Biochemical analysis of the biosynthetic pathway of an anticancer tetracycline SF2575

Article abstract of DOI:10.1021/ja907852c

SF2575 1 is a tetracycline polyketide produced by Streptomyces sp. SF2575 and displays exceptionally potent anticancer activity toward a broad range of cancer cell lines. The structure of SF2575 is characterized by a highly substituted tetracycline aglyco

Full text of DOI:10.1021/ja907852c

Published on Web 11/12/2009
Biochemical Analysis of the Biosynthetic Pathway of an
Anticancer Tetracycline SF2575
Lauren B. Pickens,† Woncheol Kim,† Peng Wang,† Hui Zhou,† Kenji Watanabe,‡
Shuichi Gomi,§ and Yi Tang*,†
Department of Chemical and Biomolecular Engineering, UniVersity of California, Los Angeles,
School of Pharmaceutical Sciences, UniVersity of Shizuoka, Shizuoka 422-8526, Japan, and
Medicinal Chemistry Research Laboratories, Pharmaceutical Research Center, Meiji Seika
Kaisha, Ltd., 760 Morooka-cho, Kohoku-ku, Yokohama 222-8567, Japan
Received September 15, 2009; E-mail: yitang@ucla.edu
Abstract: SF2575 1 is a tetracycline polyketide produced by Streptomyces sp. SF2575 and displays
exceptionally potent anticancer activity toward a broad range of cancer cell lines. The structure of SF2575
is characterized by a highly substituted tetracycline aglycon. The modifications include methylation of the
C-6 and C-12a hydroxyl groups, acylation of the 4-(S)-hydroxyl with salicylic acid, C-glycosylation of the
C-9 of the D-ring with D-olivose and further acylation of the C4′-hydroxyl of D-olivose with the unusual
angelic acid. Understanding the biosynthesis of SF2575 can therefore expand the repertoire of enzymes
that can modify tetracyclines, and facilitate engineered biosynthesis of SF2575 analogues. In this study,
we identified, sequenced, and functionally analyzed the ssf biosynthetic gene cluster which contains 40
putative open reading frames. Genes encoding enzymes that can assemble the tetracycline aglycon, as
well as installing these unique structural features, are found in the gene cluster. Biosynthetic intermediates
were isolated from the SF2575 culture extract to suggest the order of pendant-group addition is C-9
glycosylation, C-4 salicylation, and O-4′ angelylcylation. Using in vitro assays, two enzymes that are
responsible for C-4 acylation of salicylic acid were identified. These enzymes include an ATP-dependent
salicylyl-CoA ligase SsfL1 and a putative GDSL family acyltransferase SsfX3, both of which were shown
to have relaxed substrate specificity toward substituted benzoic acids. Since the salicylic acid moiety is
critically important for the anticancer properties of SF2575, verification of the activities of SsfL1 and SsfX3
sets the stage for biosynthetic modification of the C-4 group toward structure-activity relationship studies
of SF2575. Using heterologous biosynthesis in Streptomyces lividans, we also determined that biosynthesis
of the SF2575 tetracycline aglycon 8 parallels that of oxytetracycline 4 and diverges after the assembly of
4-keto-anhydrotetracycline 51. The minimal ssf polyketide synthase together with the amidotransferase
SsfD produced the amidated decaketide backbone that is required for the formation of 2-naphthacenecar-
boxamide skeleton. Additional enzymes, such as cyclases C-6 methyltransferase and C-4/C-12a dihy-
droxylase, were functionally reconstituted.
Introduction
Other common features include the planar phenyldiketone
arrangement and the tricarbonylmethane moiety in the A ring.
Bacterial aromatic polyketide natural products comprise a
group of molecules that displays diverse structures and bioac-
tivities yet share common biosynthetic origins. The poly-ꢀ-
ketone backbone is synthesized by a minimal polyketide
synthase (PKS) consisting of a ketosynthase (KS or KSR), a
chain-length factor (CLF or KSꢀ), and an acyl carrier protein
(ACP).1 Additional tailoring enzymes are required to fix the
regioselectivity of sequential cyclization steps and perform a
multitude of decorating reactions to afford the various polycyclic
bioactive compounds. A small, yet extremely important group
of aromatic polyketides is the tetracycline family. Tetracyclines
are characterized by the linearly fused four-ring structure and a
heavily oxidized 2-naphthacenecarboxamide carbon skeleton.
This family is exemplified by the well-known tetracyclines
chlorotetracycline 5 and oxytetracycline 42 (Scheme 1). Dis-
covered in 1948 from Streptomyces aureofaciens3 and in 1950
from S. rimosus,4 respectively, these compounds were com-
mercialized as broad-spectrum antibiotics and used successfully
until bacterial resistance prompted the need for the creation of
analogues to combat drug resistant strains.5 The natural
tetracycline scaffold has since been used to create second- and
third-generation tetracyclines semisynthetically. The third-
generation tetracycline tigecycline gained FDA approval in
(2) Chopra, I.; Roberts, M. Microbiol. Mol. Biol. ReV. 2001, 65, 232–
260.
† University of California.
(3) Duggar, B. M. Ann. N.Y. Acad. Sci. 1948, 51, 177–181.
(4) Finlay, A. C.; Hobby, G. L.; P’an, S. Y.; Regna, P. P.; Routein, J. B.;
Seeley, D. B.; Shull, G. M.; Sobin, B. A.; Solomons, I. A.; Vinson,
J. W.; Kane, J. H. Science 1950, 111, 85.
‡ University of Shizuoka.
§ Meiji Seika Kaisha, Ltd.
(1) Hertweck, C.; Luzhetskyy, A.; Rebets, Y.; Bechthold, A. Nat. Prod.
Rep. 2007, 24, 162–190.
(5) Wright, G. D.; Zakeri, B. Biochem. Cell Biol. 2008, 86, 124–136.
9
10.1021/ja907852c CCC: $40.75  2009 American Chemical Society
J. AM. CHEM. SOC. 2009, 131, 17677–17689 17677

A R T I C L E S
Pickens et al.
Scheme 1
2005,6,7 demonstrating the continued utility of the tetracycline
scaffold. Remarkably, since the initial discovery of 4 and 5 over
60 years ago, there have been only a few natural tetracyclines
discovered. Among these are SF25758,9 1 and related com-
pounds, TAN-1518A 2 and TAN-1518B 3,10 chelocardin,11 and
dactylocycline.12 Each of these compounds contains the tetra-
cycline core and exhibits novel structural features not observed
in 4 or 5. Given the rarity of tetracycline natural products,
understanding the biosynthesis of these newly discovered
compounds may provide a route to the generation of new
tetracycline antibiotics through the use of combinatorial bio-
synthesis and metabolic engineering strategies.
lines tested, resulting in an average IC50 value of 11.2 nM. The
mechanism of action of several closely related SF2575 ana-
logues, 2 and 3 has been identified as inhibition of DNA
topoisomerase I.10 As these structures are nearly identical to
that of 1, it is likely that this family of compounds shares a
common molecular target. As key enzymes during DNA
replication, both DNA topoisomerases I and II are known targets
for current anticancer therapies in clinical use such as doxoru-
bicin, a topoisomerase II poison,14 and camptothecin derivatives
which target topoisomerase I.15 Elucidation of the SF2575
biosynthetic pathway can therefore be useful toward future
structure-activity relationship (SAR) studies and engineered
biosynthesis of new anticancer compounds.
SF2575 was first isolated by Hatsu et al. from the producing
strain Streptomyces sp. SF2575.9 The 2-naphthacenecarboxa-
mide carbon framework of 1 is identical to that of 4 and 5. In
addition, there are a number of unique tailoring modifications
decorating the tetracyclic aglycon including two methoxy groups
at C-6 and C-12a, a 4-(S)-salicylate that replaces the more
common 4-(R)-dimethylamine substituent, a C-9 C-glycoside
of 2-6-dideoxy-arabino-hexopyranose (D-olivose), and acylation
of the O-4′ of D-olivose with (Z)-2-methyl-2-butenoate (ange-
late). The angelate and salicylate moieties are unusual polyketide
modifications which add to the intrigue of the biosynthesis of
1. In addition to its unique structure, 1 also introduces potent
anticancer bioactivity to the tetracycline family. Screening by
Hatsu et al. demonstrated that 1 has very weak antibiotic activity,
which is most likely due to, among others, methylation of the
C-12a hydroxyl group that disrupts interactions with the bacterial
30S ribosomal subunit.13 SF2575 was, however, found to have
exceptionally potent anticancer activity both through an in vitro
cytotoxicity assay with P388 leukemia cells and during an in
vivo assay using mouse xenografts.9 More recently, a 60-cell
line screening by the National Cancer Institute demonstrated
the potent activity of 1 against nearly all types of cancer cell
In this report we have identified and sequenced the ssf gene
cluster responsible for the biosynthesis of 1. This ssf gene cluster
is only the third tetracycline family gene cluster to be identified
and sequenced following chlorotetracycline16 and oxytetracycline.17,18
The genetic information offers valuable opportunities to further
enhance our understanding of tetracycline biosynthesis and to
investigate an entirely new set of tetracycline-tailoring modifica-
tions that distinguish 1 from the previously studied 4 and 5.
Bioinformatic analysis of the gene cluster along with identifica-
tion of the possible biosynthetic intermediates from Streptomyces
sp. SF2575 fermentation extract led to the assembly of a putative
biosynthetic pathway for 1. We have identified key tailoring
enzymes involved in the attachment of salicylate to the
tetracycline aglycon and verified their function through in vitro
assays. Additionally, we have reconstituted the early portions
of the ssf pathway in a heterologous host and demonstrated the
biosynthesis of the tetracycline core of 1 parallels the biosyn-
thetic pathway of 4.18–20
Results and Discussion
Identification of SF2575 Intermediates from Crude Extract.
Comparing the core structures of 1 and 4, biosynthesis of the
naphthacenecarboxamide carbon framework of 1 is predicted
to parallel that of the oxytetracycline (oxy) biosynthetic
pathway17,18 and may share a number of common intermediates.
(6) Sum, P. E.; Lee, V. J.; Testa, R. T.; Hlavka, J. J.; Ellestad, G. A.;
Bloom, J. D.; Gluzman, Y.; Tally, F. P. J. Med. Chem. 1994, 37, 184–
188.
(7) Sum, P.-E.; Petersen, P. Bioorg. Med. Chem. Lett. 1999, 9, 1459–
1462.
(8) Hatsu, M.; Sasaki, T.; Gomi, S.; Kodama, Y.; Sezaki, M.; Inouye, S.;
Kondo, S. J. Antibiot. 1992, 45, 325–330.
(14) Kellner, U.; Sehested, M.; Jensen, P. B.; Gieseler, F.; Rudolph, P.
Lancet Oncol. 2002, 3, 235–243.
(9) Hatsu, M.; Sasaki, T.; Watabe, H.; Miyadoh, S.; Nagasawa, M.;
Shomura, T.; Sezki, M.; Inouye, S.; Kondo, S. J. Antibiot. 1992, 45,
320–324.
(15) Pommier, Y. Nat. ReV. Cancer 2006, 6, 789–802.
(16) Ryan, M. J. U.S. Patent 5989903, 1999.
(10) Horiguchi, T.; Hayashi, K.; Tsubotani, S.; Iinuma, S.; Harada, S.;
Tanida, S. J. Antibiot. 1994, 47, 545–556.
(17) Hunter, I. S., Tetracyclines. In Microbial Secondary Metabolites:
Biosynthesis, Genetics and Regulation; Martin, J. F., Ed.; Research
Signpost: Lucknow, India, 2002; pp 141-166.
(11) Mitscher, L. A.; Juvarkar, J. V.; Rosenbrook, W.; Andres, W. W.;
Schenck, J. R.; Egan, R. S. J. Am. Chem. Soc. 1970, 92, 6070–6071.
(12) Tymiak, A. A.; Aklonis, C.; Bolgar, M. S.; Kahle, A. D.; Kirsch, D. R.;
O’Sullivan, J.; Porubcan, M. A.; Principe, P.; Trejo, W. H. J. Org.
Chem. 1993, 58, 535–537.
(18) Zhang, W.; Ames, B. D.; Tsai, S. C.; Tang, Y. Appl. EnViron.
Microbiol. 2006, 72, 2573–2580.
(19) Zhang, W.; Watanabe, K.; Cai, X.; Jung, M. E.; Tang, Y.; Zhan, J.
J. Am. Chem. Soc. 2008, 130, 6068–6069.
(13) Brodersen, D. E.; Clemons, W. M., Jr.; Carter, A. P.; Morgan-Warren,
R. J.; Wimberly, B. T.; Ramakrishnan, V. Cell 2000, 103, 1143–1154.
(20) Zhang, W.; Watanabe, K.; Wang, C. C. C.; Tang, Y. J. Biol. Chem.
2007, 282, 25717–25725.
9
17678 J. AM. CHEM. SOC. VOL. 131, NO. 48, 2009

