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Cloning and characterization of a gene cluster for geldanamycin production in Streptomyces hygroscopicus NRRL 3602
FEMS Microbiology Letters 218 (2003) 223^230
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Cloning and characterization of a gene cluster for geldanamycin production in Streptomyces hygroscopicus NRRL 3602 Andreas Rascher a; , Zhihao Hu a , Nina Viswanathan a , Andreas Schirmer a , Ralph Reid a , William C. Nierman b , Matthew Lewis b , C. Richard Hutchinson b
a
a Kosan Biosciences, 3832 Bay Center Place, Hayward, CA 94545, USA The Institute for Genome Research, 9712 Medical Center Drive, Rockville, MD 20850, USA
Received 30 August 2002; received in revised form 7 November 2002; accepted 8 November 2002 First published online 12 December 2002
Abstract We illustrate the use of a PCR-based method by which the genomic DNA of a microorganism can be rapidly queried for the presence of type I modular polyketide synthase genes to clone and characterize, by sequence analysis and gene disruption, a major portion of the geldanamycin production gene cluster from Streptomyces hygroscopicus var. geldanus NRRL 3602. 4 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Ansamycin ; Antitumor; Biosynthesis ; Cancer chemotherapy; Hsp90; Polyketide synthase
1. Introduction Geldanamycin [1^4] (Fig. 1) and several other benzoquinone microbial products classi¢ed as ansamycin antibiotics [5], herbimycin A [6,7], macbecins [8], ansatrienins [9] and reblastatin [10,11] were discovered between 1970 and 2000 in screens for antibacterial, antifungal and antiviral compounds. Interest in the benzoquinone ansamycins increased greatly upon the discovery of the antitumor properties of geldanamycin [12] and herbimycin A [13], and the remarkable cytotoxic properties of the ansamitocins [14]. It was initially believed that geldanamycin and herbimycin A interfered with signal transduction pathways in tumor cells by direct inhibition of oncogenic protein tyrosine kinases. However, Neckers and co-workers [15] showed in 1994 that the principal cellular target is not a tyrosine kinase but Hsp90, a ubiquitous and abundant protein chaperone of mammalian cells [16]. Geldanamycin competes with ATP for the ATP-binding site of Hsp90 and, when bound, inhibits the ATP-dependent functions of Hsp90. A particular function is its ability to chaperone nascent protein kinases that are critical components of
* Corresponding author. E-mail address : [email protected] (A. Rascher).
signal transduction pathways, especially those in certain cancer cells [16]. In the presence of geldanamycin, herbimycin A or macbecin, the immature kinases undergo rapid degradation, as a consequence of ubiquitination and subsequent catabolism by the proteosome, and the levels of the mature kinases become depleted. This can result in a cytostatic e¡ect on a cancer cell or in some cases apoptosis and cell death. The discovery that Hsp90 and one or more of its protein kinase cohorts are overproduced in several types of human cancers has led to considerable interest in geldanamycin and its analogs as potential anti-cancer drugs [17]. Many geldanamycin analogs have been produced by replacement of the C17 O-methoxy group with substituted amines. One such drug, 17-allylamino-17-demethoxygeldanamycin, is currently undergoing Phase I clinical trials [18]. We are interested in engineering the geldanamycin polyketide synthase (PKS) genes to make novel geldanamycin analogs. To obtain the genes from the producer, Streptomyces hygroscopicus NRRL 3602, we faced a set of challenges common to the isolation of any PKS gene cluster from a streptomycete: bacteria commonly produce more than one polyketide metabolite (e.g. Streptomyces coelicolor has 10 [19] and Streptomyces avermitilis at least 13 [20] PKS gene clusters). We therefore developed a reliable method for querying the genomic DNA of the NRRL 3602 strain for the number of modular PKS genes, then
0378-1097 / 02 / $22.00 4 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-1097(02)01148-5
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OH
OH
OH CH3
15
17
HO
7
5 14
13
12
7
11
4
AHBA
CH3
21
OCH3
CH3
OCH3
CH3
11
CH3O CH3
CH 3
O 21
15
LDD:mod1
mod2
mod3
mod4
mod5
mod6
NH2
COS-ACP7
5
CH3 OCH3 4 OH CH3 NH
17
mod7
OH progeldanamycin
O
A)
O
5
5
S-ACP6
ER6
4
4
S-ACP6 1) ansamycin formation 2) C4,5-oxidation
5
C7 carbamoylation C17 hydroxylation C17 O-methylation C21 oxidation
4
OCONH2 CH3 CH3
CH3O
HNOC
OH
CH3
O
CH 3 OCH3
O
CH3
NH CH3O
O
geldanamycin Fig. 1. Main steps in the proposed biosynthesis of geldanamycin. The enzyme-bound product shown is assembled from AHBA and the carbon chain extender substrates, malonyl-CoA, 2-methoxymalonate and 2-methylmalonyl-CoA, by the PKS modules indicated beneath the product structure. The dashed line at position 4 signi¢es that the 4,5-CNC may be introduced by the PKS or afterwards as in A. Abbreviations : ACP, acyl carrier protein ; LDD, loading didomain; mod, module. The order of the four tailoring steps that convert progeldanamycin into geldanamycin is unknown.
used this method to help clone most of the geldanamycin production genes.
