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J. Biol. Chem., Vol. 278, Issue 37, 35552-35557, September 12, 2003
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From the
Department of Medicinal Chemistry and
Institute for Structural Biology and Drug Discovery, Virginia Commonwealth
University, Richmond, Virginia 23219 and
Antibiotics Laboratory, RIKEN, 2-1 Hirosawa,
Wako, Saitama 351-0198, Japan
Received for publication, May 14, 2003 , and in revised form, June 18, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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,
-unsaturated
-lactone and either a conjugated linear diene or triene chain
(1). Fostriecin
(Fig. 1) and the structurally
related PD 113,270 and PD 113,271 are three related natural products produced
by Streptomyces pulveraceus subsp. fostreus ATCC 31906
(2,
3). Fostriecin inhibits DNA
topoisomerase II (IC50, l40 mM)
(4) and is also a potent and
selective inhibitor of protein phosphatases 1
(PP1),1 PP2A, and PP4
(IC50, 45 mM, 1.5 nm, and 3.0 nm, respectively)
(1,
5). The latter activity is
attributed to the efficacious in vivo antitumor activity of
fostriecin, as well as its in vitro activity against leukemia, lung,
breast, and ovarian cancer. The same activity likely also permits fostriecin
to limit myocardial infarct size and to protect cardiomyocytes during ischemia
(6–8).
Recent evidence suggests that PP2A has a role in regulating cell death by
apoptosis, in the activation of natural killer cells and cytotoxic
T-lymphocytes involved in tumor surveillance, and even in the transcription
and replication of human immunodeficiency virus-1 (HIV1)
(9). For these reasons
selective protein phosphatase inhibition has been suggested as a clinically
unexplored novel mechanism worthy of pursuit for the introduction of a new
class of antitumor or even antiviral agents
(1,
5,
9).
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Phoslactomycins (PLMs) (also called phosphazomycins or phospholines) and leustroducsins (Fig. 1) are structurally related natural products. The principal differences from fostriecin are replacement of the C-8 methyl substituent by ethylamine and the terminal allylic alcohol by a cyclohexane ring. Like fostriecin, these compounds are produced in multiple forms (Fig. 1). PLM-B (phospholine) contains a fully reduced cyclohexyl ring, whereas the other PLMs and the leustroducsins contain a hydroxyl substituent (C-18) esterified with a wide array of carboxylic acids (ranging in length from 4 to 9 carbons) (10–13). The phoslactomycins and leustroducsins all exhibit antifungal activity (1, 10, 13, 14). More importantly, these compounds like fostriecin are potent and highly selective inhibitors of PP2A (IC50 values raging from 3.7 to 5.8 µM) as compared with PP1 (IC50 > 1 mM) (15). Most recently, the PP2A inhibition activity of phoslactomycin has been shown to inhibit tumor metastasis through augmentation of natural killer cells (16).
The unique and selective biological activity of this class of natural products has attracted considerable interest in recent years. Phase I clinical trials of fostriecin were suspended before either dose-limiting toxicities or therapeutic plasma levels were attained because of inherent drug instability and unpredictable purity in the clinical supply of the natural product (1, 4). In an attempt to address these limitations and further develop this class of novel antitumor agents, no less than six elegant total syntheses of fostriecin have been developed over a short 2-year period (5, 17–21). A complementary approach for developing these agents can come through gene manipulation of the natural biosynthetic process. In all cases, the production of numerous analogs complicates the isolation process and the purity of the product, as well as negatively impacting yields (2, 14, 22). These analogs are based on variations in the level of hydroxylation and, in the case of the PLMs and leustroducsins, esterification of the resulting secondary alcohol with a series of different carboxylic acids (this modification does not significantly impact either antifungal activity or binding to PP2A). Using a PLM-producing strain, Streptomyces sp. HK-803, we have carried out an incorporation study of [7-13C]CHC, which suggests that PLM-A and PLM C–F are derived from a hydroxylation of PLM-B. The entire 75 kb of the phoslactomycin biosynthetic gene cluster has been identified and sequenced, including a gene, plmS2, encoding the cyctochrome P-450 monooxygenase responsible for this hydroxylation. The application of a new PCR targeting technology (23) in this strain allowed rapid gene replacement of plmS2. The resulting mutant strain, NP1, produces only PLM-B, at 6-fold higher levels than the wild type strain. This mutant will serve as a starting point for engineering strains capable of producing fostriecin and PLM analogs.