Biosynthetic Pathway for Anticancer Tetracycline SF2575
A R T I C L E S
Scheme 2
As shown in Scheme 2, the putative aglycon is likely to be 8,
which exhibits the typical features of a tetracycline except
replacement of 4-(R)-dimethylamine with 4-(S)-hydroxyl, inver-
sion of stereochemistry at C-6 and O-methylations at C-6 and
C-12a. Downstream tailoring of 1 is therefore likely to occur
following assembly of 8. In order to decipher the timing of the
three unique post-PKS tailoring reactions leading to the bio-
synthesis of 1: glycosylation with D-olivose 14, O-4′ acylation
of angelic acid and C-4 acylation of salicylic acid 19, crude
extract of Streptomyces sp. SF2575 culture was prepared and
analyzed on HPLC and LC/MS to try to identify any potential
stable intermediates that may be present. Streptomyces sp.
SF2575 culture was grown on solid Bennette’s media for 10
days at 30 °C. A sample was extracted each day starting with
day 4 to analyze the metabolic profile. Using selected ion
monitoring, two potential intermediates identified were 6 ([M
+ H]+ at m/z 576, RT ) 18.3 min) and 7 ([M + H]+ at m/z
696, RT ) 26.8 min), in addition to the parent compound 1
([M + H]+ at m/z 778, RT ) 34.2 min) (Figure 1). The UV
spectrum of each compound was similar to that of 1 with a
characteristic tetracycline λmax at 358 nm, and a λmax at 302 nm
for 1 and 7 characteristic of the salicylate. The UV spectrum of
6 lacks this contribution at 302 nm, resulting in a smooth peak
at 358 nm, and is indicative of the loss of salicylate compared
to 7 as suggested by the molecular weight difference. To confirm
the identity of these compounds as shown in Figure 1, authentic
standards were prepared from base hydrolysis of purified 1 as
described by Hatsu et al.8 Treatment of 1 with 0.5 M NaOH
led to the hydrolysis of the angelate and afforded 7, while
complete conversion to 6 was obtained by treating 1 with 1.0
M NaOH for 15 h. Following purification of 6 and 7 from the
base hydrolysis reactions, proton NMR and HRMS were used
to confirm the structures by comparison to published data
(Supporting Information).8 These prepared samples were then
used as standards to verify the identities of 6 and 7 in the
fermentation extract of Streptomyces sp. SF2575 by HPLC
retention times, mass fragmentation patterns and UV spectra.
Other potential intermediates, such as 6 acylated with
angelate, or 8 acylated with salicylate, were not detected in the
crude extracts using selected ion monitoring by LC/MS. This
evidence, along with the positive detection of 6 and 7 were used
to construct a proposed aglycon-tailoring pathway of 1 as shown
in Scheme 2. Therefore, the likely order of the tailoring
modifications starts with C-glycosylation of 8 with 14 that results
in 6, followed by attachment of salicylic acid 19 at C-4 to
produce 7, and capped by the acylation of O-4′ of 7 with
angelate to produce the final product 1. It is noteworthy that
each of these isolated intermediates has already been O-
methylated at the C-12a and C-6 hydroxyl groups, which
indicates that these modifications likely take place early in the
biosynthesis, providing further support for 8 as an advanced
intermediate. While 8 was not identified in the fermentation
extract, this could be due either to the potential instability of 8
or the rate of the glycosylation reaction which may not permit
the accumulation of 8 in detectable quantities.
Bioactivity of SF2575 and Putative Biosynthetic Intermedi-
ates. Since the potent antiproliferative activity of 1 is new to
the tetracycline family, we sought to gain insight to the structural
features of 1 that may contribute to this activity. The angelate
and salicylate modifications of 1 are unique structural features
among tetracyclines and bacterial aromatic polyketides. To probe
whether these moieties are the “warheads” that contribute to
the antitumor activities of 1, we performed in vitro cytotoxicity
assays with Nalm-6 pre-B cells using 6, 7 and 1 (Supporting
9
J. AM. CHEM. SOC. VOL. 131, NO. 48, 2009 17679