2. Materials and methods 2.1. Bacterial strains and culture conditions The geldanamycin-producing strain, ¢rst described by DeBoer et al. [1,2] as S. hygroscopicus var. geldanus var. nova UC-5208, was obtained from the Northern Regional Research Laboratory of the Agricultural Research Service as S. hygroscopicus NRRL 3602. To con¢rm production of geldanamycin, spores were used to inoculate 5 ml of R2YE liquid media [38]. The culture was incubated at 28‡C for 36 h, transferred into 100 ml geldanamycin production medium [1] and the ¢nal culture was incubated at 28‡C in 500-ml Erlenmeyer £asks at 300 rpm for another 5 days. Following low-speed centrifugation, the cell pellet from the culture was extracted with methanol by stirring for 10 min. The methanol broth was clari¢ed by centrifu-
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gation (17 500Ug) and the supernatant was analyzed for the presence of geldanamycin using high-performance liquid chromatography (HPLC) under the following conditions: column Inertsil C18 (4.6U150 mm, Ansys Technologies, Inc.), mobile phase 60% acetonitrile (isocratic), £ow rate (2 ml min31 ), temperature (40‡C), detection (UV 315 nm), injection volume (10^20 Wl). Geldanamycin (SigmaAldrich) was quanti¢ed by comparing the peak area at 315 nm with that measured for a standard solution. The titer of geldanamycin was approximately 250 mg l31 . 2.2. Manipulation of DNA and organisms For genomic DNA extraction, a spore stock was used to prepare a seed culture as described above. The entire seed culture was transferred into 50 ml of the same growth medium in a 250-ml ba¥ed Erlenmeyer £ask and incubated for 48 h at 28‡C. A 20-ml portion of the cell suspension was centrifuged (10 000Ug) and the resulting pellet was washed with 10 ml bu¡er 1 (Tris, 50 mM, pH7.5 ; 20 mM EDTA). The pellet was pulverized with mortar and
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pestle under liquid nitrogen and transferred into 3.5 ml of bu¡er containing 150 Wg ml31 RNase (Sigma-Aldrich). After incubation of the mixture at 30‡C for 20 min, the salt concentration was adjusted by adding 850 Wl 5 M NaCl solution, then the mixture was extracted multiple times with phenol:chloroform:isoamylaclohol (25:24:1, vol/vol) with gentle agitation followed by centrifugation for 10 min at 3500Ug. After precipitation with 1 vol of isopropanol, the genomic DNA knot was spooled on a glass rod and redissolved in water (200 Wl). This method yielded about 1 mg DNA with a protein factor of about 2, as determined by the ratio of the UV absorbances at 260 and 280 nm. Standard agarose gel electrophoresis using 0.7% Seakem0 LE-Agarose (BioWhiaker Molecular Applications, Rockland, ME) at a voltage of 50 mV overnight revealed that the sample contained mainly high-molecular-mass DNA fragments of about 60 kb. 2.3. Genomic analysis of S. hygroscopicus NRRL 3602 The following degenerate keto synthase (KS) primers were used to scan the genomic DNA of S. hygroscopicus for PKS genes: degKS1F (5P-TTCGAYSCSGVSTTCTTCGSAT-3P), degKS2F (5P-GCSATGGAYCCSCARCARCGSVT-3P), degKS3F (5P-SSCTSGTSGCSMTSCAYCWSGC-3P), degKS5R (5P-GTSCCSGTSCCRTGSSCYTCSAC-3P), degKS6R (5P-TGSGYRTGSCCSAKGTTSSWCTT-3P) and degKS7R (5P-ASRTGSGCRTTSGTSCCSSWSA-3P). Forward (F) and reverse (R) primers were tested in all possible combinations in standard PCR reactions with annealing temperatures between 50 and 60‡C. A typical 50 Wl PCR reaction consisted of 200 ng genomic DNA, 200 pmol of each primer, 0.2 mM dNTP (containing 7-deaza-dGTP), 10% DMSO and 2.5 U Taq DNA polymerase (Roche Applied Science). Carbamoyl transferase gene fragments were ampli¢ed with degenerate forward primer degCT2F (5P-AARGTSATGGGSYTSGCSCCSTA-3P) and reverse primers degCT3R (5P-CCSARSGCSCKSGGSCCRAAYTC-3P) or degCT4R (5P-CKSGCSSWSCCRTCSACRTGSGT-3P) using an annealing temperature of 55‡C. 3-Amino-5-hydroxybenzoate (AHBA) synthase gene fragments were ampli¢ed with a set of two degenerate forward primers, degAH-F1 (5P-GTSATCGTSCCSGCSTTCACSTTC-3P) and degAH-F2 (5P-ATCATGCCSGTSCAYATGGCSGG-3P), and two reverse primers, degAHR1 (5P-GGSTBSGKGAACATSGCCATGTA-3P) and degAH-R2 (5P-CKRTGRTGSARCCASTKRCARTC-3P). Again forward and reverse primers were tested in all combinations in the described standard PCR reactions with annealing temperatures between 50 and 65‡C. A set of four gene speci¢c primers, AH-B-spF (5P-AGGACAGTGGCGCGGCAAGAA-3P), AH-B-spR (5P-GGTCGACGATCTTCGCGCGGCG-3P), AH-N-spF (-5PTCGACGTGGCTGCCGCGGCTT-3P) and AH-N-spR (5P-TGTCGACGAGGGCGTTGCGGG-3P), was used to
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distinguish between AHBA-B and AHBA-N synthase genes. PCR amplimers were gel-puri¢ed and cloned into pCR2.1-TOPO using TA cloning (Invitrogen). For each primer pair, a representative set of cloned amplimers (600^800 bp) was sequenced using a Beckman CEQ2000 with M13 forward and reverse primers. 2.4. Library construction and gene isolation A genomic library of the NRRL3602 strain was constructed at TIGR using the proprietary single-copy BAC vector pHOS3. Sheared genomic DNA was made bluntended using T4 DNA polymerase and BstXI adaptors were ligated to the blunt ends. Adaptors were removed and inserts were size-selected by three rounds of ¢eld inversion gel electrophoresis. The inserts were ligated to the vector and electroporated into Escherichia coli. A total of 4896 BAC clones were arrayed into 384-well microtiter plates and spotted in high density onto nylon ¢lters (Amplicon Express). Probes were labeled using [K-32 P]dCTP and a random prime-labeling system (rediprime II, Amersham Pharmacia Biotech). Filters were hybridized at 68‡C for 12 h using ExpressHyb hybridization solution (Clontech). After removal of the probe and hybridization solution, the ¢lter was washed twice for 30 min each time with 100 ml of bu¡er I [2USSC: 300 mM NaCl, 30 mM sodium citrate pH 7.0, 0.05% sodium dodecyl sulfate (SDS)] at room temperature and then three times for 60 min each time at 50‡C with 100 ml of bu¡er II (0.1USSC, 0.1% SDS) with continuous shaking. Finally, the ¢lter was rinsed several times with 0.05USSC and analyzed by autoradiography. BAC DNA was prepared by alkaline lysis, starting with 10 ml culture volume. The resulting DNA was ¢rst treated with RNase (Sigma-Aldrich) at 30‡C for 3 h and then with plasmid safe DNase (Epicentre Technologies) at 37‡C o/n. After heat inactivation at 70‡C for 10 min the DNA was precipitated with 1 volume isopropanol for 30 min on ice and recovered by centrifugation at 1880Ug for 45 min to separate the remaining smaller fragments from the large, intact BAC plasmids. The ¢nal pellet was washed with 70% EtOH and redissolved in 80 Wl water. This method typically yielded about 100 Wg of BAC DNA. 2.5. DNA sequence analysis BAC DNA was sequenced to completion using the standard shotgun procedures [39]. The DNA and deduced protein sequences were analyzed with Sequencer 4.1 (Gene Codes Corporation) and MacVector 6.5.3 (Accelrys) software, and compared with sequences in the public databases using the CLUSTAL W [40] and BLAST [41] programs. The sequence data for the genes displayed in Fig. 2 have been deposited with GenBank (accession number AY179507).
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Fig. 2. Geldanamycin gene cluster. The colored broad arrows indicate the genes and their direction of transcription. PKS and gdmF genes are red, 2-methoxymalonate synthesis genes are orange, the AHBA synthesis gene is green and tailoring enzyme genes are yellow. The right end of the cluster continues beyond ORF22. The domains in each of the seven modules of the gdm PKS are indicated by the abbreviations above the gdmA arrows (see Table 1 for de¢nitions). Scale beneath the genes indicates the distance in kb from the beginning of the 85-kb region sequenced.
