Biochemical and genetic insights into asukamycin biosynthesis

Article abstract of DOI:10.1074/jbc.M110.128850

Asukamycin, a member of the manumycin family metabolites, is an antimicrobial and potential antitumor agent isolated from Streptomyces nodosus subsp. asukaensis. The entire asukamycin biosynthetic gene cluster was cloned, assembled, and expressed heterolo

Full text of DOI:10.1074/jbc.M110.128850

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 32, pp. 24915–24924, August 6, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Biochemical and Genetic Insights into Asukamycin
□
S
Biosynthesis*
Received for publication, March 31, 2010, and in revised form, May 25, 2010 Published, JBC Papers in Press, June 3, 2010, DOI 10.1074/jbc.M110.128850
‡
§
§
§
‡
¶
ˇ
Zhe Rui , Katerˇina Petrˇícˇkova´ , Frantisˇek Skanta , Stanislav Pospísˇil , Yanling Yang , Chung-Yung Chen ,
Shih-Feng Tsaiʈ, Heinz G. Floss**, Miroslav Petrˇícˇek§1, and Tin-Wein Yu‡2
From the ‡Department of Biological Science, Louisiana State University, Baton Rouge, Louisiana 70803, the §Institute of
Microbiology AS CR, Vídenˇska´ 1083, 142 20 Prague 4, Czech Republic, the ¶Department of Bioscience Technology, Chung Yuan
Christian University, Chung Li 32023, Taiwan, the ʈInstitute of Genetics, National Yang-Ming University, Taipei 112, Taiwan, and the
**Department of Chemistry, University of Washington, Seattle, Washington 98195
2-amino-4-hydroxy-5,6-epoxycyclohex-2-enone (mC7N)3
core and two trans-triene polyketide chains, in which the upper
one starts with a cyclohexane ring, and the lower one termi-
nates in the five-membered ring of a 2-amino-3-hydroxycyclo-
pent-2-enone (C5N) moiety (Fig. 1) (5). Asukamycin, together
with a series of congeners A2–A7 carrying branched alkyl
groups in place of the cyclohexane moiety, were isolated from
Streptomyces nodosus subsp. asukaensis (6–8). Like in a major-
ity of the manumycin-type metabolites, the C5,C6 epoxy group
can be reduced to a 5-hydroxyethylene structure enzymatically
or nonenzymatically to form the type II asukamycins, B1–B5
(Fig. 1) (8). The epoxide group of the manumycins is considered
crucial for many biological functions, as the type II products
show weaker activities (1).
Asukamycin, a member of the manumycin family metabolites,
is an antimicrobial and potential antitumor agent isolated from
Streptomyces nodosus subsp. asukaensis. The entire asukamycin
biosynthetic gene cluster was cloned, assembled, and expressed
heterologously in Streptomyces lividans. Bioinformatic analysis
and mutagenesis studies elucidated the biosynthetic pathway at
the genetic and biochemical level. Four gene sets, asuA–D, gov-
ern the formation and assembly of the asukamycin building
blocks: a 3-amino-4-hydroxybenzoic acid core component, a
cyclohexane ring, two triene polyketide chains, and a 2-amino-
3-hydroxycyclopent-2-enone moiety to form the intermediate
protoasukamycin. AsuE1 and AsuE2 catalyze the conversion of
protoasukamycin to 4-hydroxyprotoasukamycin, which is
epoxidized at C5–C6 by AsuE3 to the final product, asukamycin.
Branched acyl CoA starter units, derived from Val, Leu, and Ile,
can be incorporated by the actions of the polyketide synthase III
(KSIII) AsuC3/C4 as well as the cellular fatty acid synthase FabH
to produce the asukamycin congeners A2–A7. In addition, the
type II thioesterase AsuC15 limits the cellular level of ␻-cyclo-
hexyl fatty acids and likely maintains homeostasis of the cellular
membrane.
Based on precursor feeding experiments, the asukamycin
mC7N core unit and the cyclohexane moiety originate from
3-amino-4-hydroxybenzoic acid (3,4-AHBA) and shikimic
acid, respectively (9–11). The identical lower and upper triene
polyketide chains are built up from 3,4-AHBA and cyclohexyl-
carbonyl CoA (CHC-CoA), respectively, by three steps of clas-
sical polyketide condensations. A gene set involved in the CHC-
CoA biosynthesis of ansatrienin has been well characterized in
Streptomyces collinus (12), but the genes involved in 3,4-AHBA
and polyketide chain assembly of asukamycin have not been
identified. The C5N ring moiety, found in several natural
products including reductiomycin, moenomycin A, and ECO-
02301, was suggested to be derived from a 5-aminolevulinate
(5-ALA) intermediate (13–16). The only reported gene product
of asuD2 (hemA-asuA), a member of the 2-oxoamine synthases,
was found to be involved in asukamycin biosynthesis and to
catalyze the condensation of succinyl-CoA and glycine (15).
Protoasukamycin C1, the polyketide assembly product, is fur-
ther oxygenated to give the final product asukamycin (1, 10, 17).
The upper polyketide chain assembly of the congeners A2–A7
recruits the branched acyl CoA starters, which are predomi-
nantly used for fatty acid biosynthesis in S. nodosus subsp. asu-
kaensis (7). Alternatively, the CHC-CoA starter of asukamycin
The metabolites of the manumycin family are known to have
a broad range of antibiotic functions including antibacterial,
anticoccidial, and antifungal activities (1). Manumycin com-
pounds also display a strong activity against farnesyltransferase,
I␬B kinase ␤, interleukin-1␤-converting enzymes, and acetyl-
cholinesterase and are considered as drug candidates to treat
cancers, inflammation, and Alzheimer disease (1–4). Asuka-
mycin A1, a manumycin-type metabolite, contains a unique
* Thisworkwassupported, inwholeorinpart, byNationalInstitutesofHealth
Grant CA76461 (to T.-W. Y.). This work was also supported by Czech Minis-
try of Education Grant 2B06154, Institutional Scientific Program
AV0Z50200510 (to M. P.), a Louisiana State University Faculty Startup Fel-
lowship (to T.-W. Y.), and a Louisiana State Economic Development Assis-
tantship (to T.-W. Y. and Z. R.).
□
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental text, Tables S1–S3, and Figs. S1–S6.
3 The abbreviations used are: mC7N, 2-amino-4-hydroxy-5,6-epoxycyclohex-
2-enone; 3,4-AHBA, 3-amino-4-hydroxybenzoic acid; C5N, 2-amino-3-hy-
droxycyclopent-2-enone; 5-ALA, 5-aminolevulinate; ACP, acyl carrier pro-
tein; FAS, fatty acid synthase; ALAS, 5-aminolevulinate synthase; CHC,
cyclohexylcarbonyl; HPLC, high pressure liquid chromatography; LC-MS,
liquid chromatography-mass spectrometry; KS, polyketide synthase; PDB,
Protein Data Bank.
1 To whom correspondence may be addressed: Inst. of Microbiology AS CR,
Vídenˇska´ 1083, 142 20 Prague 4, Czech Republic. Fax: 420-24106-2347;
E-mail: petras@biomed.cas.cz.
2 To whom correspondence may be addressed: 678 Life Science Bldg., Louisi-
ana State University, Baton Rouge, LA 70803. Fax: 225-578-2597; E-mail:
tin.laorchid@gmail.com.
AUGUST 6, 2010•VOLUME 285•NUMBER 32
JOURNAL OF BIOLOGICAL CHEMISTRY 24915

Asukamycin Biosynthetic Pathway
linearized 2B9 and 10D6 were trans-
formed separately by the same pro-
cedure. The recombinant clone was
confirmed by matching the antici-
pated EcoRI digestion pattern and
named pART1361. Intergeneric
conjugation of the recombinant
cosmids from E. coli to S. lividans
K4-114 followed the established
protocol (21).