A R T I C L E S
Pickens et al.
(Figure 2). The cluster spans a 47.2 kb region and contains 40
ORFs putatively involved in SF2575 biosynthesis, including the
essential KSR-KSꢀ heterodimer for synthesis of the polyketide
backbone. To confirm that this cluster is responsible for SF2575
biosynthesis, the ssfB gene encoding KSꢀ was inactivated by
an insertion of a kanamycin resistance gene (aphII) through a
double crossover recombination. Disruption of this gene resulted
in complete loss of SF2575 production (Supporting Information
Figure S6), hence confirming the link between 1 and the
sequenced ssf gene cluster.
A map of the ssf gene cluster is shown in Figure 2. The
putative boundaries are determined on the basis of sequence
analysis and biosynthetic logic. The ssf genes identified along
with their proposed function are listed in Table 1. Represented
within are genes likely responsible for the biosynthesis of the
aglycon 8, pendant groups 14, 19, and 25, as well as regulation
and self-resistance. Based on the putative functions of these
genes, the proposed biosynthetic pathways of 8 and the
subsequent tailoring of 8 to 1 are shown in Scheme 4 and
Scheme 2, respectively.
Genes Putatively Involved in the Biosynthesis of D-Olivose
14. Deoxysugar decoration is an important tailoring modification
of bacterial secondary metabolites and is usually essential for
the bioactivity of the natural products.22 The C-9 of 1 is modified
with a C-glycoside D-olivose, which is also found among other
polyketides such as urdamycin A23 and simocyclinone.24
Putative enzymes encoded in the ssf gene cluster that can convert
glucose to NDP-D-olivose are SsfS1 (NDP-glucose synthase),
SsfS2 (4′,6′-dehydratase), SsfS3 (2′,3′-dehydratase), SsfS4 (C-
3′ reductase), and SsfS5 (C-4′ reductase) (Scheme 2). The
proposed pathway includes the unstable intermediate 12 which
is reduced at the 3′ position by SsfS4.25 NDP-D-olivose is
subsequently transferred to the C-9 of the aglycon 8 by a
C-glycosyltransferase to yield 6. C-glycosyltransferases are more
rare than O-glycosyltransferases, and are found in a number of
aromatic PKS biosynthetic gene clusters such as urdamycin,23
hedamycin,26 and simocyclinone.24 The putative ssf glycosyl-
transferase, SsfS6, has high sequence homology to HedJ, a
C-glycosyltransferase from the hedamycin biosynthetic path-
way.26 Since SsfS6 is the first glycosyltransferase that can
modify the tetracycline scaffold, it can potentially be a useful
enzyme toward increasing the structural diversity of the D-ring,
and in particular the C-9 position, of tetracycline compounds.
Figure 1. Production of 1 and biosynthetic intermediates by Streptomyces
sp. SF2575. The data were collected on the Shimadzu LC/MS. (A) LC trace
(358 nm) for a mixture of standards 6, 7, and 1. (B) LC trace (358 nm) of
the extract after 7 days growth on solid Bennette’s media. (C) Selected ion
monitoring was used to confirm the identification of 7 ([M + H]+ m/z )
696) and 6 ([M + H]+ m/z ) 576). The UV spectra of these compounds
are shown for comparison.
Information Figure S5). While the parent compound 1 had a
potent IC50 of 8.8 nM, removal of the angelate resulted in a
significantly attenuated IC50 of 327 nM for 7. Further hydrolysis
of the salicylate as in 6 led to an additional 15-fold decrease of
potency with an IC50 of 5.2 µM. Similar trends were observed
when other cell lines, including MCF-7, HeLa, and M249 were
subjected to the cytotoxicity assays (Supporting Information
Figure S5). Hence, both pendant groups are critically important
for the bioactivity of 1 and are attractive targets for SAR studies.
Identification of the enzymes responsible for these additions may
therefore provide useful biosynthetic approaches toward func-
tionalizing these positions, especially considering the densely
functionalized tetracycline core may be difficult to assess
synthetically.
Identification and Sequencing of the ssf Biosynthetic Gene
Cluster. To construct a blueprint of SF2575 biosynthesis, the
ssf gene cluster was identified by screening a cosmid library
prepared from Streptomyces sp. SF2575 genomic DNA using
degenerative primers published by Wawrik et al. for screening
the type II polyketide KSR gene.21 Several PCR products from
different cosmids were sequenced and were found to encode
an identical KS fragment, which indicates that there is likely
only one aromatic PKS gene cluster present in the Streptomyces
sp. SF2575 genome. Three overlapping cosmids were sequenced
by a combination of shotgun sequencing and primer walking
Genes Putatively Involved in the Biosynthesis of Salicylate
19. The C-4 salicylate substitution in 1 is unusual not just among
tetracyclines but quite rare among aromatic polyketides. One
known example is that of thermorubin, in which the salicylate
moiety is thought to be incorporated as a starter unit.27 A related
modification is the addition of a 6-methylsalicylic acid which
is typically synthesized via a dedicated type I PKS, such as
(22) Salas, J. A.; Me´ndez, C. Trends Microbiol. 2007, 15, 219–232.
(23) Faust, B.; Hoffmeister, D.; Weitnauer, G.; Westrich, L.; Haag, S.;
Schneider, P.; Decker, H.; Kunzel, E.; Rohr, J.; Bechthold, A.
Microbiology 2000, 146, 147–154.
(24) Trefzer, A.; Pelzer, S.; Schimana, J.; Stockert, S.; Bihlmaier, C.;
Fiedler, H. P.; Welzel, K.; Vente, A.; Bechthold, A. Antimicrob. Agents
Chemother. 2002, 46, 1174–1182.
(25) Draeger, G.; Park, S.-H.; Floss, H. G. J. Am. Chem. Soc. 1999, 121,
2611–2612.
(26) Bililign, T.; Hyun, C.-G.; Williams, J. S.; Czisny, A. M.; Thorson,
J. S. Chem. Biol. 2004, 11, 959–969.
(21) Wawrik, B.; Kerkhof, L.; Zylstra, G. J.; Kukor, J. J. Appl. EnViron.
Microbiol. 2005, 71, 2232–2238.
(27) Aragozzini, F.; Craveri, R.; Maconi, E.; Ricca, G. S.; Scolastico, C.
J. Chem. Soc., Perkins Trans. 1 1988, 1865–1867.
9
17680 J. AM. CHEM. SOC. VOL. 131, NO. 48, 2009

Biosynthetic Pathway for Anticancer Tetracycline SF2575
A R T I C L E S
Figure 2. Organization of the ssf biosynthetic gene cluster. Genes are categorized according to their proposed role. The gene cluster spans 47.2 kb and
contains 40 ORFs. Details of proposed functions are shown in Table 1.
Table 1. Putatively Assigned Functions of Enzymes Encoded in the ssf Biosynthetic Gene Cluster
name
closest homologue
putative role
size (kDa)
identity (%)/similarity (%)
accession number
SsfA
SsfB
SsfC
SsfD
SsfE
SsfF
SsfG
SsfH
OxyA
OxyB
OxyC
OxyD
ChlJ
Npun_F4742
ChlB3
MbtI
PlmI
SAV_2786
Athe_1156
SdgA
OxyH
OxyT
DauK
MtmMII
OxyF
PpzT
OxyS
OxyL
CmmQ
Lct52
Krad_0275
StrD
CalS3
Sim20
ChlC4
NanG4
HedJ
SnorA
SAML0351
OxyJ
ZhuC
ketosynthase
45
43
9.5
68
58
34
36
48
45
28
26
58
56
36
35
37
37
31
55
59
29
43
58
39
36
53
33
37
41
31
23
27
34
8.5
36
40
34
33
31
13
12
17
46
66
14
35
76/86
64/77
54/73
68/79
81/89
55/70
37/56
42/57
56/67
61/75
38/59
75/84
51/67
48/62
38/53
38/55
51/68
46/63
42/54
57/70
49/54
84/90
33/51
72/83
69/80
58/72
49/59
42/53
40/55
44/61
48/62
76/86
54/64
50/60
64/72
48/61
57/67
56/68
29/42
66/76
84/94
89/96
90/94
60/69
78/88
82/90
CAA80985
AAZ78326
AAZ78327
AAZ78328
AAZ77684
ACC83094
DQ116941
Q7D785
chain length factor
acyl carrier protein
amidotransferase
carboxytransferase
ketoreductase
ketosynthase III
salicylate synthase
DAHP-7-phosphate synthase
enoyl CoA hydratase/isomerase
3-ketoacyl-(ACP) reductase
Salicylyl-AMP ligase
acyl-CoA ligase
O-methyltransferase
O-methyltransferase
methyltransferase
C-methyltransferase
ketosynthase III
SsfI
SsfJ
SsfK
AY354515
BAC70497
ACM60257
BAC78380
DQ143963
DQ143963
AAB16938
CAK50790
AAZ78330
CAX48662
AAZ78342
AAZ78335
YP_118212
ABX71135
ABS01765
BAG22759
AAM94770
AF322256
AAZ77681
AAP42863
AAP85354
CAA12016
CAJ89338
AAZ78333
AAG30190
AAZ77683
CAE51179
AAK83171
AAZ78334
AAD20271
CAE17552
AAZ78332
BAG22430
EDY55253
EDY65440
EEP13034
NP_627922
EDY56197
SsfL1
SsfL2
SsfM1
SsfM2
SsfM3
SsfM4
SsfN
SsfO1
SsfO2
SsfP
oxygenase
oxygenase
dehydrogenase
SsfQ
SsfR
SAM synthetase
MFS transporter
dTDP glucose synthase
glucose-4,6-dehydratase
hexose-2,3-dehydratase
hexose-3-ketoreductase
hexose-4-ketoreductase
glycosyltransferase
regulation
regulation (TetR family)
ketoreductase
acyltransferase
unknown function
carbohydrate kinase
acyltransferase
aromatase/cyclase
cyclase
cyclase
cyclase
unknown
FeS assembly protein
cysteine desulferase
unknown
unknown
AraC transcriptional regulator
SsfS1
SsfS2
SsfS3
SsfS4
SsfS5
SsfS6
SsfT1
SsfT2
SsfU
SsfV
SsfX1
SsfX2
SsfX3
SsfY1
SsfY2
SsfY3
SsfY4
Orf-1
Orf-2
Orf-3
Orf1
ChlI
RemJ
AviX9
OxyK
NcnE
CmmQ
OxyI
hypothetical protein SGR_5601
SSEG_08767
SSEG_03816
hypothetical conserved protein
hypothetical protein SCO3803
SSEG_08979
Orf2
Orf3
those found in the chlorothricin28 or polyketomycin29 pathways.
Sequence analysis of ssf gene cluster revealed a likely mech-
anism of salicylate 19 synthesis from the shikimate pathway
(Scheme 2), similar to that utilized in the biosynthesis of
siderophores yersinobactin30 and mycobactin.31 SsfH was found
to be homologous to the salicylate synthase genes Irp930 and
(28) Jia, X.-Y.; Tian, Z.-H.; Shao, L.; Qu, X.-D.; Zhao, Q.-F.; Tang, J.;
Tang, G.-L.; Liu, W. Chem. Biol. 2006, 13, 575–585.
(29) Daum, M.; Peintner, I.; Linnenbrink, A.; Frerich, A.; Weber, M.;
Paululat, T.; Bechthold, A. ChemBioChem 2009, 10, 1073–1083.
(30) Kerbarh, O.; Ciulli, A.; Howard, N. I.; Abell, C. J. Bacteriol. 2005,
187, 5061–5066.
9
J. AM. CHEM. SOC. VOL. 131, NO. 48, 2009 17681

