aloesaponarin II (Fig. 5, reac-
tion B), which would then undergo spontaneous air oxida-
tion to form aloesaponarin II.
Function of the actIlI gene in polyketide formation. Malpar-
tida et al. (22) hypothesized that the actIII gene encodes a
polyketide reductase which reduces a keto group of the
actinorhodin polyketide intermediate (Fig. 4B). After this
reduction a hydroxyl group would be lost from this position
during ring closure and aromatization, and the resulting
compound would lack an oxygen function in that position
(Fig. 4). If no reduction were to occur at C-9 of the
actinorhodin precursor polyketide before ring formation and
aromatization, then the corresponding compound would
contain a hydroxyl group in that position (Fig. 4). Hallam et
al. (15) recently determined the DNA sequence for the actIII
gene and have found it to be similar to the sequences of the
ribitol dehydrogenase gene from Klebsiella aerogenes and
alcohol dehydrogenase gene from Drosophila melanogaster.
Considerable data also have indicated that the actI locus
encodes a polyketide synthase function (13, 23, 24) and that
actI and actIII mutants are unable to cross-feed or cosyn-
thesize to form actinorhodin (30). Considering that these two
genes operate at the beginning of the actinorhodin biosyn-
thesis pathway, along with the sequence data indicating that
the actIII gene product is likely a dehydrogenase, Hallam et
J. BACTERIOL.
Downloaded from http://jb.asm.org/ on June 23, 2017 by guest
Mutactin
0 OH
Aloesaponarin 11
al. (15) proposed that actIII encodes a ,3-keto reductase
which reduces the 0-keto group on the forming polyketide
chain once it reaches a chain length of 10 carbons. Sherman
et al. (31) also have recently shown that S. violaceoruber
Tu22, a strain that produces the benzoisochromane quinone
antibiotic granaticin, contains two actIII-homolog genes
which displayed a high sequence similarity to actIII.
Our data indicate that the actIII gene of S. coelicolor, as
well as an analogous gene of S. galilaeus 31133, encodes an
activity which is responsible for the absence of a hydroxyl
group at C-2 of anthracyclinones(Fig. 2D) and C-3 of the
anthraquinone (Fig. 2A and B), aloesaponarin II. S. gali-
laeus 31671, the 2-hydroxyanthracycline producer, was de-
rived by mutation and screening from a strain that did not
produce 2-hydroxyanthracyclines (26). This finding suggests
that S. galilaeus 31671 was missing a function or functions
contained in the parent strain, S. galilaeus ATCC 31133.