Construction
of
pART1391,
pART1361E3, and pALS4-S83T—
To construct pART1391, a 1.5-
kb DNA fragment from pIJ778
was PCR-amplified using primers
AsuRED-4 and -6 (supplemental
Table S3) to carry a spectinomycin
resistance gene cassette plus two
50-bp nucleotides, designed to be
identical to the boundaries of the
targeted region on pART1361
(supplemental Fig. S5) (20). To
FIGURE 1. Structures of asukamycin and related metabolites. Compounds are grouped according to the
main A, B, C, and D core units.
obtain the recombinant strain, the
gel-purified PCR fragment was
polyketide chain assembly can also be used to form ␻-cyclo-
hexyl fatty acids as a part of cellular membrane components. As
the membrane fluidity and permeability are strongly deter-
mined by the fatty acid composition, the biosynthetic regula-
tion of each fatty acid component is crucial for membrane
homeostasis in bacteria (18, 19). The content of ␻-cyclohexyl
fatty acids, a byproduct generated during the peak period of
asukamycin biosynthesis, is maintained to be as low as 3% (7).
This implies that the organism has a control mechanism to
balance the metabolic flux between the production of mem-
brane fatty acids and asukamycin. Herein, we report the clon-
ing, mutagenic analysis, and characterization of the biosyn-
thetic genes, which provides insight into the biosynthetic
assembly of the asukamycins in S. nodosus subsp. asukaensis.
Possible cross-talk between the biosynthesis of the fatty acids
and the asukamycins is also discussed.
introduced into E. coli BW25113/pART1361/pIJ790 by electro-
poration, and the transformants were screened for spectinomy-
cin resistance. The deletion in the resulting clone was con-
firmed by matching up to the expected EcoRI digestion pattern
and verified by PCR. The pART1361E3 construction followed
the same strategy as described above but using primers
AsuRED-E3F and -E3R (supplemental Table S3).
To insert the point mutation that modifies the TCC (Ser)
codon into ACC (Thr) in the asuD2 gene, the internal fragment
containing the BamHI-KpnI sites was PCR-amplified using
primers S83TF and S83TR (supplemental Table S3). The
BamHI-KpnI-cleaved fragment was then used to replace the
corresponding region in the pALS3 plasmid. The further steps
were identical with the pALS4 construction (15).
HPLC and LC-MS Analysis of the Asukamycin Metabolites—
S. nodosus subsp. asukaensis, S. lividans K4-114, and the
derived strains were cultivated as described (10, 21). Feeding
experiments with 3,4-AHBA (100 ␮g/ml) and 4-hydroxypro-
toasukamycin (20 ␮g/ml) were conducted by adding these
compounds to 1-day-old cultures and harvesting 1 day later.
Fermented cultures were mixed thoroughly with an equal
volume of ethyl acetate. The mixture was separated by centri-
fugation, and the ethyl acetate layer was collected and evapo-
rated to near dryness. The crude extract was dissolved in an
equal volume of methanol and analyzed using a P680 HPLC
system (Dionex, Sunnyvale, CA) and an XTerra RP18 column
(4.6 ϫ 250 mm; Waters) at a flow rate of 0.5 ml/min at 25 °C.
Twenty ␮l of crude extract were injected, equilibrated with 65%
solvent B (acetonitrile with 0.1% formic acid) in solvent A
(water with 0.1% formic acid) for 5 min, developed with a gra-
dient of 65–90% solvent B in solvent A for 20 min, and washed
with 90% solvent B in solvent A for 10 min. To analyze the
EXPERIMENTAL PROCEDURES
Bacterial Strains and Chemicals—S. nodosus subsp. asukaen-
sis ATCC 29757 was obtained from the American Type Culture
Collection. Streptomyces lividans K4-114 was kindly provided
by Dr. C. Khosla. Escherichia coli strains were obtained from
the John Innes Center (Norwich, UK). The media and chemi-
cals were purchased from Difco, Sigma-Aldrich, and EMD
Chemicals.
Genomic Library and Mutant Construction—See supplemen-
tal text.
In Vivo Recombination and Heterologous Expression of the
Cloned asu Gene Cluster—The cosmids 2B9 and 10D6 were
linearized by SpeI and EcoRI, respectively, equally mixed, and
transformed into E. coli BW25113/pIJ790 competent cells by
electroporation (20). The cells were then added to 1 ml of SOC
medium (20), recovered at 37 °C for 3 h, and plated out onto LB
agar with 100 ␮g/ml apramycin. For the negative control, the asuD1–D3 mutants, a gradient of 50–70% solvent B in solvent
24916 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 32•AUGUST 6, 2010

Asukamycin Biosynthetic Pathway
FIGURE 2. Open reading frames identified in the asu cluster. The putative genes were divided into eight groups based on the functional characterization.
Group A, 3,4-AHBA biosynthesis and adenylation; Group B, CHC-CoA biosynthesis; Group C, polyketide chain assembly; Group D, C5N moiety biosynthesis;
Group E, oxygenation; Group R, transcriptional regulation. AsuM1, efflux protein. The relative genomic regions and overlapping inserts of three cosmid clones,
2B9, 10D6, and pART1391, are indicated with bold lines.
A was applied instead. The extraction and HPLC analysis of (dd, 1H, J ϭ 14.9, 11.5 Hz, H-8), 6.38 (d, 1H, J ϭ 15.0 Hz, H-12),
3,4-AHBA followed the described protocol (22).
6.12 (d, 1H, J ϭ 15.2 Hz, H-7), 3.85 (m, 1H, H-5), 2.71 (dd, 1H,
High resolution MS and LC-MS analyses were carried out J ϭ 16.8, 3.2 Hz, H-6ax), 2.59 (dd, 1H, J ϭ 16.5, 6.07 Hz, H-6eq),
on a 6210-TOF LC-MS system (Agilent Technologies Inc., 1.90 (s, 3H, H-2Ј). 13C-NMR (700 MHz, Me2SO-d6) ␦C 192.6
Santa Clara, CA) in which the LC was performed using a (C-1), 169.5 (C-1Ј), 165.0 (C-13), 140.3 (C-11), 138.8 (C-9),
XDB C18 (4.6 ϫ 50 mm, 1.8-micron particle size) high 138.4 (C-7), 131.5 (C-8), 130.8 (C-2), 129.6 (C-3), 129.5 (C-10),
throughput column (Agilent Technologies, Inc.) with the 124.5 (C-12), 73.0 (C-4), 71.3 (C-5), 40.1 (C-6), 23.9 (C-2Ј).
same elution program as the HPLC analysis mentioned above.
RESULTS
The exact masses of the identified compounds are listed in
supplemental Table S1.
Cloning of the asu Cluster—To clone the asukamycin biosyn-
thetic gene cluster, a cosmid genomic library of S. nodosus
Fatty Acid Analysis—See supplemental text.