A R T I C L E S
Pickens et al.
Scheme 3
from the reaction mixture by HPLC and mass analysis confirmed
the identity ([M + H]+ at m/z ) 888, Supporting Information
Figure S8).
Figure 3. In vitro assay of SsfL1 activity and substrate specificity. (A)
Synthesis of salicylyl-AMP as indicated by the release of PPi in the presence
of ATP, salicylate 19 and the salicylyl-CoA ligase SsfL1. (i) all reaction
components; (ii) no 19; 9iii) no ATP; (iv) no SsfL1; and (v) pyrophosphate
reagent only. (B) Utilization of substituted benzoic acids by SsfL1 as
indicated by PPi release assay. The structures of compounds indicated by
number here are shown in Scheme 3.
For the ligation of 20 to 6, no clear enzyme candidate emerged
during the bioinformatic analysis. One of the initially unassigned
enzymes, SsfX3, has high sequence homology to only one
enzyme from a published natural product biosynthetic pathway,
which is AviX9 from the avilamycin gene cluster (56%/44%
similarity/identity). However, the role of AviX9 in avilamycin
biosynthesis has not been identified.35 Several proteins with
lower homology to SsfX3 were reported as hypothetical proteins
in the GDSL lipase/acyl-hydrolase family (PF00657). Charac-
teristic features of this class of acyltransfer enzymes are (i) the
active site motif GDSL, (ii) the lack of a nucleophilic elbow
formed by the GXSXG motif of typical hydrolases, and (iii) a
flexible substrate binding pocket.36,37 Despite the low sequence
homology, residues strictly conserved in this family (Ser, Gly,
Asn, His)36 can be predicted to be S174, G209, N236, and H338
in SsfX3. Residues which form the catalytic triad were also
identified by sequence homology and are predicted to be S174,
D333, and H338. Therefore, SsfX3 was considered as a potential
acyltransferase that may catalyze the acyltransfer of either 20
or an activated form of angelate during the biosynthesis of 1.
To probe the potential role of SsfX3, we first assayed the
reverse hydrolysis reaction using different products. 1 was
treated with purified SsfX3 overnight, and the assay mixture
was analyzed by LC/MS. The result showed the presence the
parent compound 1 and a second compound with mass corre-
sponding to the loss of 19 ([M + H]+ at m/z ) 658, Supporting
Information Figure S9). A control reaction without SsfX3 did
not afford this compound, as determined by selected ion
monitoring. The UV of this compound showed λmax of 358 nm
and the loss of the shoulder at 302 nm, consistent with the
hydrolysis of 19. Despite the low conversion, this result was
the first indication that SsfX3 may be involved in C-4 salicy-
lation. To examine if the intermediate 7 may be the endogenous
product of SsfX3, we repeated the same hydrolysis reaction
using purified 7. Analysis of the product mixture showed that
nearly all 7 was converted to 6 in the presence of SsfX3, while
no 6 was recovered in the absence of the enzyme (Supporting
Information Figure S9). To confirm the SsfX3-catalyzed acyl-
transfer reaction, an in vitro assay was performed in which
SsfX3 and SsfL1 were added to 6 with 19, ATP, and free CoA.
MtbI31 from yersinobactin and mycobactin biosynthesis respec-
tively, which convert chorismate 18, a byproduct of the
shikimate pathway, to 19 in a two step reaction that proceeds
through an isochorismate intermediate.30 In addition, the ssf gene
cluster contains ssfI which encodes 3-deoxy-d-arabino-heptu-
losonate-7-phosphate (DAHP) synthase that condenses eryth-
rose-4-phosphate 16 and phosphoenolpyruvate 15 into DAHP
17,32 which is the first step in the shikimate pathway. Genes
encoding the remainder of the shikimate pathway that converts
17 to 18 are absent from the ssf gene cluster and are likely shared
with the endogenous metabolism of Streptomyces sp. SF2575.
The ssfI gene is therefore likely an extra copy dedicated for the
biosynthesis of 1 and serves to direct carbon flow through the
shikimate pathway when SF2575 biosynthesis is induced.
Genes Involved in the Transfer of Salicylate to C-4. Attach-
ment of 19 to 6 is proposed to occur through an ATP-dependent
activation of 19 to salicylyl-CoA 20 by a salicylyl-CoA ligase,
and subsequent transfer catalyzed by an acyltransferase. SsfL1
has high sequence homology to NcsB233 and MdpB2,34 which
catalyze formation of the CoA-ester of naphthoic acid and 3,6-
dimethylsalicylic acid in the enediyne biosynthetic pathways
of neocarzinostatin and maduropeptin, respectively. To verify
that SsfL1 is involved in the activation of 19, the enzyme was
expressed from Escherichia coli BL21(DE3) as an N-terminal
hexahistidine tag fusion protein and purified using Ni-NTA
chromatography (∼5 mg/L, Supporting Information Figure S7).
ATP and 19 were added to SsfL1 and the release of pyrophos-
phate (PPi) was monitored spectrophotometrically at 340 nm
using a coupled assay that oxidizes NADH. As shown in Figure
3A, significant PPi release was observed only in the presence
of SsfL1, ATP and 19. To demonstrate the CoA ligase properties
of SsfL1, enzymatic synthesis of 20 was performed by introduc-
ing CoA into the assay. After overnight incubation, a new
compound consistent with the retention time of 20 was purified
(31) Zwahlen, J.; Kolappan, S.; Zhou, R.; Kisker, C.; Tonge, P. J.
Biochemistry 2007, 46, 954–964.
(35) Weitnauer, G.; Mu¨hlenweg, A.; Trefzer, A.; Hoffmeister, D.; Su¨ssmuth,
R. D.; Jung, G.; Welzel, K.; Vente, A.; Girreser, U.; Bechthold, A.
Chem. Biol. 2001, 8, 569–581.
(32) Knaggs, A. R. Nat. Prod. Rep. 1999, 16, 525–560.
(33) Cooke, H. A.; Zhang, J.; Griffin, M. A.; Nonaka, K.; Van Lanen, S. G.;
Shen, B.; Bruner, S. D. J. Am. Chem. Soc. 2007, 129, 7728–7729.
(34) Van Lanen, S. G.; Oh, T.-j.; Liu, W.; Wendt-Pienkowski, E.; Shen,
B. J. Am. Chem. Soc. 2007, 129, 13082–13094.
(36) Akoh, C. C.; Lee, G.-C.; Liaw, Y.-C.; Huang, T.-H.; Shaw, J.-F. Prog.
Lipid Res. 2004, 43, 534–552.
(37) Upton, C.; Buckley, J. T. Trends Biochem. Sci. 1995, 20, 178–179.
9
17682 J. AM. CHEM. SOC. VOL. 131, NO. 48, 2009

Biosynthetic Pathway for Anticancer Tetracycline SF2575
A R T I C L E S
acids (27-29), as well as 4-aminosalicylic acid 34 were
significantly better substrates than 19, indicating substantial
plasticity in the binding pocket of SsfL1.
Acyltransferases from the GSDL family are often found to
have broad substrate specificity, which is proposed to be the
result of a flexible substrate binding pocket.36 Therefore, SsfX3
may be similarly tolerant and use different aryl-CoAs as
substrates. Given nearly all substrates shown in Scheme 3 can
be activated by SsfL1, we tested whether new variants of 7 can
be synthesized in a coupled assay using both SsfL1 and SsfX3.
To do so, 2 mM of each analogue was combined with SsfL1
(15 µM), and SsfX3 (1.5 µM) in an in vitro assay containing 2
mM free CoA, 2 mM ATP, 10 mM MgCl2, and 20 µM 6 in 50
mM HEPES buffer pH 7.9. The reaction was incubated at room
temperature for 30 min and analyzed by LC/MS for emergence
of analogues of 7 (Figure 4B). The CoA-activated forms of the
dihydroxybenzoic acids 27-29 were readily recognized by
SsfX3 and led to the synthesis of new analogues of 7. This
demonstrates that the additional hydroxyl group is well tolerated
at all three positions and this finding may be used to produce
more hydrophilic derivatives of 1. However, the trihydroxy-
benzoic acid 30, which was a poor substrate in the SsfL1
activation assay, only afforded trace amounts of the adduct in
the coupled assay as revealed by selected ion monitoring. The
coupled assay also reveals SsfL1 and SsfX3 have significant
differences in substrate tolerance. Whereas 34 and 35 were each
well-activated by SsfL1, neither compound was transacylated
to 6 by SsfX3. To test if SsfX3 can accept membrane permeable
aryl thioesters, we mixed benzoyl-S-N-acetylcysteamine (ben-
zoyl-SNAC) with 6 and SsfX3. The resulting extract showed
that the C-4 benzoyl analogue of 7 was synthesized in similar
yield as that using 26 and CoA in the coupled-enzyme system.
This demonstrated SsfX3 also has relaxed substrate tolerance
toward the acyl carrier. Taken together, these results indicate
SsfL1 and SsfX3 are attractive enzymes in the biocatalytic
synthesis of analogues of 1 for structural-activity relationship
studies. Mutasynthesis of 1 containing different substituents at
C-4 may also be accomplished by inactivating the endogenous
salicylic biosynthetic pathways, such as deletion of ssfH.
Figure 4. In vitro assay of SsfX3 activity and substrate specificity. (A)
The tandem actions of SsfL1 and SsfX3 transfer 19 to the aglycon substrate
6 to yield 7. The assays are performed in 50 mM HEPES, pH 7.9 and 10
mM MgCl2. (i) the semisynthetic 6 standard; (ii) the semisynthetic 7
standard; (iii) Complete reaction containing 50 mM HEPES pH 7.9, 10
mM MgCl2, 2 mM ATP, 2 mM free CoA, 2 mM 19, 20 µM 6, 1.5 µM
SsfX3 and 15 µM SsfL1. Control reactions were performed as (iii) with
the following exclusions: (iv) no SsfL1; (v) no ATP; (vi) no 19; (vii) no
SsfX3; and (viii) no CoA. The reactions were examined with HPLC (358
nm). (B) Synthesis of analogues of 7 using salicylic acid analogues, SsfL1
and SsfX3. All reactions were performed at 25 °C for 30 min, extracted
with organic solvent and analyzed by LC/MS (358 nm, positive ionization).
Genes Putatively Involved in the Biosynthesis of Angelyl-
CoA 25. Little has been reported on the biosynthesis of angelic
acid among bacterial natural products. Angelate is commonly
found to be associated with plant metabolites and is biosyn-
thesized from the isoleucine catabolic pathway.38,39 No genes
were found in the ssf cluster that indicate a plant-like pathway
for this unusual substitution in 1. Instead, a set of genes encoding
enzymes involved in the formation and tailoring of a short-
chain acyl group was found. These genes include SsfE (car-
boxytransferase), SsfJ (enoyl-CoA hydratase/isomerase), SsfN
(KSIII), and SsfK (3-ketoacyl-ACP reductase). These genes
resemble those found in fatty acid biosynthesis and may be
responsible for synthesizing angelyl-CoA 25 from a propionyl-
CoA 21, or other short-chain acyl building blocks. One possible
pathway is shown in Scheme 2.
The reaction was incubated for 30 min at room temperature
and analyzed by HPLC (Figure 4A). The reaction containing
all components showed nearly quantitative conversion of 6 to
7 (Figure 4A, trace iii). Omission of any of the enzyme,
substrate, or cofactor component of the assay did not lead to
the synthesis of 7, as shown in Figure 4A, traces iv-viii.
Together, these results confirmed that SsfX3 is responsible for
the conversion of 6 to 7 using 20 as the acyl donor.
Substrate Specificity of SsfL1 and SsfX3. Exploiting the
substrate tolerance of SsfX3 and SsfL1 may be useful toward
diversifying the C-4 functionality of 1. To probe this, differently
substituted benzoic acids (Scheme 3) were tested for activation
and acyltransfer by SsfL1 and SsfX3, respectively. Since SsfL1
is the initial gatekeeper in these reactions, the different substrates
were first examined for recognition by SsfL1 using the PPi
release assay as described above (Figure 3B). Interestingly,
nearly all the substrates examined showed levels of PPi release
above background (except 33), demonstrating SsfL1 has relaxed
substrate specificity. The 2-OH substituent is clearly not essential
for binding, as benzoic acid 26, 2-chlorobenzoic acid 31 and
2-methoxy benzoic acid 35 all supported similar rates of
pyrophosphate release. Surprisingly, three dihydroxybenzoic
SsfE contains a carboxyltransferase domain (PF01039) and
is homologous to biotin-dependent methylmalonyl-CoA decar-
boxylase and propionyl-CoA carboxylase. Several of these
homologues are present in polyketide gene clusters such as
(38) McGaw, B. A.; Woolley, J. G. Phytochemistry 1979, 18, 1647–1649.
(39) Seigler, D. S. Plant Secondary Metabolism; Kluwer Academic
Publishers: Norwell, MA, 1998; p 759.
9
J. AM. CHEM. SOC. VOL. 131, NO. 48, 2009 17683