2.6. Disruption of the gdmH gene The gdmH gene disruption vector was made as follows. The aphII neomycin/kanamycin resistance gene from Tn5 was excised as a StuI^SmaI fragment from SuperCos-1 (Stratagene), then inserted into the MscI site within gdmH carried in a 4-kb BstXI fragment, containing the gdmN, gdmH and gdmI genes, and cloned in pOJ260 [42] to give pKOS246-33. The XbaI^EcoRI fragment from pKOS246-33 was excised and cloned into the XbaI^EcoRI sites of pKC1139 [42] to give pKOS279-37. The gdmH gene was disrupted by introducing pKOS279-37 into the S. hygroscopicus NRRL3602 strain by conjugation from E. coli ET12567/pUB307 according to a published method [43]. Exconjugants resistant to apramycin (pKC1139 carries the accIV(3) gene) and kanamycin were isolated and one of them was grown at 30‡C in 6 ml of R5 liquid medium [38] supplemented with 100 Wg ml31 of kanamycin for 2 days in 50-ml culture tubes at 200 rpm. Approximately 5% of this culture was transferred into 6 ml of fresh R5/apramycin liquid medium and the culture was grown at 37‡C for 3 days in order to force chromosomal integration of pKOS279-37. After recovery of the mycelia by centrifugation, cells were plated on tomato paste medium containing 100 Wg ml31 kanamycin and grown at 30‡C for sporulation. Spores collected from these plates were diluted and replated on the same medium for single colonies. Among 100 colonies screened, 20 of them were apramycin sensitive and kanamycin resistant when assayed on plates containing apramycin or kanamycin, using 60 or 50 Wg ml31 of antibiotic, respectively. Genomic DNA was isolated from 11 of these 20 colonies by an established method [38] and probed by Southernblot hybridization [38] with the aphII gene to determine
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that all kanamycin resistant recombinant strains had the restriction fragment pattern upon digestion with PstI^ EcoRV expected for integration of the aphII gene into the gdmH locus by a double crossover recombination (hybridizing bands at 2.9 and 3.2 kb that were absent in the NRRL3602 strain). To determine geldanamycin production, each of the 11 strains was individually cultured in 35 ml of the geldanamycin production medium [1] as described above. After 4 days, 500 Wl of broth from each £ask was mixed with 500 Wl of methanol, the mixture was centrifuged at 12,000 rpm in a desktop microcentrifuge for 5 min to remove mycelia and other insoluble ingredients, then the supernatant fraction was analyzed by HPLC/MS. The results showed that geldanamycin was present (retention time and low-resolution MS data were identical to the reference standard) and that two new compounds were present with molecular masses and formulas of 518.2759 (C28 H40 NO8 [M-H]3 ) and 520.2916 (C28 H42 NO8 [M-H]3 ), calculated on the basis of high-resolution MS data. These data are consistent with 4,5-dihydro-7-descarbamoyl-7-hydroxygeldanamycin and its hydroquinone form.
3. Results and discussion 3.1. Survey of modular PKS genes in S. hygroscopicus NRRL 3602 Geldanamycin biosynthesis can be depicted as shown in Fig. 1 on the basis of isotope-labeling studies by the Rinehart laboratory [4,21]. That work forecast the involvement of a PKS that would use AHBA [22^25] as the chain initiation substrate for a seven-module type I PKS, and
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malonate, 2-methylmalonate and 2-methoxymalonate, as chain extender substrates to produce the putative progeldanamycin (Fig. 1). Geldanamycin can be formed from this polyketide by four tailoring steps, as shown. For the initial approach we used standard bioinformatics methods to choose six highly conserved yet recognizably di¡erent regions of the amino acid sequence of KS domains among 20 known modular PKSs. Three primer combinations (see Section 2) consistently gave correctly sized PCR products with all organisms tested (data not shown) and were chosen to survey the complexity of PKS genes in the genome of S. hygroscopicus NRRL 3602. Of the 90 KS amplimers cloned and sequenced, 63 represented unique type I PKS KS gene fragments, but when they were analyzed further by comparison with the KS sequences of the ansamitocin [23] and rifamycin [24] PKSs, the evidence that they were part of the geldanamycin PKS genes was not conclusive. Homologs of the genes for formation of AHBA and for the C7 carbamoylation step were then chosen to design a new set of PCR primers that were used to screen the genomic DNA for AHBA synthase [23^25] and carbamoyltransferase (CT) [23,26,27] homologs. We analyzed 56 AHBA amplimers and identi¢ed two di¡erent DNA sequences that would encode AHBA synthases that are 75% identical, AHBA-B and AHBA-N. The data, when analyzed by standard bioinformatics methods (Section 2), strongly suggest that one AHBA synthase homolog belongs to the family associated with the biosynthesis of benzoquinone ansamycins (AHBA-B) [23] and the other with naphthoquinone ansamycins (AHBA-N) [24,25]. All 20 CT amplimers analyzed were identical, which let us conclude that there is only one CT gene in this strain. 3.2. Cloning and sequence analysis of geldanamycin production genes The AHBA-B synthase and CT amplimers were used as probes for primary screening of a genomic library made in a single-copy BAC vector, pHOS3, a derivative of pBeloBAC11 [28] modi¢ed for BstXI cloning. We screened 4896 BACs with average insert sizes of 45 kb, which was equivalent to ca. 20U coverage of this genome, and identi¢ed 36 AHBA synthase and 16 CT positive BACs. AHBA-B and AHBA-N synthase containing BACs were distinguished by PCR analysis, which established that about half of these BACs belonged to each of the AHBA-B and AHBA-N types. Interestingly, when these were analyzed for the presence of PKS genes by PCR analysis, we found that none of the 20 AHBA-B synthase containing BACs contained PKS genes, whereas 14 out of 17 AHBAN synthase containing BACs also had PKS genes. We thus focused on the CT containing BACS to identify the geldanamycin PKS genes. Nine out of 15 CT BACs also contained PKS genes and from these CT+PKS BACs, we subcloned four unique KS amplimers using the degenerate
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PCR primers. These putative geldanamycin KS sequences (one of which was contained in the 63 KS amplimers described above) were then used as probes in a secondary screen of the BAC library at high stringency to identify seven additional overlapping BACs. Two overlapping clones, pKOS256-107-3 and pKOS256154-1, spanning a V85-kb region, were fully sequenced by the shotgun method, and the resident genes were assigned to geldanamycin production on the basis of database comparisons (Fig. 2). Twenty-three open reading frames (ORF) and the deduced functions of their products are listed in Table 1. The CT gene probe matched the sequence of gdmN, and only one homolog of the AHBA synthase genes was found (gdmO). An additional 15 ORFs downstream of ORF16 were sequenced and analyzed (not shown) but, on the basis of their deduced functions, are not believed to be involved in geldanamycin production. (There are incomplete reports about cloning the geldanamycin genes from other streptomycetes isolated independently from the 3602 strain [29,30].) 