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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Analyses of PLM Production—The culture filtrates and mycelial extracts of Streptomyces sp. HK-803 wild type and the mutant NP1 were worked up as described previously (13). HPLC was performed on a system equipped with a WatersTM 600 pump and a Waters 996 photodiode array detector under the following conditions: column, Senshu Pak PEGASIL ODS (4.6 x 250 mm for analytical HPLC and 20 x 250 mm for preparative HPLC); solvent, acetonitrile, 0.05% HCO2H (40/60); flow rate, 1 ml/min for analytical HPLC and 9 ml/min for preparative HPLC; detection at UV 235 nm. Pure PLM standards were used to generate a standard curve for determining PLM production in both the wild type and NP1 mutant. The PLMs were also identified by co-injections with the corresponding standards and by LC-MS analyses. LC-MS spectra (positive turbo-ion spray ionization mode; HPLC, Hewlett-Packard Series 1100; column, 150 x 2.1 mm RP 183.5-mm column from Waters; mobile phase, 40:60 CH3CN-water (containing 0.05% HCO2H) at a flow rate 0.2 ml/min) were taken on a PerkinElmer Life Sciences SCIEX API 2000 pneumatically assisted electrospray triple quadrupole mass spectrometer.
Incorporation of [7-13C]CHC into
PLMs—[7-13C]CHC was synthesized from cyclohexanone and
K13CN via cyclohexanecarbonitrile as described previously
(25,
26): 1H NMR (270
MHz, CDCl3) 1.20–2.00 (10H, m), 2.33 (1H, tdt,
JH-H = 15.1, 2JC-H = 7.1,
JH-H = 3.6 Hz), 11.4 (1H, bs); 13C-NMR (67.5
MHz, CDCl3) 25.4, (d, 3JC-C
= 3.85 Hz, C-3), 25.8, 28.8 (d, 2JC-C = 1.08
Hz, C-2), 43.0 (1JC-C = 55.0 Hz, C-1), 182.6
(enhanced signal, C-7).
The labeled CHC (10 mg) was dissolved in EtOH (1 ml) and added directly to each of 10 flasks containing 70 ml of production medium at 30 h after inoculation. LC-MS analyses (see "Experimental Procedures") of the resulting PLMs allowed an estimation of the level of incorporation of [7-13C]CHC. For each PLM the ratio of the [M+1] and [M+2] relative to [M], the abundance of the molecular ion (m/z), was determined. A similar analysis was carried out with unlabeled PLMs and allowed the contributions of naturally abundant 13C and [7-13C]CHC to be separated in the standard manner. The amount of each 13C-labeled PLM derived from [7-13C]CHC was determined as a percentage of that entire analog pool. The location of the 13C label was confirmed by 13C NMR analyses of HPLC purified PLM-B, -E, and -F (see "Experimental Procedures") using previously published 13C assignments (10).
Cloning and Sequencing of the PLM Biosynthetic Gene Cluster—The total genomic DNA of Streptomyces sp. HK-803 (grown on SY medium) was prepared according to the cetyltrimethylammonium bromide procedure for the isolation of genomic DNA in the manual of Practical Streptomyces genetics (27). A genomic library was constructed by using Supercos-1 cosmid vector as recommended in the manufacturer's protocol (Stratagene). About 2000 cosmid clones were probed by using the DNA of a digoxigenin-labeled chcA gene (28). Preparation of the digoxigenin probes and the subsequent hybridization and detection were performed as recommended in the manufacturer's protocol (Roche Applied Science). The overlapping cosmid clones identified by the chcA probe were sequenced to completion using the TOPO shotgun subcloning kit (Invitrogen). Automated DNA sequencing was performed on an ABI Prism 3700 DNA sequencer at the DNA core facility of the Medical College of Virginia, Virginia Commonwealth University. The DNA sequences were assembled using SeqMan II (DNAStar Inc.). The assembled DNA and deduced protein sequences were analyzed with MacVector 7.0 software (Accelrys) and FramePlot (29) and were compared with sequences in the public data bases using the BLAST suite of programs (30).