Anderson et al. (1) recently showed that cell extracts pre-
pared from the fungus Pyrenochaeta terrestris could reduce
a trihydroxyanthraquinone, emodin, to a dihydroxyan-
thraquinone, chrysophanol, by an NADPH-linked enzyme
reaction. Their work, which described an aromatic counter-
part to deoxygenation in polyketide biosynthesis (1), raises
the possibility that the actIII gene, as well as the analogous
gene in S. galilaeus 31133, might encode a phenol reductase
(1) rather than one which operates on the open polyketide
chain (15). We tested the possibility that S. galilaeus ATCC
31133 or S. galilaeus 31671(pANT45) might show phenol
reductase activity, by reducing emodin to chrysophanol and
desoxyerythrolaccin to aloesaponarin II, that would be
VOL. 172, 1990
absent in S. galilaeus 31671. We never observed chrysoph-
anol or aloesaponarin II as products when emodin or desox-
yerythrolaccin, respectively, were incubated with NAD(P)H
and cell extracts of S. galilaeus ATCC 31133, S. galilaeus
31671(pANT45), or S. galilaeus ATCC 31671 (included as a
negative control; data not shown). Thus, we could not
demonstrate any phenol reductase activity by S. galilaeus
31133 or in S. galilaeus 31671(pANT45) which could account
for the deoxygenation at C-2 of 2-hydroxyaklavinone or at
C-3 of desoxyerythrolaccin. Although these are negative
data, they are consistent with the hypothesis of Hopwood
and colleagues (15, 22) that the actIII gene product encodes
a reductase that acts on the polyketide chain before ring
formation. Finally, we have recently shown that cell extracts
of S. galilaeus 31671 synthesize aklavinone from aklanonic
acid, the apparent earliest stable intermediate of aklavinone
biosynthesis, in the presence of NADPH (and S-adenosyl-
methionine), which also indicates that the deoxygenation of
C-2 takes place before the formation of aklanonic acid, i.e.,
at the stage of an open polyketide chain (N. C. Connors,
P. L. Bartel, and W. R. Strohl, J. Gen. Microbiol., in press).
Although there is some evidence showing that the reduc-
tion and dehydration steps in polyketide biosynthesis may
occur at defined stages of chain assembly (5, 32, 36), the
results presented in this study suggest rather that the poly-
ketide chain is completely assembled before reduction of the
keto group takes place. In the formation of both aklavinone
and aloesaponarin II, the carbonyl group which is reduced
by the actIII gene product represents the ninth carbon from
the carboxy terminus of the theoretical polyketide chains
(Fig. 4A and C, respectively), the same relative location as
that of the proposed activity in actinorhodin biosynthesis
(15; Fig. 4B). According to the theories of polyketide forma-
tion (3, 11, 32), these compounds are formed by the succes-
sive additions of acetyl groups (from malonyl-CoA) to a
starter unit. Thus, in actinorhodin, aloesaponarin II, and
aklavinone formation, chain growth starts at the methyl
terminus and proceeds to the carboxyl terminus. The fact
that the activity of the actIII gene product occurs at the ninth
carbon from the carboxyl terminus, and not at a given
distance from the methyl terminus (Fig. 4), implies that this
enzyme does not reduce the carbonyl group until the entire
chain is assembled. Hypothetically, then, the polyketide
reductase might "measure" the distance from the carboxyl
end of the completely assembled chain and reduce the
proper keto group. On the other hand, not all polyketides are
reduced at the ninth carbon from the carboxyl end (e.g.,
erythromycin and tylosin), and many polyketides are re-
duced at multiple sites (macrolides and polyethers). For
example, the macrolide milbemycin is reduced at multiple
positions, including C-9. Nevertheless, the strains that pro-
duce each of these polyketides contain actIII homolog
genes, as evidenced by DNA-DNA hybridizations (22). It is
possible that a single ,-keto reductase could reduce multiple
positions in a manner analogous to reduction of a nascent
fatty acid chain. This would require that certain keto groups
on the polyketide chain, which are not reduced, must be
protected. Thus, the polyketide synthase may present only
the proper keto group to the associated 3-keto reductase for
reduction as the polyketide chain is being formed. This
hypothesis is supported by studies showing that intact re-
duced polyketide fragments are incorporated into final mac-
rolide molecules (5, 36). The operation of the actIII gene
product as a ,-keto reductase (15, 22) is also supported by
the theory that polyketide biosynthesis is closely analogous
HYBRID ANTHRAQUINONE BIOSYNTHESIS 4825
to, and perhaps derived from, fatty acid biosynthesis (3, 11,
31).
The data presented in this study are consistent with the
hypothesis that the actIll gene product operates as a keto
reductase (15). The fact that this reduction occurs at C-9
during the formation of actinorhodin, aloesaponarin II, and
aklavinone may be a coincidence. In both cases studied
here, actinorhodin (or aloesaponarin II) and anthracycline
formation, the reduction occurs at a carbon which is part of
the first ring formed during the biosynthesis of these various
polyketide compounds. The reduction may be required for
the proper or efficient folding of the polyketide chain to form
the benzo- or naphthacene quinones. However, compounds
such as tetracenomycin (37) and 2-hydroxyaklavinone (26)
are produced by strains apparently lacking this keto reduc-
tase activity (22; data presented herein), indicating that this
reduction step is not a strict requirement for anthracycline
formation. Interestingly, S. galilaeus 31671 produces much
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smaller quantities of anthracyclines than does its parent
strain, S. galilaeus 31133 (Bartel, unpublished data), possi-
bly because of the absence of the polyketide reductase
function.
Desoxyerythrolaccin and 1-O-methyldesoxyerythrolaccin as
hybrid molecules. In the presence of only the actl and actVII
loci, certain gene functions present in S. galilaeus strains
31133 and 31671, and a function absent from S. galilaeus
31671, determine which products are synthesized. Desoxy-
erythrolaccin and 1-0-methyldesoxyerythrolaccin, products
of such a cloning venture, were not produced by any S.
coelicolor strain tested. Our data also indicate that the actIll
gene product, and its equivalent in S. galilaeus 31133, can
react with differentpolyketides such as the precursors for
actinorhodin or aloesaponarin II and for aklavinone. Meder-
rhodins A and B were produced as a result of cloning DNA
encoding part of the actinorhodin pathway, including the
actV locus, in the medermycin producer Streptomyces sp.
strain AM-7161 (17). We have used a completely different
segment of the actinorhodin pathway DNA (24) to produce
desoxyerythrolaccin and 1-0-methyldesoxyerythrolaccin.
Unless these two examples are highly coincidental, these
successes suggest that the formation of hybrid natural prod-
ucts via interspecies cloning of streptomycete antibiotic
biosynthesis DNA may be a generally applicable process for
the formation of new chemical structures.
ACKNOWLEDGMENTS
We sincerely thank David A. Hopwood for the gift of bacterial
strains and plasmids and for sharing unpublished information with
us. We also thank Donald Ordaz for assistance with the HPLC
analyses, Akira Yagi for an authentic sample of aloesaponarin II,
and John Fry for his assistance with purification of compounds. We
thank the Frederick Cancer Research Center, Rh6ne-Poulenc, and
Adria for reference compounds.
This material is based on work supported by the National Science
Foundation under grant DMB-8607619.
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