3.3. Key features of the geldanamycin biosynthesis genes and their deduced products 3.3.1. The PKS A seven-module PKS encoded by gdmA1-A3 and immediately followed by the gdmF gene encoding an amide synthase, as found for the rifamycin and ansamitocin PKSs [23,24], is entirely consistent with the biosynthetic hypothesis shown in Fig. 1. The N-terminal region of module 1 of the GdmA1 protein contains two domains strongly resembling their counterparts in the rifamycin and ansamitocin PKSs. Both of the latter enzymes use AHBA as the starter unit, and the RifA loading module recently has been shown to load the AHBA onto the acyl carrier protein (ACP0) domain through formation of an intermediate acyl-adenylate catalyzed by the acyl ligase domain [31]. The starter unit is transferred intramolecularly from ACP0 to the KS1 of the following module for reaction with the 2-methylmalonate chain extender unit bound to ACP1 [32]. Modules 2 and 5 of the GdmA1 and GdmA2 proteins, respectively, have acyltransferase (AT) domains with the speci¢c motifs similar to other ATs that utilize 2-methoxymalonate (unpublished data). The substrate for AT2 and AT5 is presumed to be provided by the products of the gdmG-gdmK genes, given the recent genetic and functional characterization of their homologs from the FK520 [33] and ansamitocin [34,35] gene clusters. In contrast, the AT domain of module 6 of GdmA3 clearly falls within the class that uses malonylCoA, and those of GdmA1 module 1 and module 3, GdmA2 module 4 and GdmA3 module 7 strongly resemble those that use 2-methylmalonyl-CoA [36,37]. Finally, the presence of enoyl reductase (ER) domains in modules 1, 2 and 3, dehydratase (DH) domains in modules 1, 2, 4, 6 and 7, and keto reductase (KR) domains in all the mod-
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ules ¢t the requirements for the predicted functionality of progeldanamycin (Fig. 1). The GdmA PKS has one notable feature. The ER domain in module 6 is apparently functional yet geldanamycin has a C4 cis-double bond. Consequently, it is plausible that the PKS reduces this double bond and that the C4 cis-double bond is introduced oxidatively either before or after formation of progeldanamycin (Fig. 1A). 3.3.2. Tailoring genes The predicted functions for most of these genes are consistent with the requirements for conversion of progeldanamycin (or its 4,5-dihydro form) into geldanamycin (Fig. 1). Disruption of the gdmH gene, carried out by insertion of a kanamycin resistance gene (Section 2), resulted in strain K279-37 that produced geldanamycin together with a compound whose high-resolution mass spectral data are consistent with 4,5-dihydro-7-descarbamoyl7-hydroxygeldanamycin. This result con¢rms the function assigned to gdmN (the partial inactivation of this gene is believed to be the consequence of read-through expression) and shows that gdmH is dispensible or that its mutation is compensated in trans by a paralog. Distinction among the three genes, gdmL, gdmM and gdmP, likely to
govern C17 hydroxylation, C21 oxidation and C4,5 unsaturation, if that actually occurs, will have to await studies of mutants or the expressed and puri¢ed enzymes. The putative C17 O-methyltransferase gene presumably lies outside the currently sequenced region. 3.3.3. Other genes Possible regulatory genes (gdmR1, gdmR2, ORF19 and ORF20) and a gene for the amino dehydroquinate synthase needed for AHBA biosynthesis (gdmO) are listed in Table 1 as part of the gdm gene cluster because their homologs have been found in other clusters of bacterial secondary metabolism genes. ORF16 and ORF17 are candidates for putative geldanamycin resistance and/or export genes. All but one of the remaining genes for AHBA biosynthesis in the geldanamycin pathway, as deduced from the sequences of homologs involved in the biosynthesis of other benzoquinone ansamycins [22^24], are clustered elsewhere in the genome, more than 20 kb from the end of the BAC that contains ORF22 (data not shown). This is consistent with the observation that three of the AHBA biosynthesis genes for ansamitocin production and the remaining asm genes are in a subcluster separated from all the other genes for AHBA biosynthesis by 30 kb [23].
Table 1 Gene designations and functions Predicted polypeptide
Amino acids
Homologous gene(s)
Putative function
ORF16 ORF17 ORF18 ORF19 ORF20 GdmL GdmX GdmA1 Loading Module 1 Module 2 Module 3 GdmA2 Module 4 Module 5 GdmA3 Module 6 Module 7 GdmF GdmM GdmN GdmH GdmI GdmJ GdmK GdmG GdmR2 GdmO GdmP GdmR1 ORF22
227 300 263 294 253 479 156 6842
RhtB family transporters SC3C8.01 SCF1.09 AraC family TetR family Rif19 JadX; MmyY
E¥ux Secreted protein Hydrolase Transcriptional regulation Transcriptional regulation Unknown Unknown PKS (modules 0^3) AL X ACP KS AT DH ER KR ACP KS AT DH ER KR ACP KS AT KR ACP PKS (modules 4^5) KS AT DH KR ACP KS AT KR ACP PKS (modules 6^7) KS AT DH ER KR ACP KS AT DH KR ACP Amide synthase Unknown CT Methoxymalonyl-ACP biosynthesis Methoxymalonyl-ACP biosynthesis Methoxymalonyl-ACP biosynthesis Methoxymalonyl-ACP biosynthesis Methoxymalonyl-ACP biosynthesis Transcriptional regulation AminoDHQ synthase P450 Transcriptional regulation Hydrolase
3435
3896
257 545 682 370 370 91 288 218 926 354 397 962 237
RifF Rif19 NovN FkbH FkbI FkbJ FkbK FkbG LuxR family Asm47 PikC LuxR family SC2G4.19
Abbreviations for PKS domain functions: ACP, acyl carrier protein; AL, acyl-CoA ligase ; AT, acyltransferase ; CT, carbamoyltransferase ; DH, dehydratase; ER, enoyl reductase ; KR, keto reductase; KS, keto synthase; X, unknown.
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Acknowledgements We thank David Hopwood, Robert McDaniel, Chris Reeves and Peter Revill for helpful comments during preparation of the manuscript. This research was supported in part by a Small Business Innovative Research grant from the National Institute of Health (R43 CA96262 and AL38947).
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A. Rascher et al. / FEMS Microbiology Letters 218 (2003) 223^230 with substrate tolerance for substituted benzoates. Biochemistry 40, 6116^6123. Admiraal, S.J., Walsh, C.T. and Khosla, C. (2002) The loading and initial elongation modules of rifamycin synthetase collaborate to produce mixed aryl ketide products. Biochemistry 41, 5313^5324. Wu, K., Chung, L., Revill, W.P., Katz, L. and Reeves, C.D. (2000) The FK520 gene cluster of Streptomyces hygroscopicus var. ascomyceticus (ATCC 14891) contains genes for biosynthesis of unusual polyketide extender units. Gene 251, 81^90. Kato, Y., Bai, L., Xue, Q., Revill, W.P., Yu, T.W. and Floss, H.G. (2002) Functional expression of genes involved in the biosynthesis of the novel polyketide chain extension unit, methoxymalonyl-acyl carrier protein, and engineered biosynthesis of 2-desmethyl-2-methoxy-6deoxyerythronolide B. J. Am. Chem. Soc. 124, 5268^5269. Carroll, B.J., Moss, S.J., Bai, L., Kato, Y., Toelzer, S., Yu, T.W. and Floss, H.G. (2002) Identi¢cation of a set of genes involved in the formation of the substrate for the incorporation of the unusual ‘glycolate’ chain extension unit in ansamitocin biosynthesis. J. Am. Chem. Soc. 124, 4176^4177. Haydock, S.F., Aparicio, J.F., Molnar, I., Schwecke, T., Khaw, L.E., Konig, A., Marsden, A.F., Galloway, I.S., Staunton, J. and Leadlay, P.F. (1995) Divergent sequence motifs correlated with the substrate speci¢city of (methyl)malonyl-CoA :acyl carrier protein transacylase domains in modular polyketide synthases. FEBS Lett. 374, 246^248. Kakavas, S.J., Katz, L. and Stassi, D. (1997) Identi¢cation and characterization of the niddamycin polyketide synthase genes from Streptomyces caelestis. J. Bacteriol. 179, 7515^7522.