Purification and NMR Analysis of D1—A reliable yield of the subsp. asukaensis was constructed and screened with two 32P-
novel intermediate 4-hydroxyprotoasukamycin D1 was ob- labeled DNA probes. ChcA, encoding the 1-cyclohexenylcar-
tained in S. lividans K4-114 carrying pART1361E3, which was bonyl CoA reductase of ansatrienin biosynthesis in S. collinus
generated from pART1361 with the ␭-Red PCR strategy by (12), identified two cosmids, 2B9 and 10D6, which revealed a
replacing asuE3 with a spectinomycin resistance gene. S. livi- 1.8-kb overlapping region of the cloned DNA inserts. AsuD2,
dans K4-114/pART1361E3 was inoculated into 500 ml of MS encoding a 2-oxoamine synthase, hybridized only with cosmid
medium in 2-liter flasks with a coil and fermented on a rotary 10D6 (15). Cosmids 2B9 and 10D6 were mapped and sequenced
shaker at 250 rpm and 28 °C for 4–5 days. One liter of harvested to disclose a total combined 63,922 bp of DNA inserts
culture was extracted three times with an equal volume of ethyl (GenBankTM accession number GQ926890). DNA sequence
acetate, and the combined ethyl acetate extract was dried, con- analysis revealed 36 potential open reading frames, tentatively
centrated, and purified on a silica gel column (100–200 mesh), assigned as the asu genes, encoding gene products potentially
eluting with dichloromethane/methanol (100:3). The com- involved in asukamycin biosynthesis and regulatory activities
pound D1 fraction was collected and further purified by (Fig. 2 and Table 1).
semi-preparative C18 reverse phase HPLC (Dionex), eluting
Heterologous Expression of the asu Cluster—To confirm that
with 75–85% solvent C (methanol with 0.1% formic acid) in the cloned region contains all of the genes in control of asuka-
solvent A, to give 20 mg of pure compound D1. Its 1H-NMR, mycin biosynthesis, regulation, and self-immunity (Table 1), a
13C-NMR, HSQC, and HMBC spectra were recorded in series of heterologous expression experiments was conducted
Me2SO-d6 on a Varian System 700 spectrometer (sup- in the asukamycin nonproducing S. lividans K4-114. Because
plemental Table S2).
neither 2B9 nor 10D6 carries a full set of the predicted biosyn-
Purification and NMR Analysis of B8—The asuC2 mutant thetic genes, we modified the conventional ␭-Red recombina-
was inoculated into 50 ml of YMG medium in 250-ml flasks tion strategy (20, 23–25) and reassembled the cosmids 2B9
with coils and fermented at 250 rpm and 28 °C for 3 days. One and 10D6 into a single clone (Fig. 3). The resulting cosmid
liter of harvested culture was extracted three times with an pART1361 was introduced into S. lividans by conjugation from
equal volume of ethyl acetate. The combined ethyl acetate E. coli. Brightly yellowish metabolites were observed in the cul-
extract was dried, concentrated, and purified on a silica gel col- ture and were confirmed to be asukamycins A1–A7 by high
umn (100–200 mesh), eluting with dichloromethane/methanol resolution LC-MS. Cosmid pART1391 was further constructed
(90:10). The compound B8 fraction was further purified by from pART1361 to eliminate a 25.4-kb left-hand side insert
semi-preparative C18 reverse phase HPLC (Dionex), and elut- region, in which the identified open reading frames were mainly
ing with 35–50% solvent C in solvent A, and 2 mg of pure com- related to primary metabolic functions in Streptomycetes.
1
pound B8 was obtained. H NMR (700 MHz, Me2SO-d6) ␦H LC-MS analysis confirmed that S. lividans K4-114/pART1391
8.91 (s, 1H, NH-14), 8.88 (s, 1H, NH-0Ј), 7.27 (d, 1H, J ϭ 7.8 Hz, is fully capable of producing asukamycins A1–A7 (Fig. 4d).
H-3), 7.12 (dd, 1H, J ϭ 15.0, 11.5 Hz, H-11), 6.67 (dd, 1H, J ϭ These expression results indicate that the cloned 36 asu genes
15.1, 10.9 Hz, H-9), 6.47 (dd, 1H, J ϭ 15.2, 11.0 Hz, H-10), 6.43 in pART1391 are sufficient for asukamycin production.
AUGUST 6, 2010•VOLUME 285•NUMBER 32
JOURNAL OF BIOLOGICAL CHEMISTRY 24917

Asukamycin Biosynthetic Pathway
TABLE 1
Deduced functions of open reading frames in the asukamycin biosynthetic gene cluster
Protein
Amino acid
Homolog (accession no.)
Identified or proposed function
AsuA1
AsuA2
AsuA3
AsuB1
AsuB2
AsuB3
AsuB4
AsuC1
AsuC2
AsuC3
AsuC4
AsuC5
AsuC6
AsuC7
AsuC8
AsuC9
AsuC10
AsuC11
AsuC12
AsuC13
AsuC14
AsuC15
AsuR1
AsuR2
AsuR3
AsuR4
AsuR5
AsuH1
AsuR6
AsuE1
AsuE2
AsuE3
AsuD1
AsuD2
AsuD3
AsuM1
372
471
278
983
394
277
667
219
269
338
361
84
GriH (YP_001825760.1)
ACMS I (AAD30111.1)
GriI (BAF36651.1)
3,4-AHBA synthase
3,4-AHBA carboxyl group adenylation
Condensation of L-aspartate-4-semialdehyde and dihydroxyacetone phosphate
5-Enolpyruvylshikimate-3-phosphate synthase/CHC-CoA ligase
Acyl-CoA dehydrogenase
1-Cyclohexenylcarbonyl CoA reductase
2,4-DienoylCoA reductase
Phosphopantetheinyl transferase
Arylamine N-acyltransferase
Asukamycin ketosynthase III
Asukamycin ketosynthase III
Asukamycin KSIII associated ACP
Thioesterase
PlmJK (AAQ84158.1)
PlmL (AAQ84159.1)
ChcA (AAC44655.1)
PlmM (AAQ84161.1)
Sfp (PDB 1QR0)
GdmF (AAO06919.1)
ZhuH (PDB 1MZJ)
ZhuH (PDB 1MZJ)
ZhuG (AAG30194.1)
PaaI (P76084)
151
234
152
150
94
Sco1815 (PDB 2NM0)
HadC (NP_215151.1)
HadB (YP_001281933.1)
Putative open reading frame
ZhuN (AAG30201.1)
AcmACP (AAD30112.1)
FabF (PDB 2GQD)
Ketoreductase
Acyl dehydratase
Acyl dehydratase
Unknown
91
KSI/II associated ACP
87
3,4-AHBA carrier protein
Asukamycin ketosynthase I/II
Asukamycin ketosynthase I/II
Type II thioesterase
397
240
260
242
191
307
141
254
384
280
407
185
371
531
409
468
512
FabF (PDB 2GQD)
RifR (PDB 3FLB)
GerE (PDB 1FSE)
Transcriptional regulation
Transcriptional regulation
Transcriptional regulation
Transcriptional regulation
Transcriptional regulation
Unknown
TetR family protein
Membrane protein
DNA-binding protein
FarR3 (BAG74713.1)
Putative open reading frame
GerE (PDB 1FSE)
Transcriptional regulation
Protoasukamycin 4-hydroxylase
Flavin reductase
PHBH (PDB 1YKJ)
PheA2 (PDB 1RZ0)
LimB (Q9EUT9.1)
4-Hydroxyprotoasukamycin epoxidase
Amide synthase
NovL (AAF67505.1)
MoeC4 (ABJ90148.1)
MoeA4 (ABJ90146.1)
EmrB (YP_001507963.1)
2-Oxoamine synthase
5-Aminolevulinate CoA ligase
Asukamycin exporter
dihydroxyacetone phosphate into the grixazone 3,4-AHBA
moiety in Streptomyces griseus (22). Mutants disrupted in
asuA1 and asuA3, respectively, were constructed to confirm
their roles in 3,4-AHBA formation. Neither 3,4-AHBA nor
asukamycin were detected in the cultures of these mutants, but
the addition of 3,4-AHBA restored asukamycin production in
both mutants (supplemental Fig. S1a). However, the new minor
accumulated products E1 and E3/E4, which possess a C5N
moiety amide-linked with the triene upper chains correspond-
ing to A1 and A3/A4, respectively, were detected by LC-MS
analysis (supplemental Fig. S6).