A R T I C L E S
Pickens et al.
chlorothricin28 (ChlJ), jadomycin40 (JadN), simocyclinone24
(SimA12), medermycin41 (med-ORF22), and others. One pos-
sible role for SsfE is to synthesize methylmalonyl-CoA 22 from
21. The next set of steps in the biosynthesis of 25 are proposed
to be catalyzed by ssfK, ssfN, and ssfJ, which are cotranscribed
as a tricistronic cassette on the same operon. SsfN is homologous
to KS III enzymes such as FabH, which catalyzes the condensa-
tion between acetyl-CoA and malonyl-ACP to initiate fatty acid
biosynthesis.42 FabH homologues have been found in a number
of polyketide gene clusters such as in R1128,43 frenolicin,43
and daunorubicin44 to catalyze the decarboxylative condensation
of short acyl groups. SsfN may therefore catalyze the condensa-
tion of 22 with acetyl-CoA to form 2-methyl-acetoacetyl-CoA
23. ꢀ-Ketoreduction of 23 catalyzed by SsfK, which is
homologous to 3-oxoacyl-ACP reductases such as FabG,45
yields 3-hydroxyl-2-methyl-butyryl-CoA 24. Stereospecific de-
hydration of 24 by SsfJ, which is a member of the enoyl-CoA
hydratase/isomerase family (PF00378), results in 25. As an
alternative start to this pathway, SsfN may directly condense
malonyl-CoA with acetyl-CoA to form acetoacetyl-CoA. The
R-methyl group may then be incorporated by SsfM3, which is
homologous to C-methyltransferases, to afford 23.
Transfer of 25 to 7 is predicted to be catalyzed by an
acyltransferase using similar mechanisms as for other acylated
deoxysugars present in chromomycin46 or mannopeptimycin.47
Those belong to a large acyltransferase family (PF01757) that
contains proteins with O-acetyltransferase activity, including the
deoxyhexose O-acyltransferases MdmB.48 Unexpectedly, no
similar acyltransferase is present in the ssf gene cluster. One
acyltransferase homologue, SsfV, however, remains unassigned.
SsfV has homologues in many type II polyketide gene clusters
including OxyP from the oxy pathway and ZhuC from the R1128
pathway. A Pfam homology search revealed that it contains an
acyltransferase domain (PF00698) from a family of proteins that
includes bacterial malonyl-CoA-ACP transacylase (MAT). In
vitro experiments with homologue ZhuC showed that it did
indeed have malonyl-CoA ACP transacylase activity, however
it was much slower than endogenous MAT from the fatty acid
biosynthetic pathway.49,50 Additional in vitro studies demon-
strated that ZhuC could efficiently hydrolyze acetyl and pro-
pionyl primed ACPs and therefore acts to ensure that the R1128
PKS is primed with the correct medium chain length acyl-
ACPs.50 Similarly, SsfV may be involved in ensuring the
biosynthesis of 1 is initiated with malonamyl starter unit and is
hence unlikely to be involved in angelate attachment.
biosynthetic cluster.28 The role of these genes in the processing
and attachment of the 5-chloro-6-methyl-O-methylsalicylic acid
during chlorothricin biosynthesis has recently been published.51
ChlB3 is utilized to transfer 6-methylsalicylic acid from the type
I PKS ChlB1 to a discrete ACP for further tailoring. ChlB6 is
shown to transfer the tailored 5-chloro-6-methyl-O-methylsali-
cylic acid to D-olivose of desmethylsalicyl chlorothricin to form
chlorothricin.51 Considering SsfG displays high sequence simi-
larity to ChlB6 (52%) and the same deoxysugar substrate
D-olivose is involved, SsfG therefore likely catalyzes the
attachment of angelate in the ssf pathway. Instead of using a
discrete ACP as the acyl carrier, the CoA-activated angelate 25
may directly serve as the substrate of SsfG.
Genes Encoding Enzymes That Synthesize the Polyketide
Backbone 40. The ssf minimal PKS consists of SsfA (KSR), SsfB
(KSꢀ or CLF), and SsfC (ACP) that are highly homologous to
the oxy minimal PKS.18 The amidotransferase SsfD, which is
responsible for producing the malonamate starter unit unique
to tetracyclines, is found adjacent to the ssf minimal PKS. The
C-2 amidated starter unit is one of the signature moieties of
tetracyclines, and the revelation of SsfD as an OxyD homologue
with 68% sequence identity was one of the earliest convincing
pieces of evidence that this gene cluster indeed encodes a
tetracycline compound. Like OxyD, SsfD is highly similar to
class II asparagine synthases that utilize ATP to convert aspartate
to asparagine using glutamine as the nitrogen source.52 Zhang
and co-workers demonstrated that the “extended minimal PKS”,
which includes OxyABCD, is sufficient for producing the
amidated polyketide chain 38.18 In addition, SsfU, which is 76%
identical to OxyJ, is predicted to be the C-8 (or C-9* using
biosynthetic carbon numbering as shown in Scheme 4) ketore-
ductase that regioselectively reduces the nascent backbone to
yield 40.20
To verify the function of the ssf minimal PKS, genes encoding
SsfA, SsfB and SsfC, SsfD, and SsfU were placed into a pRM5-
derived Streptomyces-E. coli shuttle vector53 (Table 2). Shuttle
plasmid pLP27 was subsequently transformed into S. liVidans
strain K4-114.54 The resulting organic extract was analyzed by
HPLC and is shown in Figure 5A. The product profile of K4-
114/pLP27 was indistinguishable from that of K4-114/pWJ35,
which contained the corresponding enzymes from the oxy
biosynthetic pathway.18 By comparison to an authentic standard,
the major product of the extract (RT ) 13.5 min) was confirmed
to be the isoquinolone 42 (20 mg/L), which can form via the
spontaneous cyclization of the C-9* reduced amidated polyketide
40.18 We further investigated whether the oxy and ssf extended
minimal PKS components are functionally interchangeable.
Three additional shuttle vectors were prepared with a combina-
tion of oxy and ssf genes as shown in Table 2 and transformed
into K4-114. Each of these host/vector combinations was
capable of producing 42 in similar quantities as K4-114/pLP27,
indicating that each of the ssf components is functionally
compatible and equivalent to its oxy counterpart. These results
confirm (i) the extended ssf minimal PKS (SsfABCD) is capable
of synthesizing the full length amidated polyketide precursor
38, (ii) the hypothesis that the carbon backbone of 1 is
The ssf gene cluster encodes another KS III homologue SsfG,
which resembles ChlB3 and ChlB6 from the chlorothricin
(40) Wang, L.; McVey, J.; Vining, L. C. Microbiology 2001, 147, 1535–
1545.
(41) Ichinose, K.; Ozawa, M.; Itou, K.; Kunieda, K.; Ebizuka, Y. Micro-
biology 2003, 149, 1633–1645.
(42) Magnuson, K.; Jackowski, S.; Rock, C. O.; Cronan, J. E. Microbiol.
ReV. 1993, 57, 522–542.
(43) Tang, Y.; Lee, T. S.; Kobayashi, S.; Khosla, C. Biochemistry 2003,
42, 6588–6595.
(44) Grimm, A.; Madduri, K.; Ali, A.; Hutchinson, C. R. Gene 1994, 151,
1–10.
(45) Heath, R. J.; Rock, C. O. J. Biol. Chem. 1996, 271, 1833–1836.
(46) Mene´ndez, N.; Nur-e-Alam, M.; Bran˜a, A. F.; Rohr, J.; Salas, J. A.;
Me´ndez, C. Chem. Biol. 2004, 11, 21–32.
(51) He, Q.-L.; Jia, X.-Y.; Tang, M.-C.; Tian, Z.-H.; Tang, G.-L.; Liu, W.
ChemBioChem 2009, 10, 813–819.
(47) Magarvey, N. A.; Haltli, B.; He, M.; Greenstein, M.; Hucul, J. A.
Antimicrob. Agents Chemother. 2006, 50, 2167–2177.
(48) Hara, O.; Hutchinson, C. R. J. Bacteriol. 1992, 174, 5141–5144.
(49) Meadows, E. S.; Khosla, C. Biochemistry 2001, 40, 14855–14861.
(50) Tang, Y.; Koppisch, A. T.; Khosla, C. Biochemistry 2004, 43, 9546–
9555.
(52) Boehlein, S. K.; Richards, N. G.; Schuster, S. M. J. Biol. Chem. 1994,
269, 7450–7457.
(53) McDaniel, R.; Ebert-Khosla, S.; Hopwood, D. A.; Khosla, C. Science
1993, 262, 1546–1550.
(54) Ziermann, R.; Betlach, M. C. Biotechniques 1999, 26, 106–110.
9
17684 J. AM. CHEM. SOC. VOL. 131, NO. 48, 2009