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[38] Kieser, T., Bibb, M.J., Buttner, M.J., Chater, T. and Hopwood, D.A. (2000) Practical Streptomyces Genetics: A Laboratory Manual. The John Innes Foundation, Norwich. [39] Lin, X., Kaul, S., Rounsley, S., Shea, T.P., Benito, M., Town, C.D., Fujii, C.Y., Mason, T., Bowman, C.L., Barnstead, M., Feldblyum, T.V., Buell, C.R., Ketchum, K.A., Ronning, C.M., Koo, H.L., Moffat, K.S., Cronin, L.A., Shen, M., Pai, G., Van Aken, S., Umayam, L., Tallon, L.J., Gill, J.E., Lee, J., Adams, M.D., Carrera, A.J., Creasy, T.H., Goodman, H.M., Somerville, C.R., Copenhaver, G.P., Preuss, D., Nierman, W.C., White, O., Eisen, J.A., Salzberg, S.L., Fraser, C.M. and Venter, J.C. (1999) Sequence and analysis of chromosome 2 of Arabidopsis thaliana. Nature 402, 761^768. [40] Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-speci¢c gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673^4680. [41] Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403^ 410. [42] Bierman, M., Logan, R., O’Brien, K., Seno, E.T., Rao, R.N. and Schoner, B.E. (1992) Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene 116, 43^49. [43] Flett, F., Mersinias, V. and Smith, C.P. (1997) High e⁄ciency intergeneric conjugal transfer of plasmid DNA from Escherichia coli to methyl DNA-restricting streptomycetes. FEMS Microbiol. Lett. 155, 223^229.
Cyaan Magenta Geel Zwart
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Cloning and characterization of a gene cluster for geldanamycin production in Streptomyces hygroscopicus NRRL 3602 Andreas Rascher a; , Zhihao Hu a , Nina Viswanathan a , Andreas Schirmer a , Ralph Reid a , William C. Nierman b , Matthew Lewis b , C. Richard Hutchinson b
a
a Kosan Biosciences, 3832 Bay Center Place, Hayward, CA 94545, USA The Institute for Genome Research, 9712 Medical Center Drive, Rockville, MD 20850, USA
Received 30 August 2002; received in revised form 7 November 2002; accepted 8 November 2002 First published online 12 December 2002
Abstract We illustrate the use of a PCR-based method by which the genomic DNA of a microorganism can be rapidly queried for the presence of type I modular polyketide synthase genes to clone and characterize, by sequence analysis and gene disruption, a major portion of the geldanamycin production gene cluster from Streptomyces hygroscopicus var. geldanus NRRL 3602. 4 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Ansamycin ; Antitumor; Biosynthesis ; Cancer chemotherapy; Hsp90; Polyketide synthase
1. Introduction Geldanamycin [1^4] (Fig. 1) and several other benzoquinone microbial products classi¢ed as ansamycin antibiotics [5], herbimycin A [6,7], macbecins [8], ansatrienins [9] and reblastatin [10,11] were discovered between 1970 and 2000 in screens for antibacterial, antifungal and antiviral compounds. Interest in the benzoquinone ansamycins increased greatly upon the discovery of the antitumor properties of geldanamycin [12] and herbimycin A [13], and the remarkable cytotoxic properties of the ansamitocins [14]. It was initially believed that geldanamycin and herbimycin A interfered with signal transduction pathways in tumor cells by direct inhibition of oncogenic protein tyrosine kinases. However, Neckers and co-workers [15] showed in 1994 that the principal cellular target is not a tyrosine kinase but Hsp90, a ubiquitous and abundant protein chaperone of mammalian cells [16]. Geldanamycin competes with ATP for the ATP-binding site of Hsp90 and, when bound, inhibits the ATP-dependent functions of Hsp90. A particular function is its ability to chaperone nascent protein kinases that are critical components of
* Corresponding author. E-mail address : [email protected] (A. Rascher).
signal transduction pathways, especially those in certain cancer cells [16]. In the presence of geldanamycin, herbimycin A or macbecin, the immature kinases undergo rapid degradation, as a consequence of ubiquitination and subsequent catabolism by the proteosome, and the levels of the mature kinases become depleted. This can result in a cytostatic e¡ect on a cancer cell or in some cases apoptosis and cell death. The discovery that Hsp90 and one or more of its protein kinase cohorts are overproduced in several types of human cancers has led to considerable interest in geldanamycin and its analogs as potential anti-cancer drugs [17]. Many geldanamycin analogs have been produced by replacement of the C17 O-methoxy group with substituted amines. One such drug, 17-allylamino-17-demethoxygeldanamycin, is currently undergoing Phase I clinical trials [18]. We are interested in engineering the geldanamycin polyketide synthase (PKS) genes to make novel geldanamycin analogs. To obtain the genes from the producer, Streptomyces hygroscopicus NRRL 3602, we faced a set of challenges common to the isolation of any PKS gene cluster from a streptomycete: bacteria commonly produce more than one polyketide metabolite (e.g. Streptomyces coelicolor has 10 [19] and Streptomyces avermitilis at least 13 [20] PKS gene clusters). We therefore developed a reliable method for querying the genomic DNA of the NRRL 3602 strain for the number of modular PKS genes, then
0378-1097 / 02 / $22.00 4 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-1097(02)01148-5
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OH
OH
OH CH3
15
17
HO
7
5 14
13
12
7
11
4
AHBA
CH3
21
OCH3
CH3
OCH3
CH3
11
CH3O CH3
CH 3
O 21
15
LDD:mod1
mod2
mod3
mod4
mod5
mod6
NH2
COS-ACP7
5
CH3 OCH3 4 OH CH3 NH
17
mod7
OH progeldanamycin
O
A)
O
5
5
S-ACP6
ER6
4
4
S-ACP6 1) ansamycin formation 2) C4,5-oxidation
5
C7 carbamoylation C17 hydroxylation C17 O-methylation C21 oxidation
4
OCONH2 CH3 CH3
CH3O
HNOC
OH
CH3
O
CH 3 OCH3
O
CH3
NH CH3O
O
geldanamycin Fig. 1. Main steps in the proposed biosynthesis of geldanamycin. The enzyme-bound product shown is assembled from AHBA and the carbon chain extender substrates, malonyl-CoA, 2-methoxymalonate and 2-methylmalonyl-CoA, by the PKS modules indicated beneath the product structure. The dashed line at position 4 signi¢es that the 4,5-CNC may be introduced by the PKS or afterwards as in A. Abbreviations : ACP, acyl carrier protein ; LDD, loading didomain; mod, module. The order of the four tailoring steps that convert progeldanamycin into geldanamycin is unknown.
used this method to help clone most of the geldanamycin production genes.
