Identification of the Post-Polyketide Synthase Modification Enzymes

Apr 15, 2013 - Beijing Biostar Technologies, Ltd., Beijing, 101111, People,s Republic of China. •S Supporting Information. ABSTRACT: Fostriecin (FST...
0 downloads 0 Views 858KB Size
Download PDF
Article pubs.acs.org/jnp

Identification of the Post-Polyketide Synthase Modification Enzymes for Fostriecin Biosynthesis in Streptomyces pulveraceus Xue-jiao Liu,†,§ Ri-xiang Kong,†,§ Ming-shan Niu,† Rong-guo Qiu,†,‡ and Li Tang*,†,‡ †

Research Center for Molecular Medicine, Faculty of Chemical, Environmental and Biological Science and Technology, Dalian University of Technology, Dalian, 116024, People’s Republic of China ‡ Beijing Biostar Technologies, Ltd., Beijing, 101111, People’s Republic of China S Supporting Information *

ABSTRACT: Fostriecin (FST, 1) is a natural product with promising antitumor activity produced by Streptomyces pulveraceus. Its antitumor activity is associated with the selective inhibition of protein phosphatase activities. The biosynthetic gene cluster for FST has recently been cloned and sequenced. To better understand the post-polyketide synthase (PKS) modification steps in the biosynthetic pathway of FST, we constructed and characterized three post-PKS modification gene mutants of fosG, fosK, and fosM by knockout inactivation in S. pulveraceus. As a result, we determined that a fosKencoded cytochrome P450 monooxygenase is responsible for C-18 hydroxylation, formation of an unsaturated lactone is dependent upon FosM, and the fosG gene product is involved in hydroxylation at C-4 after the action of FosM to yield PD 113,271 from FST. The accumulated analogues from the ΔfosK and ΔfosM mutant strains possessed a malonyl ester moiety that suggested that all the post-PKS modification steps in FST biosynthesis occur with the polyketide chain bearing a malonyl ester at the C-3 position, with formation of the unsaturated six-membered lactone as the last step in FST biosynthesis.

P

olyketides are complex natural products produced by bacteria and fungi. Their applications range from antibiotics and immunosuppressants to antitumor agents.1 The important medicinal and agrochemical properties of polyketides have attracted considerable interest.2 Some polyketides are difficult to synthesize or modify chemically. Recently, combinatorial biosynthesis methods have attracted intensive attention and have emerged as a powerful tool to diversify this class of novel antitumor agents.3 One valuable class of polyketides is fostriecin (FST, 1) and its closely related analogues. FST (CI-920), along with two structurally related compounds, PD 113,270 (2) and PD 113,271 (3) (Figure 1A), is produced by Streptomyces pulveraceus subspecies fostreus ATCC 31906.4,5 It exhibits in vitro activity against a wide range of tumor cell lines such as leukemia, lung cancer, and breast cancer cell lines.6 FST inhibits DNA topoisomerase II (IC50 = 40 μM)7 and is also a potent inhibitor of protein serine/threonine phosphatases.8 Its antitumor activity is accounted for by its highly selective inhibition of protein phosphatases PP2A and PP4 (IC50 = 1.5 and 3.0 nM, respectively).9,10 The chemical and biological activities of FST have been reviewed.11 Owing to storage instability and unpredictable purity of FST preparations, clinical trials of FST were halted in early phase I.6,7,11 In nature, FST is first synthesized by modular FST polyketide synthases (PKSs) and then modified by post-PKS enzymes. Once a polyketide intermediate is released from the PKS assembly line, it is subjected to the post-PKS modifying © 2013 American Chemical Society and American Society of Pharmacognosy

Figure 1. Structure and biosynthetic gene cluster of FST. (A) Structures of FST and its related analogues. (B) Biosynthetic gene cluster for FST. PKS genes are shown in gray; post-PKS modification genes are represented by a diagonal grid pattern; other genes are in white.