Biosynthetic Pathway for Anticancer Tetracycline SF2575
A R T I C L E S
9
J. AM. CHEM. SOC. VOL. 131, NO. 48, 2009 17685

A R T I C L E S
Pickens et al.
Table 2. Plasmids and Major Metabolites Produced by S. lividans
(67% sequence similarity) and is predicted to be responsible
for D ring cyclization to afford 43. SsfY4 is similar to OxyI,
itself similar to fourth ring cyclase MtmX from the mithramycin
pathway. However OxyI was found to be unnecessary in the
biosynthesis of 47.20 Although SsfY2 has low sequence homol-
ogy (25% similarity and 14.5% identity) to the second ring
cyclase (OxyN) from the oxy pathway, it displays stronger
homology to second ring cyclases from benzoisochromane-
quinone (BIQ) pathways such as granaticin (68% similarity, 56%
identity)56 and medermycin (66% similarity, 55% identity).41
SsfY2 is therefore predicted to be the second ring cyclase in
the ssf pathway. Intriguingly, an additional cyclase, SsfY3, also
displays sequence similarity to first ring cyclase/aromatases, with
42% similarity to CmmQ46 and 39% similarity to OxyK.
Completion of the biosynthesis of 49 requires C-6 methylation
of 47, which is catalyzed by the OxyF homologue SsfM4.
To deduce the role, or lack thereof, of each of these cyclases
in the biosynthesis of 49, we heterologously expressed different
combinations of cyclases found in the ssf gene cluster with
SsfABCDU. We first constructed a pSET152-derived integrating
vector57 containing ssfY1 and cotransformed the resulting
plasmid pLP77 with pLP27 into K4-114. We detected the
complete disappearance of 42 in the extract and emergence of
a single major product that corresponds to the benzopyrone
WJ78 44 (Figure 5B), which is the shunt product of the amidated
polyketide intermediate 43 that has been regioselectively
cyclized at the D ring.58 Cotransformation of K4-114 with
pLP27 and an integrating vector containing ssfY3, did not lead
to the recovery of 44, indicating SsfY3 cannot substitute for
SsfY1 and likely serves a different role in the biosynthesis
of 1.
K4-114 Transformed with Each Plasmid
plasmid
ssf genes
oxy genes
major products
pLP24
ssfD
ssfDC
ssfABD
oxyABCJ
oxyABJ
oxyCJ
41,42
41,42
41,42
41,42
44
pLP25
pLP26
pLP27
ssfABCDU
pLP27/pLP77 ssfABCDUY1
pLP27/pLP113 ssfABCDUY1Y2M4
pLP27/pLP115 ssfABCDUY1Y2M4Y4
pLP27/pLP118 ssfABCDUY1Y2M4Y4Y3
PLP27/pLP126 ssfABCDUY1Y2M4L2
46
46
46
50
pLP36
pLP75
ssfO2
ssfO2
oxyABCDJKNF
oxyABCDJKNFQT
52
57
biosynthesized via a tetracycline-like pathway, and (iii) SsfU
as the C-9* KR that affords 40. Also detected from these extracts
was the acetate-primed product RM20b 41 (RT ) 17.7 min),
consistent with the previous report of oxy minimal PKS being
able to initiate polyketide biosynthesis with the acetate starter
unit.55
Genes Encoding Enzymes That Synthesize 6-Methylpretet-
ramid 49. Sequential cyclizations of the four rings are the next
steps following biosynthesis of the full length polyketide chain.
In the oxy cluster three cyclases were identified on the basis of
sequence homology; however, only two were shown to be
required to yield the intermediate pretetramid 47.20 Diverging
from the oxy cluster, the ssf gene cluster contains four genes
that have homology to cyclases. SsfY1 is highly similar to OxyK
We next set out to reconstitute the biosynthesis of 49 in K4-
114 using entirely ssf genes. SsfM4 was chosen to be the likely
C-6 methyltransferase because of its high sequence homology
to OxyF. The integrating vector pLP113 bearing ssfY1Y2M4
was cotransformed with pLP27 and resulting extract was
intensely yellow and contained the anthraquinone carboxylic
acid WJ83Q2a 46. This confirmed SsfY2 was responsible for
the cyclization of the second (or C) ring, while the third ring is
likely spontaneously cyclized. However, to our surprise, the
expected tetracyclic product 49, which can be isolated in the
oxidized form WJ119 50, was not found. This observation is
different from what was observed for the oxy pathway, in which
the equivalent set of oxy enzymes yielded 50.20 To determine
if the ssf pathway requires the additional cyclases found in its
gene cluster toward the formation of 47, pLP115 and pLP118
were constructed, which contained in addition to genes encoded
in pLP113, ssfY4, and ssfY3Y4, respectively. K4-114 contrans-
formed with pLP27, and these constructs continued to produce
46 only, suggesting that for the ssf pathway, different enzyme(s)
may be needed to facilitate cyclization of the fourth ring. We
tested this possibility by inserting additional ssf genes juxtaposed
to the ssf cyclase genes in the cluster into K4-114 and examined
the metabolic profiles. Fortuitously, upon insertion of ssfL2 into
pLP113 to afford pLP126, and cotransformation of pLP27 and
Figure 5. Reconstitution of tetracycline intermediates using ssf genes
expressed in S. liVidans K4-114. (A) HPLC analysis (245 nm) of the K4-
114/pLP27 extract shows the amidated, reduced polyketide 42 is the major
product, confirming the biosynthesis of the polyketide backbone 40 by
SsfABCD and the subsequent C-9* reduction by SsfU. (B) HPLC analysis
(253 nm) of the K4-114/pLP27/pLP77 extract shows addition of the putative
cyclase SsfY1 leads to complete cyclization and aromatization of the D
and formation of the shunt benzopyrone 44. (C) HPLC analysis (430 nm)
of K4-114/pLP27/pLP126 extract shows 50, the oxidized form of 49, as
the dominant product. Biosynthesis of 49 using entirely ssf genes
(SsfABCDUY1Y2M4L2) indicates the tetracycline nature of the ssf
biosynthetic pathway. (D) HPLC analysis (395 nm) of K4-114/pLP36 extract
confirms the function of SsfO2 as the oxygenase that dihydroxylated C-4
and C-12a of 49. The resulting product 51 undergoes spontaneous
degradation to afford the observed product 52.
(55) Fu, H.; Ebert-Khosla, S.; Hopwood, D. A.; Khosla, C. J. Am. Chem.
Soc. 1994, 116, 6443–6444.
(56) Ichinose, K.; Bedford, D. J.; Tornus, D.; Bechthold, A.; Bibb, M. J.;
Peter Revill, W.; Floss, H. G.; Hopwood, D. A. Chem. Biol. 1998, 5,
647–659.
(57) Bierman, M.; Logan, R.; O’Brien, K.; Seno, E. T.; Nagaraja Rao, R.;
Schoner, B. E. Gene 1992, 116, 43–49.
(58) Zhang, W.; Wilke, B. I.; Zhan, J.; Watanabe, K.; Boddy, C. N.; Tang,
Y. J. Am. Chem. Soc. 2007, 129, 9304–9305.
9
17686 J. AM. CHEM. SOC. VOL. 131, NO. 48, 2009