2. Materials and methods 2.1. Bacterial strains and culture conditions The geldanamycin-producing strain, ¢rst described by DeBoer et al. [1,2] as S. hygroscopicus var. geldanus var. nova UC-5208, was obtained from the Northern Regional Research Laboratory of the Agricultural Research Service as S. hygroscopicus NRRL 3602. To con¢rm production of geldanamycin, spores were used to inoculate 5 ml of R2YE liquid media [38]. The culture was incubated at 28‡C for 36 h, transferred into 100 ml geldanamycin production medium [1] and the ¢nal culture was incubated at 28‡C in 500-ml Erlenmeyer £asks at 300 rpm for another 5 days. Following low-speed centrifugation, the cell pellet from the culture was extracted with methanol by stirring for 10 min. The methanol broth was clari¢ed by centrifu-
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gation (17 500Ug) and the supernatant was analyzed for the presence of geldanamycin using high-performance liquid chromatography (HPLC) under the following conditions: column Inertsil C18 (4.6U150 mm, Ansys Technologies, Inc.), mobile phase 60% acetonitrile (isocratic), £ow rate (2 ml min31 ), temperature (40‡C), detection (UV 315 nm), injection volume (10^20 Wl). Geldanamycin (SigmaAldrich) was quanti¢ed by comparing the peak area at 315 nm with that measured for a standard solution. The titer of geldanamycin was approximately 250 mg l31 . 2.2. Manipulation of DNA and organisms For genomic DNA extraction, a spore stock was used to prepare a seed culture as described above. The entire seed culture was transferred into 50 ml of the same growth medium in a 250-ml ba¥ed Erlenmeyer £ask and incubated for 48 h at 28‡C. A 20-ml portion of the cell suspension was centrifuged (10 000Ug) and the resulting pellet was washed with 10 ml bu¡er 1 (Tris, 50 mM, pH7.5 ; 20 mM EDTA). The pellet was pulverized with mortar and
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pestle under liquid nitrogen and transferred into 3.5 ml of bu¡er containing 150 Wg ml31 RNase (Sigma-Aldrich). After incubation of the mixture at 30‡C for 20 min, the salt concentration was adjusted by adding 850 Wl 5 M NaCl solution, then the mixture was extracted multiple times with phenol:chloroform:isoamylaclohol (25:24:1, vol/vol) with gentle agitation followed by centrifugation for 10 min at 3500Ug. After precipitation with 1 vol of isopropanol, the genomic DNA knot was spooled on a glass rod and redissolved in water (200 Wl). This method yielded about 1 mg DNA with a protein factor of about 2, as determined by the ratio of the UV absorbances at 260 and 280 nm. Standard agarose gel electrophoresis using 0.7% Seakem0 LE-Agarose (BioWhiaker Molecular Applications, Rockland, ME) at a voltage of 50 mV overnight revealed that the sample contained mainly high-molecular-mass DNA fragments of about 60 kb. 2.3. Genomic analysis of S. hygroscopicus NRRL 3602 The following degenerate keto synthase (KS) primers were used to scan the genomic DNA of S. hygroscopicus for PKS genes: degKS1F (5P-TTCGAYSCSGVSTTCTTCGSAT-3P), degKS2F (5P-GCSATGGAYCCSCARCARCGSVT-3P), degKS3F (5P-SSCTSGTSGCSMTSCAYCWSGC-3P), degKS5R (5P-GTSCCSGTSCCRTGSSCYTCSAC-3P), degKS6R (5P-TGSGYRTGSCCSAKGTTSSWCTT-3P) and degKS7R (5P-ASRTGSGCRTTSGTSCCSSWSA-3P). Forward (F) and reverse (R) primers were tested in all possible combinations in standard PCR reactions with annealing temperatures between 50 and 60‡C. A typical 50 Wl PCR reaction consisted of 200 ng genomic DNA, 200 pmol of each primer, 0.2 mM dNTP (containing 7-deaza-dGTP), 10% DMSO and 2.5 U Taq DNA polymerase (Roche Applied Science). Carbamoyl transferase gene fragments were ampli¢ed with degenerate forward primer degCT2F (5P-AARGTSATGGGSYTSGCSCCSTA-3P) and reverse primers degCT3R (5P-CCSARSGCSCKSGGSCCRAAYTC-3P) or degCT4R (5P-CKSGCSSWSCCRTCSACRTGSGT-3P) using an annealing temperature of 55‡C. 3-Amino-5-hydroxybenzoate (AHBA) synthase gene fragments were ampli¢ed with a set of two degenerate forward primers, degAH-F1 (5P-GTSATCGTSCCSGCSTTCACSTTC-3P) and degAH-F2 (5P-ATCATGCCSGTSCAYATGGCSGG-3P), and two reverse primers, degAHR1 (5P-GGSTBSGKGAACATSGCCATGTA-3P) and degAH-R2 (5P-CKRTGRTGSARCCASTKRCARTC-3P). Again forward and reverse primers were tested in all combinations in the described standard PCR reactions with annealing temperatures between 50 and 65‡C. A set of four gene speci¢c primers, AH-B-spF (5P-AGGACAGTGGCGCGGCAAGAA-3P), AH-B-spR (5P-GGTCGACGATCTTCGCGCGGCG-3P), AH-N-spF (-5PTCGACGTGGCTGCCGCGGCTT-3P) and AH-N-spR (5P-TGTCGACGAGGGCGTTGCGGG-3P), was used to
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distinguish between AHBA-B and AHBA-N synthase genes. PCR amplimers were gel-puri¢ed and cloned into pCR2.1-TOPO using TA cloning (Invitrogen). For each primer pair, a representative set of cloned amplimers (600^800 bp) was sequenced using a Beckman CEQ2000 with M13 forward and reverse primers. 2.4. Library construction and gene isolation A genomic library of the NRRL3602 strain was constructed at TIGR using the proprietary single-copy BAC vector pHOS3. Sheared genomic DNA was made bluntended using T4 DNA polymerase and BstXI adaptors were ligated to the blunt ends. Adaptors were removed and inserts were size-selected by three rounds of ¢eld inversion gel electrophoresis. The inserts were ligated to the vector and electroporated into Escherichia coli. A total of 4896 BAC clones were arrayed into 384-well microtiter plates and spotted in high density onto nylon ¢lters (Amplicon Express). Probes were labeled using [K-32 P]dCTP and a random prime-labeling system (rediprime II, Amersham Pharmacia Biotech). Filters were hybridized at 68‡C for 12 h using ExpressHyb hybridization solution (Clontech). After removal of the probe and hybridization solution, the ¢lter was washed twice for 30 min each time with 100 ml of bu¡er I [2USSC: 300 mM NaCl, 30 mM sodium citrate pH 7.0, 0.05% sodium dodecyl sulfate (SDS)] at room temperature and then three times for 60 min each time at 50‡C with 100 ml of bu¡er II (0.1USSC, 0.1% SDS) with continuous shaking. Finally, the ¢lter was rinsed several times with 0.05USSC and analyzed by autoradiography. BAC DNA was prepared by alkaline lysis, starting with 10 ml culture volume. The resulting DNA was ¢rst treated with RNase (Sigma-Aldrich) at 30‡C for 3 h and then with plasmid safe DNase (Epicentre Technologies) at 37‡C o/n. After heat inactivation at 70‡C for 10 min the DNA was precipitated with 1 volume isopropanol for 30 min on ice and recovered by centrifugation at 1880Ug for 45 min to separate the remaining smaller fragments from the large, intact BAC plasmids. The ¢nal pellet was washed with 70% EtOH and redissolved in 80 Wl water. This method typically yielded about 100 Wg of BAC DNA. 2.5. DNA sequence analysis BAC DNA was sequenced to completion using the standard shotgun procedures [39]. The DNA and deduced protein sequences were analyzed with Sequencer 4.1 (Gene Codes Corporation) and MacVector 6.5.3 (Accelrys) software, and compared with sequences in the public databases using the CLUSTAL W [40] and BLAST [41] programs. The sequence data for the genes displayed in Fig. 2 have been deposited with GenBank (accession number AY179507).