Presumably, 3,4-AHBA is activated by adenylation and
then tethered to a specific aroyl carrier protein for the down-
stream chain extensions as in the aromatic polyketide and
polypeptide biosynthetic systems employing an aryl carbox-
ylate starter (26–28). AsuA2 is homologous to the actinomy-
cin synthetase I, which initiates actinomycin formation in
Streptomyces chrysomallus by activating 4-methyl-3-hy-
droxyanthranilate (4-MHA) in the presence of ATP (26).
The asuA2 mutant failed to produce any asukamycins but
accumulated 3,4-AHBA, as confirmed by HPLC comparison
with standard 3,4-AHBA as well as by restoring asukamycin
production upon co-culturing with the 3,4-AHBA assembly-
deficient asuA1 mutant (supplemental Fig. S1b). AsuC11
and AsuC12 both belong to the acyl carrier protein (ACP)
family. AsuC12 is distinctly related to AcmACP, a specific
aroyl carrier protein of the actinomycin pathway, which is
FIGURE 3. Assembly of two cosmids with overlapping inserts. a, cosmids
2B9 and 10D6 were linearized by SpeI and XbaI, respectively. Homologous
recombination occurred at two overlapping insert and vector regions, indi-
cated by heavy lines. b, the restriction map of 2B9, 10D6, and pART1361. The
inserts are shown with solid lines. The EcoRI fragment sizes are indicated (kb).
c, comparison of the EcoRI digestion patterns by DNA gel electrophoresis.
3,4-AHBA and Lower Polyketide Chain Formation—Two
genes in the cloned asu cluster, asuA1 and asuA3, are closely
related to the characterized griH and griI, respectively, which charged by the priming molecule 4-MHA-AMP and for-
are involved in the assembly of L-aspartate-4-semialdehyde and wards it to the peptide synthase AcmII for chain initiation
24918 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 32•AUGUST 6, 2010

Asukamycin Biosynthetic Pathway
terium tuberculosis H37Rv (35). Coincidently, AsuC8 is highly
related to both HadA and HadC. As asuC7 and asuC8,C9 are
the only genes in the cluster that could be involved in ␤-keto
reduction and dehydration, we suggest that they operate in the
assembly of both the lower and upper trans-triene chains.
Upper Polyketide Chain Formation—Unlike other manumy-
cins, formation of the asukamycin upper polyketide chain
starts with cyclohexylcarbonyl-CoA (11). Four clustered genes,
asuB1–B4, are homologous to two gene sets, ansJKLM/chcA
from the ansatrienin producer S. collinus and plmJKLM/chcA
from the phoslactomycin producer Streptomyces sp. HK-803,
which are involved in CHC-CoA biosynthesis (12, 36). The
asuB1 mutant failed to produce asukamycin, but levels of the
previously identified asukamycin congeners A2–A7 were
increased 3-fold compared with the wild type strain (Fig. 4a).
LC-MS analysis further identified three new compounds, A8,
B8, and C8 (Fig. 1). The NMR analysis of B8 revealed a B1-like
structure except that the upper polyketide chain was replaced
by an acetyl group. Based on the molecular weight, compounds
C8 and A8 are likely the proto-form and type I form of B8,
respectively. These products probably result from the absence
of CHC-CoA plus an oversupply of the 3,4-AHBA-lower chain-
ACP intermediate.
FIGURE 4. HPLC analysis of asukamycin metabolites. a, 20 ␮l of crude cul-
ture extract of the asuB1 mutant. b, the asuC3C4 double mutant. c, the asuC15
mutant. d, S. lividans K4-114/pART1391. e, S. nodosus subsp. asukaensis wild
type strain. The related peaks of asukamycins A1–A5 and B1–B4 are indi-
cated. The y axis indicates the absorbance abundance. Panel b was calculated
based on a 10ϫ injection amount.
To initiate fatty acid or polyketide biosynthesis, acyl-CoA
starters are recruited and condensed with a (methyl)-malonyl-
ACP by the action of fatty acid synthase FabH or KSIII. This is
distinct from the action of KSI/II, joining two ACP-bound acyl
substrates in the subsequent chain extensions in many micro-
(26). The mutant in which both asuC11 and asuC12 were organisms (29, 30). The adjacent asuC3 and asuC4 are homol-
truncated showed a similar phenotype as the asuA2 mutant, ogous to numerous genes encoding bacterial FabHs and KSIIIs
i.e. 3,4-AHBA accumulation and no asukamycin production including the zhuH of Streptomyces. sp. R1128 (37). Both
(supplemental Fig. S1b).
AsuC3 and AsuC4 feature a putative CoA-binding site and a
AsuC13 has strong similarity to a group of type II iterative conserved Cys-His-Asn catalytic triad. As AsuC3 and AsuC4
fatty acid synthase (FAS)/polyketide synthase (29, 30). AsuC13 are highly similar to each other, we assume that they might act
was confirmed to be a KSI/II polyketide synthase (31, 32) together as a heterodimer to condense CHC-CoA and malonyl-
involved in the lower polyketide chain assembly, as the asuC13 ACP to give a 3-cyclohexyl-3-oxopropanoyl-ACP intermediate
mutant failed to produce any asukamycins and accumulated for the downstream polyketide chain assembly. Interestingly, a
3,4-AHBA, similar to the asuA2 and asuC11C12 mutants double mutation of asuC3 and asuC4 did not completely elim-
(supplemental Fig. S1b). Notably, trace amounts of E1 and inate asukamycin production. The asuC3C4 mutant retained
E3/E4 were also found in this mutant culture as observed in the 0.9% asukamycin A1 and 30.0% congeners A2–A7 relative to
asuA1 and asuA3 mutants. The asuC14 gene, immediately the wild type strain yield (Fig. 4b), possibly because of the func-
downstream of asuC13, encodes a protein homologous to tional complementation by the cellular FabH of fatty acid bio-
AsuC13, except for a truncated N terminus and lack of the synthesis. AsuC5, presumably organized in the same transcrip-
characteristic Cys-His-His catalytic triad. This unusual tion unit with asuC3,C4, is a zhuG homolog (38). As ZhuG acts
asuC13-asuC14 pair in which an intact polyketide synthase together with ZhuH to initiate the assembly of the antitumor
gene accompanies a partially truncated partner is also found in polyketide R1128 (38), AsuC5 may play a crucial role in the
several uncharacterized gene clusters from the genomic beginning round of asukamycin upper chain assembly. So far,
sequencing projects of Streptomyces hygroscopicus (ZP_ the KSI/II gene(s) required for the second and third round
05513707-8) and Salinispora arenicola (YP_001535964-5 and extensions of the upper chain remain to be identified.