Targeted Disruption of plmS2—The plmS2 gene of the PLM biosynthetic gene cluster was disrupted by using the recently developed PCR-targeted Streptomyces gene replacement method (23). The aac (3)IV resistance marker and oriT were amplified from the pIJ 773 disruption cassette (23) using the primers PLMS2-F 5'-CCGCCGCCCCGAGCACGA-AGAGACGGCGTCCTCCGCATGattccggggatccgtcgacc-3' and PLMS2-R 5'-CCAGG-ACGACCGGGCCGGTTCAGCGTGCGGGAACGCTCAtgtaggctggagctgcttc-3' (pIJ773 homologous sequence is in lowercase letters). The resulting PCR product was used to replace plmS2 first in cosmid clone 3A11 and then in Streptomyces sp. HK-803 following the established methodologies (23), replacing mannitol soy agar with SY agar. Allelic replacement of the plmS2 gene in the NP1 mutant was confirmed by Southern hybridization analysis, and PCR amplification and sequencing.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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139.9 ppm) and C-1' ( 177.3 ppm), indicating that
CHC is the precursor of both cyclohexane moieties. Strong and exclusive
enrichments of the C-15 ( 138.2 ppm) was observed in 13C NMR
analyses of purified PLM-B and PLM-F. The level of enrichment in all cases was
comparable with that determined from LC-MS analyses. These observations
demonstrate that a coenzyme A-activated CHC (CHC-CoA) is the likely starter
unit for the biosynthesis of all PLMs. A pathway from shikimic acid to CHC-CoA
has been delineated in the ansatrienin producer, Streptomyces
collinus (31). The
corresponding biosynthetic genes have recently been identified
(33,
34).
Isolation and Characterization of the PLM Biosynthetic Gene
Cluster—The PLM biosynthetic gene cluster from the
Streptomyces sp. HK-803 was identified from a cosmid library of
genomic DNA using the chcA gene
(28) from CHC-CoA biosynthetic
gene cluster of S. collinus as a probe. A cosmid clone, 3A11, thus
identified was used as a probe to identify two overlapping cosmids, 10B4 and
3E5 (Fig. 2). These three
cosmids spanning 97 kb of the Streptomyces sp. HK-803 genome were
sequenced and analyzed. This sequence analysis revealed a 75-kb region
containing 29 open reading frames (ORFs) that appear to be associated with PLM
biosynthesis. The flanking region contained a series of ORFs that are highly
homologous (>80% identity at the amino acid sequence level) with and have
an organization similar to the primary metabolic genes identified through
sequencing of the S. coelicolor genome
(35), which aided in
delimiting the PLM biosynthetic gene cluster. The sequence analysis revealed
the existence of the complete set of highly conserved CHC-CoA biosynthetic
genes (plmJK, plmL, chcA, plmM)
(28,
33) as well as plmI
(encoding a 3-deoxy-D-arabino-heptulosonate-7-phosphate
synthase) responsible for formation of the CHC-CoA starter unit. As in S.
collinus, the chcB gene encoding the 
2,3-enoyl
CoA isomerase responsible for catalyzing the penultimate step in the CHC-CoA
pathway does not appear to be located close to the other CHC-CoA biosynthetic
genes or within the antibiotic biosynthetic gene cluster
(36). A total of six large
ORFs encoding the core PLM PKS were also identified
(Fig. 3). PLM1 contains an
initiation domain, presumably responsible for loading the CHC-CoA, and the
first extension module (Fig.