Biosynthetic Pathway for Anticancer Tetracycline SF2575
A R T I C L E S
pLP126 into K4-114, the formation of 50 was detected as shown
in Figure 5C. SsfL2 has homology to long-chain fatty acyl-
CoA ligases as well as putative acyl-CoA ligases from several
aromatic polyketide pathways such as CmmLII from the
chromomycin gene cluster,46 PokL from the polyketomycin gene
cluster,29 MtmL from the mithramycin gene cluster,59 as well
as OxyH.18 The role of these enzymes in their respective
biosynthetic pathways has not been determined and has been
suggested to function as an accessory protein.46 Interestingly,
biosyntheses of these compounds all require the equivalent
Claisen cyclization of the A rings. The mechanism of SsfL2 in
the heterologous biosynthesis of 49 with ssf PKS components
is yet to be determined.
Tailoring Enzymes Putatively Involved in the Production
of the Aglycon 8. During biosynthesis of 4, the ring A of 49 is
doubly hydroxylated by OxyL at C-12a and C-4 to produce the
cyclohexenone-containing 51, which is unstable and degrades
into observed product WJ135 52.19 SsfO2, which is found at
the boundary of the ssf gene cluster, is an OxyL homologue,
and is most likely to perform this role in the ssf pathway.
Compound 51 is likely the last common intermediate between
the oxy and the ssf biosynthetic pathways. In the oxy pathway,
reductive amination of the C-4 ketone group by OxyQ to yield
4-amino-ATC 56 and subsequent dimethylation by OxyT afford
the stable intermediate anyhydrotetracycline 57.19 The C-4
position in 1 lacks the dimethylamino group and is instead
acylated with a salicylic acid group. This requires ketoreduction
of 51 at C-4 to produce 4-hydroxyl-ATC 53 for the downstream
salicylation reaction.
To determine whether this hydroxyl is directly installed by
SsfO2 or whether an additional ketoreduction is required, the
oxyL gene was replaced with ssfO2 in the shuttle plasmid
pWJ135, which contains all the oxy genes required to synthesize
51 (Table 2).19 K4-114 transformed with the resulting plasmid
pLP36 produced the tricyclic ketone 52 (Figure 5D), which is
a spontaneously degraded product of 51 as observed previ-
ously.19 To further confirm that the product of SsfO2 contains
the C-4 ketone functionality as in 51, we examined if SsfO2
can complement the role of OxyL in the biosynthesis of 57,
which requires reductive amination of the C-4 ketone in 51.
The genes oxyQ and oxyT were introduced to pLP36 to afford
pLP75 (Table 2). As expected, K4-114/pLP75 produced ∼10
mg/L of 57 as the dominant product, demonstrating that OxyL
and SsfL2 catalyze the identical transformation of 49 to 51.
These results support the proposal that a dedicated C-4
ketoreductase is required to reduce 51 to 53. SsfF is a
ketoreductase with sequence homology to NADPH-dependent
oxidoreductases with specificity for aromatic and nonpolar
substrates (PF00248) and is therefore assigned as a candidate
to catalyze this step.
opposite that of OxyS, based on X-ray crystallographic structure
of 1.8 Two O-methyltransferases are then needed to methylate
the C-6 and the C-12a hydroxyl groups. Three methyltrans-
ferases (other than the C-6 methyltransferase SsfM4) are present
in the ssf gene cluster. SsfM1 and SsfM2 bear strong resem-
blance to O-methyltransferases, while SsfM3 is similar to both
O-methyltransferases and C-methyltransferases. The C12a-OH
methylation modification can take place either prior to SsfO1
oxidation (using either 51 or 53 as substrate) or after C-6
oxidation (as shown in Scheme 4). The gene encoding SsfM2
is transcribed from the same operon that encodes ssfO1 and is
therefore the likely C-6 O-methyltransferase. In the same operon
is the gene ssfQ, which encodes an S-adenosylmethionine (SAM)
synthases and likely serves in an auxiliary role to produce
sufficient SAM for the methyltransfer reactions. It is known
that 6-deoxy analogues of tetracyclines, such as doxycycline,
have enhanced stability toward acid and base degradation.62 The
C-6-OH methylation may have a similar stabilizing effect on
1. Once identified, the C-6 O-methyltransferase may potentially
be useful to generate C-6-methoxytetracycline analogues that
have enhanced stability.
The last step in the biosynthesis of 4 is the reduction of
5a,11a-dehydrooxytetracycline 59 to 4. This reductive modi-
fication required for the biosynthesis of 4 and 5 has been
postulated to involve a gene located outside their respective
gene clusters.63 The tchA gene encodes a flavin-dependent
oxidoreductase with reported cofactor 7,8-didemethyl-8-
hydroxy-5-deazariboflavin (FO) and has been identified in
S. aureofaciens to be involved for the biosynthesis of 5.64
An identical reduction step is also needed to complete the
biosynthesis of aglycon 8 from, for example, 55. One
candidate for this reaction in the ssf gene cluster is a putative
dehydrogenase SsfP. SsfP shares 54% sequence similarity
to a putative F420-dependent methylenetetrahydromethanop-
terin reductase, but shares no sequence resemblance to TchA.
Experimental evidence will be needed to determine if this
reduction step is catalyzed by an enzyme encoded in the ssf
gene cluster such as SsfP or if an external gene is required
as in chlorotetracycline biosynthesis.
Genes Encoding Regulatory and Resistance Determinants.
The ssf gene cluster encodes two regulatory proteins, SsfT1 and
SsfT2, and a resistance protein SsfR. SsfT2 is predicted to be
a TetR family65 repressor protein with a DNA-binding
helix-turn-helix motif at the N-terminus. SsfR is an efflux
protein from the major facilitator superfamily (MFS) class of
ATP-independent transporters which utilizes a chemiosmotic
gradient to transport small molecules (PF07690).66 The SsfT2
gene is located directly adjacent to ssfR with opposite polarity.
Therefore the transcription of ssfR may be controlled by SsfT2
in a similar fashion as the TetR/TetA pair.65 The second
regulatory protein SsfT1 is a positive regulator in the ssf gene
cluster, and is predicted to be a member of the pathway-specific
The last tailoring enzyme in the ssf pathway that shares
resemblances to enzymes found in oxy pathway is SsfO1 which
has 54% similarity to OxyS. OxyS is known to be responsible
for the C-6 hydroxylation of 57 to form 5a,11a-dehydrotetra-
cycline 58,60,61 and SsfO1, therefore, likely plays the parallel
role to oxidize the aromatic C-ring in 53 to 54. SsfO1, however,
most likely catalyzes the C-6 hydroxylation with stereochemistry
(62) Brown, J. R.; Ireland, D. S. Structural Requirements for Tetracycline
Activity. In AdVances in Pharmacology and Chemotherapy; Garattini,
S., Goldin, A., Hawking, F., Kopin, I. J., Eds.; Academic Press: New
York, 1978; Vol. 15, pp 161-202.
(63) Butler, M. J.; Friend, E. J.; Hunter, I. S.; Kaczmarek, F. S.; Sugden,
D. A.; Warren, M. Mol. Gen. Genet. 1989, 215, 231–238.
(64) Nakano, T.; Miyake, K.; Endo, H.; Dairi, T.; Mizukami, T.; Katsumata,
R. Biosci. Biotechnol. Biochem. 2004, 68, 1345–1352.
(59) Prado, L.; Lombo´, F.; Bran˜a, A. F.; Me´ndez, C.; Rohr, J.; Salas, J. A.
Mol. Gen. Genet. 1999, 261, 216–225.
(65) Ramos, J. L.; Martinez-Bueno, M.; Molina-Henares, A. J.; Teran, W.;
Watanabe, K.; Zhang, X.; Gallegos, M. T.; Brennan, R.; Tobes, R.
Microbiol. Mol. Biol. ReV. 2005, 69, 326–356.
(60) Binnie, C.; Warren, M.; Butler, M. J. J. Bacteriol. 1989, 171, 887–
895.
(61) Vancurova´, I.; Volc, J.; Flieger, M.; Neuzil, J.; Novotna´, J.; Vlach,
J.; Beˇhal, V. Biochem. J. 1988, 253, 263–267.
(66) Pao, S. S.; Paulsen, I. T.; Saier, M. H., Jr. Microbiol. Mol. Biol. ReV.
1998, 62, 1–34.
9
J. AM. CHEM. SOC. VOL. 131, NO. 48, 2009 17687

A R T I C L E S
Pickens et al.
mL baffled Erlenmeyer flask at 30 °C and 250 rpm for 3 days.
One milliliter of precultures were inoculated on plates (4 L) of
modified Bennett’s agar in 150 mm × 10 mm Petri dishes and
incubated at 30 °C for 8 days. The cultured agar was harvested
and extracted with the same volume of ethyl acetate twice. Organic
solvent was dried in Vacuo to obtain 875.2 mg of oily crude extract.
The crude extract was fractionated with an open silica column
(200-430 mesh, 50 mm i × 300 mm) using a methanol and
chloroform gradient. Fractions containing 1 were collected and
purified by preparative HPLC to obtain 22 mg of 1. HPLC
purification was performed using an HPLC Alltech Alltima reverse-
phase column (5 µm, 10 mm × 250 mm) with an isocratic gradient
of 75% CH3CN in water (0.1% TFA).
Preparation of Authentic Standards 6 and 7. 7 was prepared
by a 2-h incubation of a 10 mg/mL solution of 1 with 0.5 M NaOH.
Complete conversion of 1 to 6 was obtained by incubation of a 10
mg/mL solution of 1 in 1.0 M NaOH for 15 h. To isolate 6 and 7,
the reaction mixture for each was adjusted to pH 7 by addition of
HCl, and the products were extracted with ethyl acetate/acetic acid
(99%/1%). Following the evaporation of the solvent, the product
was resuspended in methanol and purified by HPLC (Alltech
Alltima reverse-phase column 5 µm, 10 mm × 250 mm) with an
isocratic gradient of 78% CH3CN in water (0.1% TFA). Purified 6
and 7 were characterized by LC/MS, HRMS, and proton NMR (see
Supporting Information).
Streptomyces antibiotic regulatory protein (SARP) family of
transcriptional activators.67 These activators have been the
subject of metabolic engineering efforts as evidenced by the
recent publication on the fredericamycin pathway which dem-
onstrated a greater than 5 fold increase in antibiotic yield by
overexpression of FdmR1.68
Conclusions
We have identified the ssf biosynthetic gene cluster and
biochemically characterized key steps in the biosynthesis of 1.
Biosynthesis of 1 from the aglycon 8 requires a new set of
tetracycline-tailoring enzymes including the C-9 glycosyltrans-
ferase SsfS6 and the C-4 salicylyl transferase SsfX3. In vitro
assays with SsfL1 and SsfX3 have shown that both enzymes
have relaxed substrate flexibility toward different aromatic
substrates, a feature that was exploited in the chemoenzymatic
synthesis of several analogues of 7. Using heterologous bio-
synthesis we have also verified the tetracycline nature of the
gene cluster by reconstituting the biosynthesis of key intermedi-
ates. The identification of the ssf gene cluster and formulation
of a putative biosynthetic pathway will therefore enable
engineered biosynthesis of new tetracycline analogues.
Bioactivity Assays. Cell proliferation was determined by (3-
(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sul-
fophenyl)-2H-tetrazolium) (MTS) assay for adherent HeLa, MCF-
7, and M249 cells. Five thousand cells per well (4000 HeLa) were
plated in 100 µL media in a 96-well plate and allowed to incubate
for 18 h. Following incubation, 10 mM stock solution of 1, 6, or 7
solubilized in DMSO was added to media to prepare dilutions
containing three times the final concentration. Fifty microliters of
the appropriate dilution was added to each well. Cell proliferation
was assayed 72 h after treatment using CellTiter 96 AQueous
OneSolution cell proliferation assay reagent (Promega). Direct cell
counting was used for Nalm-6 suspension cells by Vi-CELL
analyzer (Beckman). In a 24-well plate were plated 400 µL cells
(500,000 cells/mL). After 20 h 400 µL of media containing twice
the desired final drug concentration was added. Cells were analyzed
72 h after treatment. All cells were cultured according to ATCC or
DSMZ guidelines. Prism (GraphPad) was used for analysis.
Identification and Sequencing of the ssf Gene Cluster. A
fosmid library containing Streptomyces sp. SF2575 genomic DNA
was prepared using the CopyControl Fosmid library production kit
from Epicenter. Fosmids were screened using degenerate primers
for the KSR gene as reported by Wawrik et al.21 Fosmids 5F15,
11A12 and 19B2 were sequenced by a combination of shotgun
sequencing and primer walking. ORFs were identified using
Frameplot software (http://www.nih.go.jp/∼jun/cgi-bin/frameplot.
pl), and functions of the encoded proteins were assigned by
sequence similarity using NCBI protein-protein BLAST. Pfam
protein family database was also used to identify functional
domains.70 The sequence has been deposited in the GenBank
database under accession number GQ409537.
Materials and Methods
Bacterial Strains and DNA Manipulation. Streptomyces sp.
SF2575 was obtained from Meiji Co. E. coli XL-1 Blue (Stratagene)
was used for the manipulation of plasmid DNA. E. coli strain
BL21(DE3) was used for protein expression. Fosmids 11A12 and
5F15 were used as templates to amplify DNA by PCR. PCR
fragments were cloned into pCR-Blunt vector obtained from
Invitrogen and sequenced (Laragen). All restriction enzymes were
purchased from New England Biolabs. PCR reagents for pfx DNA
polymerase were purchased from Invitrogen. PCR Primers are listed
in Supporting Information, Table S1. S. liVidans strain K4-11454
was used at a host for the transformation of shuttle vector constructs
containing ssf genes. Protoplast preparation and PEG-mediated
DNA transformation techniques were performed as published by
Hopwood et al.69
Spectroscopic Analysis. NMR of compounds was obtained on
a Bruker ARX400 at the University of California Department of
Chemistry and Biochemistry NMR facility. Accurate high resolution
mass spectrometry was performed at the University of California
Riverside Mass Spectrometry Facility. HPLC spectra were obtained
on a Beckman Gold HPLC using a reverse-phase C18 column
(Alltech Apollo C18 5 µm column; 250 mm × 4.6 mm) and a
linear gradient of 5% CH3CN in water (0.1% trifluoroacentic acid
[TFA]) to 95% CH3CN in water (0.1% TFA) in 30 min at a flow
rate of 1 mL/min. LC/MS spectra were obtained on a Shimadzu
2010 EV liquid chromatography mass spectrometer using positive
and negative electrospray ionization and a Phenomenex Luna 5 µm,
2.0 mm × 100 mm C18 reverse-phase column. Samples were
separated on a linear gradient of 5 to 95% CH3CN in water (0.1%
formic acid) for 30 min at a flow rate of 0.1 mL/min unless
otherwise noted.
Inactivation of ssfB gene. For ssfB (CLF) disruption, ap-
proximately 1 kb of the 5′ region of ssfB gene was amplified from
genomic DNA of Streptomyces sp. SF2575 by PCR using a forward
primer KS2-LEK-S which introduces an EcoRI site, and a reverse
primer KS2-LEK-A2 which introduces a KpnI site. The PCR
product obtained was cloned into pCR-Blunt vector to produce pCR-
KSII-LEK. Approximately 1 kb of the 3′ region of ssfB was also
amplified by PCR using primer set KS2-RPH-S and KS2-RPH-A
to add PstI and HindIII site to its 5′ and 3′ends, respectively. The
PCR product obtained was cloned to produce pCR-KSII-RPH. The
Cultivation of Streptomyces sp. SF2575 and Purification of
1. Streptomyces sp. SF2575 was cultured in R5 media for subculture
and seed culture. Modified Bennett’s agar (per 1 L, beef extract
1 g, bacto-peptone 2 g, yeast extract 1 g, glucose 10 g, agar 20 g)
was used as a production media. For isolation of 1, Streptomyces
sp. SF2575 was precultured in 50 mL of R5 liquid media in a 250
(67) Wietzorrek, A.; Bibb, M. Mol. Microbiol. 1997, 25, 1181–1184.
(68) Chen, Y.; Wendt-Pienkowski, E.; Shen, B. J. Bacteriol. 2008, 190,
5587–5596.
(69) Kieser, T.; Bibb, M. J.; Buttner, M. J.; Chater, K. F.; Hopwood, D. A.
Practical Streptomyces Genetics. John Innes Foundation: Norwich,
UK, 2000.
(70) Finn, R. D.; Tate, J.; Mistry, J.; Coggill, P. C.; Sammut, S. J.; Hotz,
H.-R.; Ceric, G.; Forslund, K.; Eddy, S. R.; Sonnhammer, E. L. L.;
Bateman, A. Nucleic Acids Res. 2008, 36, D281–288.
9
17688 J. AM. CHEM. SOC. VOL. 131, NO. 48, 2009