YP_001537504-5). Mutation studies are in progress, but the
Asukamycin C5N Moiety Formation—Three adjacent genes,
AsuC14 function remains unknown for the moment. Two addi- asuD1, asuD2, and asuD3, appear to share one transcription
tional sequential reactions, a ␤-keto reduction and a dehydra- unit. AsuD2, previously identified as HemA-AsuA, belongs to a
tion, are expected for the lower chain assembly. AsuC7 displays group of pyridoxal phosphate-dependent 2-oxoamine syn-
high homology to several polyketide ketoreductases (33, 34). thases, including 5-aminolevulinate synthase (ALAS), FUM8,
Downstream of asuC7, two putative dehydratase genes, asuC8 L-serine palmitoyltransferase, and 8-amino-7-oxononanoate
and asuC9, were found next to each other. AsuC9 is closely synthase, which catalyze the decarboxylative condensation
related to HadB, which forms a heterodimer with either HadA between an amino acid and an acyl-CoA (15, 39–42). Despite
or HadC to function in mycolic acid biosynthesis in Mycobac- high overall sequence similarity, AsuD2 possesses a Ser83
AUGUST 6, 2010•VOLUME 285•NUMBER 32
JOURNAL OF BIOLOGICAL CHEMISTRY 24919

Asukamycin Biosynthetic Pathway
instead of a strictly invariant Thr83 of the ALAS subgroup. asuE2 mutant produced asukamycin A1 in considerably lower
AsuD2 functions differently from the reported ALAS forming yield than the wild type strain but accumulated major amounts
5-ALA in many organisms, as no asukamycins were detected in of the protoasukamycins C1–C5 (supplemental Fig. S1d). This
the asuD2 mutant even in the presence of 5-ALA, but the implies that AsuE2 is necessary for the full catalytic function of
asukamycin production was restored by reintroducing the plas- AsuE1 as a two-component flavoprotein hydroxylase.
mid pALS4 carrying the asuD2 gene (15). The pALS4-S83T
AsuE3 is highly related to LimB, a limonene 1,2-monooxyge-
with a S83T replacement in asuD2 was constructed but failed to nase that generates limonene-1,2-epoxide in Rhodococcus
restore asukamycin production in the asuD2 truncated mutant erythropolis (49). Instead of the recognized asukamycins, a
(supplemental Fig. S2). Notably, pALS4-S83T complemented a group of bright yellowish compounds were accumulated by the
5-ALA-deficient gtr mutant of Streptomyces coelicolor A3(2), asuE3 disrupted mutant (supplemental Fig. S1d). LC-MS anal-
which can only grow with 5-ALA supplementation. AsuD3, an ysis of the major peak suggested that one oxygen atom was
acyl-CoA ligase, is proposed to act in the C5N ring formation, missing compared with asukamycin A1. 1H, 13C, HMBC, and
since disruption of the homologous moeA4 in Streptomyces HSQC NMR analysis of the purified compound showed that a
ghanaensis led to accumulation of a moenomycin A analog double bond at C5–C6 replaces the epoxide group of asukamycin
lacking the C5N moiety (14). AsuD1 is closely related to a group A1 (supplemental Table S2). This new compound was therefore
of amide synthases, including SimL for simocyclinone biosyn- named as 4-hydroxyprotoasukamycin D1. Its congeners
thesis (43, 44). All of the asuD1, D2, and D3 mutants failed to D2–D5 were also detected. D1 was converted to asukamycin
produce asukamycin but accumulated a new set of metabolites, A1 when fed to the asuA3 mutant culture, indicating that D1 is
A1a–A5a, which carry a free carboxyl group in place of the an intermediate that is further epoxidized by AsuE3 in the final
amide-linked C5N moiety of A1–A5, respectively (sup- step of asukamycin biosynthesis (supplemental Fig. S1a). Since
plemental Fig. S1c). The corresponding type II compounds, D1 was absent in the asuE2 mutant, the epoxidation may not
B1a–B5a, were also present in all three mutants. Furthermore, require AsuE2.
the asuD1–D3 operon was expressed in S. lividans, which
Thioesterase Affects the Primed CHC—AsuC15 is closely
resulted in attachment of the C5N moiety to added ferulic acid related to a group of type II thioesterases found in polyketide
to form feruloyl-N-(3-hydroxycyclopent-2-enolone)acrylam- and nonribosomal peptide biosynthetic gene clusters, including
ide, whereas no C5N moiety was detected in the absence of RifR of rifamycin biosynthesis (50). As the yields of the
asuD1, D2, or D3 (supplemental text and Fig. S2). This con- polyketide and polypeptide products were often considerably
firmed that asuD1, D2, and D3 are required and sufficient for impaired in the knock-out mutants, type II thioesterases were
C5N moiety formation.
proposed to restore the acyl or peptidyl carrier protein function
Attachment of the Upper Polyketide Chain—Asukamycin by releasing undesirable intermediates, resulting from a prim-
contains two amide bonds: the linkage between the upper chain ing or processing error (51–54). The asuC15 mutant produced
and the mC7N core and the bridge tethering the C5N moiety to a normal amount of asukamycin A1, yet the yield of the conge-
the lower polyketide chain. AsuC2 belongs to the N-acyltrans- ners A2–A7 was significantly reduced (Fig. 4c). Since CHC-
ferase family (45). An asuC2 disrupted mutant was constructed CoA is not only a building block of asukamycin but can also
to confirm its involvement in the attachment of the upper chain form ␻-cyclohexyl fatty acids as membrane components in S.
to the 3-amino group of the 3,4-AHBA primed polyketide inter- nodosus subsp. asukaensis (7), we analyzed the fatty acid com-
mediate. The production of asukamycins A1–A7 was com- positions of the asuC15 mutant and the wild type strain by
pletely blocked in the asuC2 mutant. Instead, the N-acetylated GC-MS (supplemental Fig. S3). Notably, 16.5% of ␻-cyclo-
compounds A8, B8, and C8 were accumulated as in the asuB1 hexyl-C17 fatty acid was detected in the asuC15 mutant, at least
mutant. In view of the functional deficiency of asuC2, these 5-fold higher than the 3.1% detected in the wild type strain.
shunt products presumably resulted from the action of a cellu- Moreover, 1.7% of ␻-cyclohexyl-C19 fatty acid, undetectable in
lar arylamine N-acetyltransferase, commonly found to detoxify the wild type strain, was also found.
arylamine or arylhydroxylamine metabolites (46).
Other asu Gene Functions—AsuC6, a hot dog fold thioester-
Oxygenation of Protoasukamycin—S. parvulus Tu¨ 64 fer- ase, presumably functions to hydrolyze the thioester bond and
mentation under an 18O2 atmosphere has demonstrated that remove the acyl intermediate from the polyketide synthase (55,
the hydroxyl and the epoxide oxygens at C4 and C5–C6 of 56). It could be responsible for A1a–A5a accumulation in the
manumycin A originate from molecular oxygen, by either a asuD1, D2, and D3 mutants, which are supposed to be tightly
two-step process or a dioxygenase mechanism (10, 17). Three bound to the ACP AsuC11. The asuC6 mutant has not yet been
distantly located genes, asuE1, asuE2, and asuE3, are possibly constructed, and the precise AsuC6 function remains to be
involved in the oxygenation reaction(s). AsuE1 is homologous clarified. AsuC1, a phosphopantetheinyl transferase, presum-
to p-hydroxybenzoate hydroxylase, which converts p-hydroxy- ably primes the carrier proteins AsuC5, C11, and C12, which
benzoate into 3,4-dihydroxybenzoate in Pseudomonas aerugi- are involved in the lower and upper chain biosynthesis. AsuM1,
nosa (47). Instead of producing asukamycin, the asuE1 mutant a homolog of numerous antibiotic efflux exporters, could
accumulated protoasukamycin C1 and its congeners C2–C5 release the synthesized asukamycin products from the cells.