3), whereas PLM2–3 has two modules containing the
appropriate predicted catalytic activitities for catalyzing the second and
third extension steps. Each of the remaining components, PLM4–PLM7,
contains a single module. PLM7 has a thioesterase domain, consistent with a
role in catalyzing the last extension step and subsequent release of the
polyketide chain by formation of the ,-unsaturated
-lactone (formation of a six-member lactone is unusual, as most
thioesterase domains studied to date catalyze formation of large 12–14
macrolactone structures under natural conditions)
(37,
38). Two of the PKS
polypeptides PLM4 and PLM6, contain the same catalytic domains, which
precludes unambiguous assignment in this process. One of many interesting
features of the PLM PKS cluster is that it contains only three dehydratase
domains, and yet it produces a compound with three Z- and one
E-double bonds (PLM7 does not contain the predicted dehydratase
domain). Virtually all known cases in which a PKS modules has a ketoreductase
and a dehydratase activity result in the formation of E-double bonds
(39). It has been suggested
that a stereochemical configuration of the product derived from the
ketoreductase domain may control the double bond geometry. A diagnostic Asp
residue has been proposed for predicting the stereospecificity of PKS
ketoreductase domains and, by extension, the double bond configuration for
products from cognate dehydratase domains
(39). We constructed a
multiple alignment of the seven ketoreductase domains of the PLM PKS and found
that this model alone was only partially predictive of the stereochemical
configuration of the PLM product.
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The PLM gene cluster also contains genes that encode a separate type II TE protein (plmT8), recently established to have an important role in the editing and proofreading of PKSs (40), and a crotonyl-CoA reductase (plmT7), presumably required for providing the ethylmalonyl-CoA extender unit used in the fourth and sixth extension steps. The plmT5 gene product has some sequence identity with homoserine kinases and is likely responsible for phosphorylation of the C-9 hydroxyl group and for providing biological activity to PLM. Analysis of the gene cluster also predicts an oxidoreductase (plmT8) and aminotransferase (plmT1), likely involved in the replacement of a hydroxyl group of a C-8 hydroxyethyl side chain with an amine. The gene cluster also contains numerous other genes involved in regulation and resistance. A detailed analysis of the entire PLM gene cluster will be described elsewhere.
PCR-targeted Gene Disruption of plmS2—Analysis of the PLM biosynthetic gene cluster, together with the results of the biosynthetic incorporation study, indicated that three hydroxylation steps (C-18, C-8, and the C-8 ethyl side chain) occur after assembly of the polyketide chain from the CHC-derived starter unit. Analysis of the PLM biosynthetic gene cluster revealed two ORFs, plmT4 and plmS2, that both encode proteins with high sequence similarity to each other (38% identity and 53% similarity) as well as cytochrome P-450 monooxygenases from several microorganisms, including those involved in the modification of the natural products of streptomycetes (41). We reasoned that replacement of one of these genes would provide a strain blocked in hydroxylation of C-18 of PLM. With the caveat that the other hydroxylations are catalyzed by the second monooxygenase, the mutant strain should produce PLM-B exclusively.
There are numerous different approaches for probing the secondary metabolic
gene clusters through targeted gene disruptions. These approaches typically
involve in vitro generation of suitable gene disruption vectors,
introduction of these into the host using methods such as conjugation or
transformation, and an extensive screening process to find the desired allelic
replacement (42,
43). This entire process can
take several months, even when methodologies have been developed for the
strain. For strains that have not previously been characterized or are not
genetically amenable, the process can be substantially longer
(44,
45). We applied a new
PCR-targeted gene replacement, developed for work with Streptomyces
coelicolor (23), to
efficiently and rapidly carry out targeted disruption of
plmS2 in Streptomyces sp. HK-803. The
plmS2 in cosmid 3A11 was replaced in vivo by
aac (3)IV (AprR) and the oriT
(which allows for efficient transfer to Streptomyces by RP4-mediated
intergenic conjugation). This recombination was accomplished in E.
coli BW25113/pIJ790 (expressing the 
-Red recombination functions)
using a PCR product in which these two genes and a yeast Flp-recombinase
target sequence were flanked by 39 nt of plmI and
plmS1 (genes surrounding plmS2). The
mutagenized cosmid, isolated from the resulting apramycin-resistant
transformants, was introduced by electroporation into E. coli
ET12567/pUZ8002 and was subsequently transferred into Streptomyces
sp. HK-803 by conjugation. Approximately 80% of the resulting ApraR
exconjugants were KanS, indicating a double-crossover allelic
exchange of plmS2, which was exhaustively confirmed by PCR
and Southern blot analysis (data not shown). Utilization of the same approach
by us and others (23) in
S. coelicolor have shown a frequency of 30% or less double
crossovers, suggesting that Streptomyces sp. HK-803 may have a higher
rate of homologous recombination.