Biosynthetic Pathway for Anticancer Tetracycline SF2575
A R T I C L E S
1.1 kb of aphII gene was obtained from pFD-neoS plasmid.71
Plasmid pCR-KSII-LEK was digested with EcoRI and KpnI;
plasmid pCR-KSII-RPH was digested with PstI and HindIII.
Fragments were ligated together with KpnI-PstI digested aphII gene
into plasmid pKC1139 digested with EcoRI and HindIII to produce
about 9.5 kb of pKC-SF2-KSII. The construct was delivered into
Streptomyces sp. SF2575 by conjugation with E. coli ET12567-
(pUZ8002). Intergeneric conjugation between E. coli and Strepto-
myces was performed as described previously with minor modifica-
tion.57 The transformant was resistant to both apramycin and
kanamycin and was grown in fresh R2YE/kanamycin liquid medium
at 37 °C for 1-2 days in order to force chromosomal integration
of the gene-disruption constructs. The resulting disruption mutant
(Streptomyces sp. SF2575 ∆ssfB) was selected on kanamycin-
containing plates. Gene disruption in Streptomyces sp. SF2575
∆ssfB was confirmed by PCR of total genomic DNA. Streptomyces
sp. SF2575 ∆ssfB did not produce SF2575 or intermediates when
grown on Bennett’s plates as confirmed by LC/MS analysis.
Expression and Purification of Recombinant Enzymes. SsfX3
and SsfL1 were cloned into a pET28 vector with an N-terminal
His tag and expressed in E. coli BL21(DE3) strain. For each
enzyme, a 5 mL overnight culture of BL21(DE3) transformed with
expression plasmid was grown in LB medium containing 35 mg/L
kanamycin. This overnight culture was used to inoculate a 500 mL
flask of LB containing 35 mg/L kanamycin. Cultures were grown
at 37 °C until OD600 of 0.5 at which point IPTG was added to a
final concentration of 120 µM to induce protein expression. The
induced cultures were further shaken overnight at 16 °C.
Protein purification was carried out at 4 °C. Cell pellets were
resuspended in Buffer A (50 mM Tris-HCl, pH 7.9, 10 mM
imidazole and 50 mM NaCl). Cell membranes were disrupted by
sonication. Cell lysate was centrifuged at 16,000 rpm, and the
soluble fraction was collected and incubated with Ni-NTA resin
(Qiagen) for 2 h with gentle rotation. Protein-resin mixture was
added to a gravity flow column, and buffers containing increasing
step gradients of imidazole were applied. The target proteins were
eluted in Buffer A containing 250 mM imidazole. A 30 kDa MWCO
Amicon filtration column (Millipore) was used for buffer exchange
and concentration of the protein solution. Purified enzymes were
stored in Buffer B (50 mM Tris-HCl pH 7.9, 2 mM EDTA, 2 mM
DTT). Proteins were aliquoted, flash frozen on dry ice, and stored
at -80 °C. Protein concentrations were measured by Bradford assay
using bovine serum albumin (BSA) as a standard.72
In Vitro Assay. PPi assay reagent was purchased from Sigma
(P7275) and was performed according to manufacturer instructions.
Typical reaction mix includes: 33% PPi reagent, 100 mM Tris-
HCl pH 7.5, 2 mM ATP, 2 mM salicylic acid and 10 µM SsfL1.
Reaction progress was monitored at 340 nm using a BioTek
PowerWave XS plate reader for 30 min at room temperature. All
in vitro assays containing SsfX3 were performed with the following
conditions unless otherwise specified: 50 mM HEPES pH 7.9, 10
mM MgCl2, 2 mM ATP, 2 mM free CoA, 2 mM salicylic acid, 20
µM 6, 1.5 µM SsfX3 and 15 µM SsfL1. Reaction mixture was
incubated at room temperature for 30 min. A 100 µL reaction
volume was extracted with 200 µL acetonitrile (1% acetic acid).
Organic phase was then removed, and solvent was evaporated in
Vacuo. Samples were then resuspended in 30 µL methanol and
analyzed by HPLC or LC/MS as described in Spectroscopic
Analysis.
Heterologous Biosynthesis of Polyketides Using S. liWidans.
S. liVidans K4-114 strain54 transformed with different constructs
shown in Table 2 were grown on R5 agar plus 50 µg/mL
thiostrepton. Cultures were grown at 30 °C for approximately one
week until colonies were well formed. For HPLC analysis, colonies
were streaked out on 40 mL of fresh R5 agar (plus 50 µg/mL
thiostrepton) and incubated for 7-10 days until the plate was well
pigmented. The agar was then finely chopped and extracted with
40 mL of organic solvent (99% ethyl acetate, 1% acetic acid).
Sodium sulfate was added to the extract to remove residual water,
the solvent was evaporated, and the products were resuspended in
400 µL DMSO. The extracts were analyzed by reverse-phase HPLC.
Compounds were identified by matching retention time and UV
spectra to those of authentic standards.
Acknowledgment. This work is funded by NSF CBET 0545860
and CDMRP W81XWH-08-1-0614to Y.T.; and NIH Training Grant
1T32GM067555 to L.B.P. We thank Dr. Kathleen Sakamoto for
advice on the bioactivity assays and providing the Nalm-6 cell line
and Prof. Tatiana Segura for the gift of the HeLa, MCF-7, and
M249 cell lines. We thank Prof. Michael Jung and Dr. Wenjun
Zhang for helpful discussion.
Supporting Information Available: Additional experimental
procedures and compound characterizations. This material is
available free of charge via the Internet at http://pubs.acs.org.
(71) Denis, F.; Brzezinski, R. FEMS Microbiol. Lett. 1991, 81, 261–264.
(72) Bradford, M. M. Anal. Biochem. 1976, 72, 248–254.
JA907852C
9
J. AM. CHEM. SOC. VOL. 131, NO. 48, 2009 17689