(supplemental Fig. S1d). AsuE2 is a homolog of the flavin reduc- AsuR1–R6, discretely detected at the two border regions of the
tase PheA2, which recycles oxidized FADox and provides cluster, show homology to numerous putative transcriptional
FADred to its hydroxylase partner PheA1 in the phenol regulator genes found in Gram-positive bacteria. AsuR1 and
hydroxylation in Bacillus thermoglucosidasius A7 (48). The AsuR6 are closely related to the LuxR-type transcriptional reg-
24920 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 32•AUGUST 6, 2010

Asukamycin Biosynthetic Pathway
FIGURE 5. Proposed asukamycin biosynthetic pathway. The primary metabolite substrates are shown in squares. The ACP and aroyl carrier protein are in
capsules.
ulators (57). AsuR2 is likely a TetR-like transcriptional regula-
tor (58). AsuR3 is a putative integral membrane sensor protein,
which could work together with the DNA-binding protein
AsuR4 (59). AsuR5 belongs to the Streptomyces antibiotic reg-
ulatory proteins (60). As many antibiotic biosynthesis gene
clusters contain one or multiple regulatory genes, AsuR1–R6
could have regulatory functions to control the asukamycin bio-
synthetic gene activities. AsuH1 is a putative open reading
frame with an unknown function.
Construction of the lower polyketide chain likely requires a
set of type II polyketide synthetic enzymes including the KSI/II
AsuC13,C14, the polyketide ketoreductase AsuC7, and the DH
AsuC8,C9, based on the sequencing and mutagenic analysis.
The results indicate that 3,4-AHBA is activated by AsuA2 to
form an acyl-AMP and subsequently loaded onto the aroyl car-
rier protein AsuC12 to initiate the lower polyketide assembly.
AsuC13 and AsuC14 are most likely responsible for the initia-
tion and two further rounds of polyketide chain extensions, as
the lower chain biosynthesis was abolished in the asuC13
mutant. The presence of asuC14 may not be coincidental.
AsuC13 and AsuC14 could function together to constrain the
growing triene structures and control the polyketide chain
length. Further studies on other manumycin biosynthetic
genes should provide more clues on the function of asuC14.
Since asuC11 is potentially organized in the same transcrip-
tion unit with asuC12–C14, the AsuC11 ACP could be
involved in the second and the third rounds of lower chain
polyketide extensions.
DISCUSSION
The structure of asukamycin features two triene polyketide
chains connected to a 3,4-AHBA-derived mC7N core. The
upper chain starts with a cyclohexane head group, and the
lower one ends with a C5N structural moiety. In this study, four
major groups of asu genes were identified to be involved in the
structural assembly: AsuA1 and A3 for 3,4-AHBA biosynthesis,
AsuB1–B4 for the conversion of shikimate into CHC-CoA,
AsuD1–D34 for C5N unit formation, and the AsuC group for
the lower and upper polyketide chain assembly (Fig. 5).
For the upper chain, AsuC3,C4 likely initiate the condensa-
tion between CHC-CoA and malonyl-ACP, resembling the
bacterial FabH and other KSIIIs, which are directly primed with
an acyl-CoA. In the asuC3,C4 mutant most asukamycin A1
4 During the peer review process of this manuscript, the Walsh group
reported a study that supports the proposed AsuD1–D3 function in the
C5N moiety formation (62).
AUGUST 6, 2010•VOLUME 285•NUMBER 32
JOURNAL OF BIOLOGICAL CHEMISTRY 24921

Asukamycin Biosynthetic Pathway
production was abolished, and levels of the congeners A2–A7 ␻-cyclohexyl fatty acids was increased 5-fold in the asuC15
were reduced compared with the wild type. This reflects a pos- mutant. The cellular membrane fluidity is probably affected by
sible involvement of the cellular fatty acid synthase FabH, variations in the content of ␻-cyclohexyl fatty acids, changing
which displays a substrate preference distinct from AsuC3,C4, the phase transition temperature, as observed for several
priming preferentially with branched chain acyl-CoAs rather thermo-acidophilic bacteria, which may contain up to 90% of
than CHC-CoA, to give a higher yield of A2–A7 relative to A1 ␻-cyclohexyl fatty acids (18, 19, 61). Thus AsuC15 could sup-
(7). The involvement of AsuC3,C4 in the second and third chain press excess ␻-cyclohexyl fatty acid formation and help to
elongations is less likely, as FabH catalyzes only the first con- maintain membrane homeostasis and structural integrity, par-
densation step and passes the resulting 3-ketoacyl-ACP directly ticularly when the cellular level of CHC-CoA is rising. Evi-
to another enzyme complex for further reductions and chain dently, AsuC15 can also discharge the CHC-acyl intermediate
elongations. Given that E1 and E3/E4 were found in the asuC13 from AsuC5-ACP to enhance the production of congeners
mutant, the only recognized KSI/II gene pair, asuC13,C14, may A2–A4, as the asuC15 mutant predominantly accumulates A1
not play a role in the upper chain assembly, although both the rather than an equal amount of A1 and A2–A4 observed in the
upper and lower polyketide chains of asukamycin share an wild type.
identical triene structure. It seems that an unidentified
AsuD1, D2, and D3 are necessary and sufficient for the
polyketide KSI/II is required to act together with the polyketide assembly and attachment of the C5N moiety to form protoa-
ketoreductase AsuC7 and DH AsuC8,C9 to carry out the upper sukamycin. AsuD2 bears a Ser83 in the position of the conserved
triene polyketide extension. We also cannot rule out the Thr83 of ALAS. The distinct role of AsuD2-Ser83 in C5N moiety
involvement of a cellular FabB/F-like fatty acid synthase, as the formation was confirmed by expression of AsuD2-S83T, which
successful heterologous expression in S. lividans implies that a failed to complement the asuD2 mutant but sustained the abil-
common KSI/II or FASI/II can fulfill the functional role of this ity to form 5-ALA in the gtr mutant of S. coelicolor. The fact that
unidentified ␤-keto condensation enzyme.
5-ALA is a precursor of the C5N unit but does not complement
The formation of A1–A4 only differs at the priming step of the asuD2 mutation shows that asuD2 has a second function in
the upper polyketide chain. A2–A4 employ the branched chain C5N formation (13, 15, 16). The nascent pyridoxal phosphate-
amino acid-derived starters 2-methylpropionyl-CoA and 3- bound 5-ALA or possibly a predecarboxylation intermediate
and 2-methylbutanoyl-CoA, respectively, which are predomi- 2-amino-3-oxoadipate could be retained on AsuD2 and car-
nantly used for fatty acid synthesis in Streptomyces (7). In the boxyl-activated by the action of the CoA ligase AsuD3, followed
asuB1 mutant, the absence of CHC-CoA abolished the entire by cyclization catalyzed by AsuD2 (Fig. 5). The dual role of
A1 production and redirected the biosynthetic flux to A2–A4. AsuD2 is also supported by the results of the heterologous
Since A2–A4 formation was 10-fold higher in this mutant than expression of asuD1–D3 to produce the C5N unit in the
in the asuC3,C4 mutant, the majority of A2–A4 upper absence of any other candidate cyclase gene in S. lividans. The
polyketide initiation is likely performed by the KSIII AsuC3,C4, amide synthase AsuD1 is responsible for the attachment of
and only a minor fraction of A2–A4 production in the asuB1 the C5N unit to the C terminus of the lower chain. As all the
mutant as well as the wild type strain is contributed by the FabH assumed intermediates in this process are relatively unstable
activity. The relatively small amount of A5–A7 production (10), AsuD1, D2, and D3 might work together to secure the
could result from an action of the primary FAS complex to give formation of protoasukamycin.