Enhanced and Selective Production of PLB by the plmS2
Mutant—The plmS2 mutant (NP1) was grown under
standard conditions alongside the wild type PLM producer. After 4 days of
fermentation both cultures were purified by ion exchange chromatography and
analyzed by reverse-phase HPLC. The wild type strain produced a mixture of
PLMs (A–F) with a total yield of PLMs of 7 mg/ml
(Fig. 4). In stark contrast the
NP1 mutant produced only PLM-B, at 
6-fold higher levels (10 mg/ml) than
observed for the wild type (<1.6 mg/liter). The PLM-B titer in the NP1
mutant exceeds the cumulative yield of all PLM analogs in the wild type
strain. The PLM-B was confirmed by coinjections with a standard and by LC-MS
analyses, which demonstrated the correct mass ([M+H]+ = 514). These
observations unequivocally demonstrate that the plmS2 gene
product is responsible for catalyzing hydroxylation at C-18 of PLM and is not
required for hydroxylation at other positions. Hydroxylation at C-8 and the
C-8 ethyl side chain are most likely catalyzed by the second cytochrome P-450
monooxygenase, encoded by plmT4. The efficient production
of PLM-B by the NP1 strain suggests that hydroxylation at C-18 of PLM-B and
the subsequent esterification step may be the final steps in the biosynthetic
process. The possibility that these steps might also occur prior to other
modifications of the PLM carbon backbone cannot be discounted.
Conclusion—This work represents the first cloning and analysis of a biosynthetic gene cluster for a potent and selective protein phosphatase inhibitor. Analysis and subsequent manipulation of this gene cluster has allowed for the rational engineering of a strain that produces only one analog, PLM-B, at significantly higher yields than the wild type strain. Some of the key drawbacks to continued development of this class of naturally occurring compounds, including low fermentation titers and resolution of the various analogs produced in the fermentation (both contributing to concerns about drug purity), have now been overcome. Cloning of the PLM biosynthetic gene cluster and creation of a strain producing only PLM-B also set the stage for the generation of novel compounds with improved stability, activity, and selectivity. As such, this work represents a complementary approach to the widespread synthetic approaches directed toward this class of compounds (5, 17–21). One of the key structural differences between PLMs and fostriecin is the C-8 ethylamine substituent, which may contribute to less potent activity (15). Inactivation of the plmT1 gene predicted to be responsible for incorporation of this amino group into the PLM structure should allow for the generation of a novel hybrid structure bearing features of both fostriecin and PLM. Other hybrid natural products may be obtained by combining different PKS components from the two biosynthetic gene clusters. These avenues of research are currently being pursued and will be reported in due course.
This work has also demonstrated the utility of PCR-targeted gene replacement (23) for the rapid characterization and manipulation of polyketide biosynthetic gene clusters. This methodology, demonstrated for the strain S. coelicolor, has worked effectively for us not only in Streptomyces sp. HK-803 but also Streptomyces cinnamonensis.2 The technique is potentially applicable to any other streptomycetes for which conditions for conjugation from E. coli either have or can be developed (43). As such, this new technology should revolutionize the way in which most natural product biosynthetic gene clusters are analyzed and manipulated. It represents a powerful new genetic tool for the generation of novel natural products through combinatorial biosynthesis.
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FOOTNOTES |
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* This work was supported by a Grant AI51629-01 from the National Institutes
of Health. The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
¶ Recipient of a fellowship from the Special Postdoctoral Researchers
Program.
|| To whom correspondence should be addressed: Institute for Structural Biology and Drug Discovery, 800 East Leigh St., Richmond, VA 23219. Tel.: 804-828-5679; Fax: 804-827-3664; E-mail: kareynol{at}hsc.vcu.edu.
1 The abbreviations used are: PP, protein phosphatases; PLM, phoslactomycin;
PKS, polyketide synthase; CHC, cyclohexanecarboxylic acid; ACP, acyl carrier
protein; TE, thioesterase; ORF, open reading frame.
2 N. Palaniappan, B. S. Kim, Y. Sekiyama, H. Osada, and K. A. Reynolds,
unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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