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Fig. 2. Geldanamycin gene cluster. The colored broad arrows indicate the genes and their direction of transcription. PKS and gdmF genes are red, 2-methoxymalonate synthesis genes are orange, the AHBA synthesis gene is green and tailoring enzyme genes are yellow. The right end of the cluster continues beyond ORF22. The domains in each of the seven modules of the gdm PKS are indicated by the abbreviations above the gdmA arrows (see Table 1 for de¢nitions). Scale beneath the genes indicates the distance in kb from the beginning of the 85-kb region sequenced.
2.6. Disruption of the gdmH gene The gdmH gene disruption vector was made as follows. The aphII neomycin/kanamycin resistance gene from Tn5 was excised as a StuI^SmaI fragment from SuperCos-1 (Stratagene), then inserted into the MscI site within gdmH carried in a 4-kb BstXI fragment, containing the gdmN, gdmH and gdmI genes, and cloned in pOJ260 [42] to give pKOS246-33. The XbaI^EcoRI fragment from pKOS246-33 was excised and cloned into the XbaI^EcoRI sites of pKC1139 [42] to give pKOS279-37. The gdmH gene was disrupted by introducing pKOS279-37 into the S. hygroscopicus NRRL3602 strain by conjugation from E. coli ET12567/pUB307 according to a published method [43]. Exconjugants resistant to apramycin (pKC1139 carries the accIV(3) gene) and kanamycin were isolated and one of them was grown at 30‡C in 6 ml of R5 liquid medium [38] supplemented with 100 Wg ml31 of kanamycin for 2 days in 50-ml culture tubes at 200 rpm. Approximately 5% of this culture was transferred into 6 ml of fresh R5/apramycin liquid medium and the culture was grown at 37‡C for 3 days in order to force chromosomal integration of pKOS279-37. After recovery of the mycelia by centrifugation, cells were plated on tomato paste medium containing 100 Wg ml31 kanamycin and grown at 30‡C for sporulation. Spores collected from these plates were diluted and replated on the same medium for single colonies. Among 100 colonies screened, 20 of them were apramycin sensitive and kanamycin resistant when assayed on plates containing apramycin or kanamycin, using 60 or 50 Wg ml31 of antibiotic, respectively. Genomic DNA was isolated from 11 of these 20 colonies by an established method [38] and probed by Southernblot hybridization [38] with the aphII gene to determine
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that all kanamycin resistant recombinant strains had the restriction fragment pattern upon digestion with PstI^ EcoRV expected for integration of the aphII gene into the gdmH locus by a double crossover recombination (hybridizing bands at 2.9 and 3.2 kb that were absent in the NRRL3602 strain). To determine geldanamycin production, each of the 11 strains was individually cultured in 35 ml of the geldanamycin production medium [1] as described above. After 4 days, 500 Wl of broth from each £ask was mixed with 500 Wl of methanol, the mixture was centrifuged at 12,000 rpm in a desktop microcentrifuge for 5 min to remove mycelia and other insoluble ingredients, then the supernatant fraction was analyzed by HPLC/MS. The results showed that geldanamycin was present (retention time and low-resolution MS data were identical to the reference standard) and that two new compounds were present with molecular masses and formulas of 518.2759 (C28 H40 NO8 [M-H]3 ) and 520.2916 (C28 H42 NO8 [M-H]3 ), calculated on the basis of high-resolution MS data. These data are consistent with 4,5-dihydro-7-descarbamoyl-7-hydroxygeldanamycin and its hydroquinone form.
3. Results and discussion 3.1. Survey of modular PKS genes in S. hygroscopicus NRRL 3602 Geldanamycin biosynthesis can be depicted as shown in Fig. 1 on the basis of isotope-labeling studies by the Rinehart laboratory [4,21]. That work forecast the involvement of a PKS that would use AHBA [22^25] as the chain initiation substrate for a seven-module type I PKS, and
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malonate, 2-methylmalonate and 2-methoxymalonate, as chain extender substrates to produce the putative progeldanamycin (Fig. 1). Geldanamycin can be formed from this polyketide by four tailoring steps, as shown. For the initial approach we used standard bioinformatics methods to choose six highly conserved yet recognizably di¡erent regions of the amino acid sequence of KS domains among 20 known modular PKSs. Three primer combinations (see Section 2) consistently gave correctly sized PCR products with all organisms tested (data not shown) and were chosen to survey the complexity of PKS genes in the genome of S. hygroscopicus NRRL 3602. Of the 90 KS amplimers cloned and sequenced, 63 represented unique type I PKS KS gene fragments, but when they were analyzed further by comparison with the KS sequences of the ansamitocin [23] and rifamycin [24] PKSs, the evidence that they were part of the geldanamycin PKS genes was not conclusive. Homologs of the genes for formation of AHBA and for the C7 carbamoylation step were then chosen to design a new set of PCR primers that were used to screen the genomic DNA for AHBA synthase [23^25] and carbamoyltransferase (CT) [23,26,27] homologs. We analyzed 56 AHBA amplimers and identi¢ed two di¡erent DNA sequences that would encode AHBA synthases that are 75% identical, AHBA-B and AHBA-N. The data, when analyzed by standard bioinformatics methods (Section 2), strongly suggest that one AHBA synthase homolog belongs to the family associated with the biosynthesis of benzoquinone ansamycins (AHBA-B) [23] and the other with naphthoquinone ansamycins (AHBA-N) [24,25]. All 20 CT amplimers analyzed were identical, which let us conclude that there is only one CT gene in this strain. 3.2. Cloning and sequence analysis of geldanamycin production genes The AHBA-B synthase and CT amplimers were used as probes for primary screening of a genomic library made in a single-copy BAC vector, pHOS3, a derivative of pBeloBAC11 [28] modi¢ed for BstXI cloning. We screened 4896 BACs with average insert sizes of 45 kb, which was equivalent to ca. 20U coverage of this genome, and identi¢ed 36 AHBA synthase and 16 CT positive BACs. AHBA-B and AHBA-N synthase containing BACs were distinguished by PCR analysis, which established that about half of these BACs belonged to each of the AHBA-B and AHBA-N types. Interestingly, when these were analyzed for the presence of PKS genes by PCR analysis, we found that none of the 20 AHBA-B synthase containing BACs contained PKS genes, whereas 14 out of 17 AHBAN synthase containing BACs also had PKS genes. We thus focused on the CT containing BACS to identify the geldanamycin PKS genes. Nine out of 15 CT BACs also contained PKS genes and from these CT+PKS BACs, we subcloned four unique KS amplimers using the degenerate