4-methylpentanoyl- and 5- and 4-methylhexanoyl-ACP inter-
Both one-step dioxygenase and two-step monooxygenase
mediates. These ACP-tethered thioesters could be directly mechanisms have been proposed for epoxyquinol formation (1,
recruited by an unidentified KSI/II or FASI/II to perform three 10, 11). The present study resolves this issue as two monooxy-
additional elongations and introduce the characteristic triene genations, first 4-hydroxylation of protoasukamycin C1 by
in analogy with the A1–A4 upper chain biosynthesis. These AsuE1 and AsuE2, followed by epoxidation of the resulting qui-
observations are in agreement with the FabH involvement and nol D1 by AsuE3 to form asukamycin A1 (Fig. 5). As AsuE3
suggest physiological cross-talk between polyketide and fatty specifically acts on the quinol moiety but not a phenolic struc-
acid biosynthesis.
ture, the protoasukamycin 4-hydroxylation is a prerequisite for
CHC-CoA is not only the substrate for the polyketide syn- epoxidation. Notably, replacement of the upper chain by an
thase AsuC3,C4 in A1 upper chain assembly but is also acetyl group or the absence of the C5N moiety has no impact on
recruited by the FAS complex and assembled into ␻-cyclohexyl epoxyquinol formation as evidenced by A8 and A1a,
fatty acids, which account for 3.1% of total cellular fatty acids in respectively.
S. nodosus subsp. asukaensis (7). The CHC-CoA utilization to
It seems that asukamycin biosynthesis has evolved through
form asukamycin A1 and ␻-cyclohexyl fatty acids does not multiple horizontal gene transfer events and possibly an exten-
occur in a fixed ratio, as A1 was increased to more than 80% of sive genomic rearrangement in S. nodosus subsp. asukaensis.
total asukamycins, whereas ␻-cyclohexyl fatty acids were kept The biosynthetic genes involved in C5N moiety and CHC-CoA
under 25% of total fatty acids upon feeding excess cyclohexan- assembly are closely positioned and appear to be co-tran-
ecarboxylic acid (7). Besides the distinct substrate specificities scribed, but many other functionally related asu genes are not
of FabH and AsuC3,C4, this could also be the result of type II organized in any apparent order. Recently, the ␭-Red recombi-
thioesterase AsuC15 action to release the tethered cyclohexyl- nation system has been extended to heterologous expression
propanoate from the FAS-ACP and allow the recruitment of (23–25). Taking advantage of the shared vector and overlap-
more branched chain acyl-CoA starters, as the percentage of ping inserts, we simplified this system and assembled two cos-
24922 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 32•AUGUST 6, 2010

Asukamycin Biosynthetic Pathway
25. Hu, Y., Phelan, V. V., Farnet, C. M., Zazopoulos, E., and Bachmann, B. O.
(2008) ChemBioChem 9, 1603–1608
mids in a single step by skipping the PCR cloning and multiple
“stitching” processes (Fig. 3). Since the linearized cosmids are
unable to replicate, the circular recombinant pART1361 could
propagate and form colonies under antibiotic selection. This
straightforward recombination approach is particularly useful
26. Pfennig, F., Schauwecker, F., and Keller, U. (1999) J. Biol. Chem. 274,
12508–12516
27. Zhou, Z., Lai, J. R., and Walsh, C. T. (2007) Proc. Natl. Acad. Sci. U.S.A.
104, 11621–11626
to examine cloned genes and provides a convenient platform 28. Ferreras, J. A., Stirrett, K. L., Lu, X., Ryu, J. S., Soll, C. E., Tan, D. S., and
Quadri, L. E. (2008) Chem. Biol. 15, 51–61
for further gene manipulations. For example, a reliable yield of
4-hydroxyprotoasukamycin D1 was obtained in S. lividans car-
rying the pART1361E3 by replacing asuE3 with a spectinomy-
cin resistance gene.
29. Hertweck, C. (2009) Angew. Chem. Int. Ed. Engl. 48, 4688–4716
30. Sattely, E. S., Fischbach, M. A., and Walsh, C. T. (2008) Nat. Prod. Rep. 25,
757–793
31. Huang, W., Jia, J., Edwards, P., Dehesh, K., Schneider, G., and Lindqvist, Y.
(1998) EMBO J. 17, 1183–1191
32. Olsen, J. G., Kadziola, A., von Wettstein-Knowles, P., Siggaard-Andersen,
M., Lindquist, Y., and Larsen, S. (1999) FEBS Lett. 460, 46–52
33. Tang, Y., Lee, H. Y., Tang, Y., Kim, C. Y., Mathews, I., and Khosla, C. (2006)
Biochemistry 45, 14085–14093
Acknowledgments—We are grateful to C. Khosla (S. lividans K4-114)
and John Innes Center (␭-Red recombination kit) for the gifts of strains
and plasmids. We thank Drs. J. Chen, G. Henderson, and T. K. Weld-
eghiorghis for assistance with mass spectral and NMR analyses and
M. Saboori for the asuC2 analysis. We thank the anonymous review-
ers for constructive comments.
34. Cohen-Gonsaud, M., Ducasse, S., Hoh, F., Zerbib, D., Labesse, G., and
Quemard, A. (2002) J. Mol. Biol. 320, 249–261
35. Sacco, E., Covarrubias, A. S., O’Hare, H. M., Carroll, P., Eynard, N., Jones,
T. A., Parish, T., Daffe´, M., Ba¨ckbro, K., and Que´mard, A. (2007) Proc.
Natl. Acad. Sci. U.S.A. 104, 14628–14633
REFERENCES
36. Palaniappan, N., Kim, B. S., Sekiyama, Y., Osada, H., and Reynolds, K. A.
(2003) J. Biol. Chem. 278, 35552–35557
1. Sattler, I., Thiericke, R., and Zeeck, A. (1998) Nat. Prod. Rep. 15,
37. Pan, H., Tsai, S., Meadows, E. S., Miercke, L. J., Keatinge-Clay, A. T.,
O’Connell, J., Khosla, C., and Stroud, R. M. (2002) Structure 10,
1559–1568
221–240
2. Shipley, P. R., Donnelly, C. C., Le, C. H., Bernauer, A. D., and Klegeris, A.
(2009) Int. J. Mol. Med. 24, 711–715
38. Tang, Y., Lee, T. S., Kobayashi, S., and Khosla, C. (2003) Biochemistry 42,
6588–6595
39. Astner, I., Schulze, J. O., van den Heuvel, J., Jahn, D., Schubert, W. D., and
Heinz, D. W. (2005) EMBO J. 24, 3166–3177
3. Bernier, M., Kwon, Y. K., Pandey, S. K., Zhu, T. N., Zhao, R. J., Maciuk, A.,
He, H. J., Decabo, R., and Kole, S. (2006) J. Biol. Chem. 281, 2551–2561
4. Zheng, Z. H., Dong, Y. S., Zhang, H., Lu, X. H., Ren, X., Zhao, G., He, J. G.,
and Si, S. Y. (2007) J. Enzyme Inhib. Med. Chem. 22, 43–49
5. Kakinuma, K., Ikekawa, N., Nakagawa, A., and Omura, S. (1979) J. Am.
Chem. Soc. 101, 3402–3404
40. Gerber, R., Lou, L., and Du, L. C. (2009) J. Am. Chem. Soc. 131, 3148–3149
41. Hanada, K. (2003) Biochim. Biophys. Acta 1632, 16–30
42. Webster, S. P., Alexeev, D., Campopiano, D. J., Watt, R. M., Alexeeva, M.,
Sawyer, L., and Baxter, R. L. (2000) Biochemistry 39, 516–528
43. Pacholec, M., Freel Meyers, C. L., Oberthu¨r, M., Kahne, D., and Walsh,
C. T. (2005) Biochemistry 44, 4949–4956
6. Omura, S., Kitao, C., Tanaka, H., Oiwa, R., Takahashi, Y., Nakagawa, A.,
Shimada, M., and Iwai, Y. (1976) J. Antibiot. 29, 876–881
7. Hu, Y. D., and Floss, H. G. (2006) Heterocycles 69, 133–149
8. Hu, Y., and Floss, H. G. (2001) J. Antibiot. 54, 340–348
9. Hu, Y. D., Melville, C. R., Gould, S. J., and Floss, H. G. (1997) J. Am. Chem.
Soc. 119, 4301–4302
44. Schmutz, E., Steffensky, M., Schmidt, J., Porzel, A., Li, S. M., and Heide, L.
(2003) Eur. J. Biochem. 270, 4413–4419
45. Yu, T. W., Bai, L., Clade, D., Hoffmann, D., Toelzer, S., Trinh, K. Q., Xu, J.,
Moss, S. J., Leistner, E., and Floss, H. G. (2002) Proc. Natl. Acad. Sci. U.S.A.
99, 7968–7973
10. Hu, Y. D., and Floss, H. G. (2004) J. Am. Chem. Soc. 126, 3837–3844
11. Thiericke, R., Zeeck, A., Nakagawa, A., Omura, S., Herrold, R. E., Wu,
S. T. S., Beale, J. M., and Floss, H. G. (1990) J. Am. Chem. Soc. 112,
3979–3987
46. Vineis, P., Bartsch, H., Caporaso, N., Harrington, A. M., Kadlubar, F. F.,
Landi, M. T., Malaveille, C., Shields, P. G., Skipper, P., Talaska, G., and
Tannenbaum, S. R. (1994) Nature 369, 154–156
12. Cropp, T. A., Wilson, D. J., and Reynolds, K. A. (2000) Nat. Biotechnol. 18,
980–983
47. Cole, L. J., Gatti, D. L., Entsch, B., and Ballou, D. P. (2005) Biochemistry 44,
8047–8058
13. Beale, J. M., Lee, J. P., Nakagawa, A., Omura, S., and Floss, H. G. (1986)
J. Am. Chem. Soc. 108, 331–332
48. Kirchner, U., Westphal, A. H., Mu¨ller, R., and van Berkel, W. J. (2003)
J. Biol. Chem. 278, 47545–47553
14. Ostash, B., Saghatelian, A., and Walker, S. (2007) Chem. Biol. 14,
257–267
49. van der Werf, M. J., Swarts, H. J., and de Bont, J. A. (1999) Appl. Environ.
Microbiol. 65, 2092–2102
15. Petrícek, M., Petríckova´, K., Havlícek, L., and Felsberg, J. (2006) J. Bacte-
riol. 188, 5113–5123
50. Claxton, H. B., Akey, D. L., Silver, M. K., Admiraal, S. J., and Smith, J. L.
(2009) J. Biol. Chem. 284, 5021–5029
16. Nakagawa, A., Wu, T. S., Keller, P. J., Lee, J. P., Omura, S., and Floss, H. G.
(1985) J. Chem. Soc. Chem. Comm. 519–521
51. Koglin, A., Lo¨hr, F., Bernhard, F., Rogov, V. V., Frueh, D. P., Strieter, E. R.,
Mofid, M. R., Gu¨ntert, P., Wagner, G., Walsh, C. T., Marahiel, M. A., and
Do¨tsch, V. (2008) Nature 454, 907–911
17. Thiericke, R., Zeeck, A., Robinson, J. A., Beale, J. M., and Floss, H. G. (1989)
J. Chem. Soc. Chem. Comm. 402–403
18. Kaneda, T. (1991) Microbiol. Rev. 55, 288–302
19. Zhang, Y. M., and Rock, C. O. (2008) Nat. Rev. Microbiol. 6, 222–233
20. Datsenko, K. A., and Wanner, B. L. (2000) Proc. Natl. Acad. Sci. U.S.A. 97,
6640–6645
52. Kotowska, M., Pawlik, K., Smulczyk-Krawczyszyn, A., Bartosz-Bechowski,
H., and Kuczek, K. (2009) Appl. Environ. Microbiol. 75, 887–896
53. Kim, B. S., Cropp, T. A., Beck, B. J., Sherman, D. H., and Reynolds, K. A.
(2002) J. Biol. Chem. 277, 48028–48034
21. Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F., and Hopwood, D. A.
(2000) Practical Streptomyces Genetics, The John Innes Foundation, Nor- 54. Yeh, E., Kohli, R. M., Bruner, S. D., and Walsh, C. T. (2004) ChemBioChem
wich, UK
5, 1290–1293
22. Suzuki, H., Ohnishi, Y., Furusho, Y., Sakuda, S., and Horinouchi, S. (2006)
J. Biol. Chem. 281, 36944–36951
55. Song, F., Zhuang, Z., Finci, L., Dunaway-Mariano, D., Kniewel, R., Buglino,
J. A., Solorzano, V., Wu, J., and Lima, C. D. (2006) J. Biol. Chem. 281,
11028–11038
23. Binz, T. M., Wenzel, S. C., Schnell, H. J., Bechthold, A., and Mu¨ller, R.
(2008) ChemBioChem 9, 447–454
56. Kotaka, M., Kong, R., Qureshi, I., Ho, Q. S., Sun, H., Liew, C. W., Goh, L. P.,
Cheung, P., Mu, Y., Lescar, J., and Liang, Z. X. (2009) J. Biol. Chem. 284,
15739–15749
24. Wolpert, M., Heide, L., Kammerer, B., and Gust, B. (2008) ChemBioChem
9, 603–612
AUGUST 6, 2010•VOLUME 285•NUMBER 32
JOURNAL OF BIOLOGICAL CHEMISTRY 24923

Asukamycin Biosynthetic Pathway
57. Nasser, W., and Reverchon, S. (2007) Anal. Bioanal. Chem. 387, 381–390 60. Kitani, S., Iida, A., Izumi, T. A., Maeda, A., Yamada, Y., and Nihira, T.
58. Ramos, J. L., Martínez-Bueno, M., Molina-Henares, A. J., Tera´n, W., Wa-
(2008) Gene 425, 9–16
tanabe, K., Zhang, X., Gallegos, M. T., Brennan, R., and Tobes, R. (2005) 61. Simbahan, J., Drijber, R., and Blum, P. (2004) Int. J. Syst. Evol. Microbiol.
Microbiol. Mol. Biol. Rev. 69, 326–356 54, 1703–1707
59. Groban, E. S., Clarke, E. J., Salis, H. M., Miller, S. M., and Voigt, C. A. 62. Zhang, W., Bolla, M. L., Kahne, D., and Walsh, C. T. (2010) J. Am. Chem.
(2009) J. Mol. Biol. 390, 380–393
Soc. 132, 6402–6411
24924 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 32•AUGUST 6, 2010

Microbiology:
Biochemical and Genetic Insights into
Asukamycin Biosynthesis
Zhe Rui, Katerina Petrícková, Frantisek
Skanta, Stanislav Pospísil, Yanling Yang,
Chung-Yung Chen, Shih-Feng Tsai, Heinz G.
Floss, Miroslav Petrícek and Tin-Wein Yu
J. Biol. Chem. 2010, 285:24915-24924.
doi: 10.1074/jbc.M110.128850 originally published online June 3, 2010
Access the most updated version of this article at doi: 10.1074/jbc.M110.128850
Find articles, minireviews, Reflections and Classics on similar topics on the JBC Affinity Sites.
Alerts:
• When this article is cited
• When a correction for this article is posted
Click here to choose from all of JBC's e-mail alerts
Supplemental material:
http://www.jbc.org/content/suppl/2010/06/03/M110.128850.DC1.html
This article cites 59 references, 21 of which can be accessed free at
http://www.jbc.org/content/285/32/24915.full.html#ref-list-1