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PCR primers. These putative geldanamycin KS sequences (one of which was contained in the 63 KS amplimers described above) were then used as probes in a secondary screen of the BAC library at high stringency to identify seven additional overlapping BACs. Two overlapping clones, pKOS256-107-3 and pKOS256154-1, spanning a V85-kb region, were fully sequenced by the shotgun method, and the resident genes were assigned to geldanamycin production on the basis of database comparisons (Fig. 2). Twenty-three open reading frames (ORF) and the deduced functions of their products are listed in Table 1. The CT gene probe matched the sequence of gdmN, and only one homolog of the AHBA synthase genes was found (gdmO). An additional 15 ORFs downstream of ORF16 were sequenced and analyzed (not shown) but, on the basis of their deduced functions, are not believed to be involved in geldanamycin production. (There are incomplete reports about cloning the geldanamycin genes from other streptomycetes isolated independently from the 3602 strain [29,30].) 3.3. Key features of the geldanamycin biosynthesis genes and their deduced products 3.3.1. The PKS A seven-module PKS encoded by gdmA1-A3 and immediately followed by the gdmF gene encoding an amide synthase, as found for the rifamycin and ansamitocin PKSs [23,24], is entirely consistent with the biosynthetic hypothesis shown in Fig. 1. The N-terminal region of module 1 of the GdmA1 protein contains two domains strongly resembling their counterparts in the rifamycin and ansamitocin PKSs. Both of the latter enzymes use AHBA as the starter unit, and the RifA loading module recently has been shown to load the AHBA onto the acyl carrier protein (ACP0) domain through formation of an intermediate acyl-adenylate catalyzed by the acyl ligase domain [31]. The starter unit is transferred intramolecularly from ACP0 to the KS1 of the following module for reaction with the 2-methylmalonate chain extender unit bound to ACP1 [32]. Modules 2 and 5 of the GdmA1 and GdmA2 proteins, respectively, have acyltransferase (AT) domains with the speci¢c motifs similar to other ATs that utilize 2-methoxymalonate (unpublished data). The substrate for AT2 and AT5 is presumed to be provided by the products of the gdmG-gdmK genes, given the recent genetic and functional characterization of their homologs from the FK520 [33] and ansamitocin [34,35] gene clusters. In contrast, the AT domain of module 6 of GdmA3 clearly falls within the class that uses malonylCoA, and those of GdmA1 module 1 and module 3, GdmA2 module 4 and GdmA3 module 7 strongly resemble those that use 2-methylmalonyl-CoA [36,37]. Finally, the presence of enoyl reductase (ER) domains in modules 1, 2 and 3, dehydratase (DH) domains in modules 1, 2, 4, 6 and 7, and keto reductase (KR) domains in all the mod-
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ules ¢t the requirements for the predicted functionality of progeldanamycin (Fig. 1). The GdmA PKS has one notable feature. The ER domain in module 6 is apparently functional yet geldanamycin has a C4 cis-double bond. Consequently, it is plausible that the PKS reduces this double bond and that the C4 cis-double bond is introduced oxidatively either before or after formation of progeldanamycin (Fig. 1A). 3.3.2. Tailoring genes The predicted functions for most of these genes are consistent with the requirements for conversion of progeldanamycin (or its 4,5-dihydro form) into geldanamycin (Fig. 1). Disruption of the gdmH gene, carried out by insertion of a kanamycin resistance gene (Section 2), resulted in strain K279-37 that produced geldanamycin together with a compound whose high-resolution mass spectral data are consistent with 4,5-dihydro-7-descarbamoyl7-hydroxygeldanamycin. This result con¢rms the function assigned to gdmN (the partial inactivation of this gene is believed to be the consequence of read-through expression) and shows that gdmH is dispensible or that its mutation is compensated in trans by a paralog. Distinction among the three genes, gdmL, gdmM and gdmP, likely to
govern C17 hydroxylation, C21 oxidation and C4,5 unsaturation, if that actually occurs, will have to await studies of mutants or the expressed and puri¢ed enzymes. The putative C17 O-methyltransferase gene presumably lies outside the currently sequenced region. 3.3.3. Other genes Possible regulatory genes (gdmR1, gdmR2, ORF19 and ORF20) and a gene for the amino dehydroquinate synthase needed for AHBA biosynthesis (gdmO) are listed in Table 1 as part of the gdm gene cluster because their homologs have been found in other clusters of bacterial secondary metabolism genes. ORF16 and ORF17 are candidates for putative geldanamycin resistance and/or export genes. All but one of the remaining genes for AHBA biosynthesis in the geldanamycin pathway, as deduced from the sequences of homologs involved in the biosynthesis of other benzoquinone ansamycins [22^24], are clustered elsewhere in the genome, more than 20 kb from the end of the BAC that contains ORF22 (data not shown). This is consistent with the observation that three of the AHBA biosynthesis genes for ansamitocin production and the remaining asm genes are in a subcluster separated from all the other genes for AHBA biosynthesis by 30 kb [23].
Table 1 Gene designations and functions Predicted polypeptide
Amino acids
Homologous gene(s)
Putative function
ORF16 ORF17 ORF18 ORF19 ORF20 GdmL GdmX GdmA1 Loading Module 1 Module 2 Module 3 GdmA2 Module 4 Module 5 GdmA3 Module 6 Module 7 GdmF GdmM GdmN GdmH GdmI GdmJ GdmK GdmG GdmR2 GdmO GdmP GdmR1 ORF22
227 300 263 294 253 479 156 6842
RhtB family transporters SC3C8.01 SCF1.09 AraC family TetR family Rif19 JadX; MmyY
E¥ux Secreted protein Hydrolase Transcriptional regulation Transcriptional regulation Unknown Unknown PKS (modules 0^3) AL X ACP KS AT DH ER KR ACP KS AT DH ER KR ACP KS AT KR ACP PKS (modules 4^5) KS AT DH KR ACP KS AT KR ACP PKS (modules 6^7) KS AT DH ER KR ACP KS AT DH KR ACP Amide synthase Unknown CT Methoxymalonyl-ACP biosynthesis Methoxymalonyl-ACP biosynthesis Methoxymalonyl-ACP biosynthesis Methoxymalonyl-ACP biosynthesis Methoxymalonyl-ACP biosynthesis Transcriptional regulation AminoDHQ synthase P450 Transcriptional regulation Hydrolase
3435
3896
257 545 682 370 370 91 288 218 926 354 397 962 237
RifF Rif19 NovN FkbH FkbI FkbJ FkbK FkbG LuxR family Asm47 PikC LuxR family SC2G4.19
Abbreviations for PKS domain functions: ACP, acyl carrier protein; AL, acyl-CoA ligase ; AT, acyltransferase ; CT, carbamoyltransferase ; DH, dehydratase; ER, enoyl reductase ; KR, keto reductase; KS, keto synthase; X, unknown.
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Acknowledgements We thank David Hopwood, Robert McDaniel, Chris Reeves and Peter Revill for helpful comments during preparation of the manuscript. This research was supported in part by a Small Business Innovative Research grant from the National Institute of Health (R43 CA96262 and AL38947).
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