reactions. These reactions are important for the biological activity and diversity among the polyketides produced. Recently, we cloned and sequenced the biosynthetic gene cluster for FST biosynthesis.12 On the basis of predicted functions for each post-PKS enzyme inferred from sequence similarities, it was proposed that the nascent polyketide chain Received: September 27, 2012 Published: April 15, 2013 524

dx.doi.org/10.1021/np300667r | J. Nat. Prod. 2013, 76, 524−529

Journal of Natural Products

Article

undergoes a series of post-PKS modifications including hydroxylation (C-8, C-18, or C-4), C-9 phosphorylation, and formation of a double bond between C-2 and C-3 to produce FST. In our previous work, we demonstrated that FosJ and FosH were responsible for the C-8 hydroxylation and C-9 phosphorylation in the biosynthesis of FST using in vivo gene inactivation experiments and in vitro enzyme activity assays.12 Given these results, we proposed a plausible biosynthetic mechanism for FST biosynthesis. Herein, we delineated the functions of three genes fosG, fosK, and fosM in S. pulveraceus via gene inactivation and isolated two new analogues, FOS K-1 (4) and FOS M-1 (5). Complementation experiments for these mutants led to the restoration of FST production, which further confirmed the roles of these genes. Our results verified the hypotheses that FosG and FosK functioned as two hydroxylases and FosM catalyzed an elimination reaction. They also provided a highly confident prediction of the pathway for post-PKS modification in FST biosynthesis from an experimental point of view. Furthermore, these findings will contribute to a better understanding of the process for FST biosynthesis in vivo and pave the way for the production of new FST analogues using combinatorial biosynthesis methods.

■

RESULTS AND DISCUSSION Analysis of the FST biosynthetic gene cluster along with the structures of FST and its related analogues indicated that there are three post-PKS hydroxylation steps (C-4, C-8, and C-18) after the assembly of the polyketide chain (Figure 1). Significant similarities were found among a number of bacterial cytochrome P450 hydroxylases, such as Tyll, 13 PicK (PikC),14,15 EryF,16 and PlmS2.17 All of these enzymes catalyze the hydroxylation of aliphatic macrolide structures. In addition to the fosJ gene, a sequence alignment indicated that fosG and fosK encoded another two cytochrome P450 monooxygenases, which play important roles in the biosynthesis of natural products produced by Streptomyces bacteria.18,19 In the present work, we inactivated the other two cytochrome P450 enzymes in FST biosynthesis, encoded by fosG and fosK, using the PCR targeting system.20 The ΔfosG and ΔfosK mutant strains were named STQ0705 and STQ0707 (Supporting Information, Table S1). Their genotypes were confirmed by PCR amplification using specific primers (Supporting Information, Table S2, Figures S1 and S2). We cultured the mutants STQ0705 and STQ0707 and the S. pulveraceus wild-type strain as a control. When cultured, STQ0705 (ΔfosG) failed to produce detectable amounts of 3, but accumulated FST as the major product (Figure 2A-b). This result suggested that the deletion of the fosG gene had no discernible effect on FST production, demonstrating that it is not an essential gene for FST biosynthesis, but it was involved in hydroxylation at C-4 to modify FST to yield 3, which was commonly observed in the wild-type culture. We then thought that the ΔfosK mutant should abolish FST production and generate exclusively 2. However, HPLC analysis of the fermentation broth from this mutant indeed demonstrated abolished FST production, but showed the accumulation of one major metabolite (4) (Figure 2A-d). This compound was purified following standard protocols and identified using HRMS, LC-MS/MS, and 1H NMR spectroscopic analysis (Table 1). High-resolution mass measurement for compound 4 was consistent with a formula of C22H31O12P for m/z 517.1460 ([M − H]−), a 104 mass-unit increase in molecular weight

Figure 2. FST biosynthetic intermediates accumulated in the ΔfosG, ΔfosK, and ΔfosM mutant strains STQ0705, STQ0707, and STQ0709. (A) HPLC traces of metabolite profiles from S. pulveraceus wild-type and mutant strains: (a) S. pulveraceus wild-type; (b) STQ0705 (ΔfosG mutant); (c) STQ0706 (complemented ΔfosG mutant); (d) STQ0707 (ΔfosK mutant); (e) STQ0708 (complemented ΔfosK mutant); (f) STQ0709 (ΔfosM mutant); (g) STQ0710 (complemented ΔfosM mutant). Numbers are shown above each peak consistent with FOS K1 (4) and FOS M-1 (5). (B) Structures of 4 from the ΔfosK mutant strain STQ0707 and 5 from the ΔfosM mutant strain STQ0709, as deduced on the basis of HRMS, LC-MS/MS, and 1H NMR.

compared to 2. Combined with the structural analysis of compounds 6,12 7,12 and M-PLMB,21 our data suggested that a malonyl ester moiety may exist in the molecular structure of 4. In the HRESIMS/MS analysis of compound 4, two fragments, [M − CO2]− and [M − COCH2CH2 − H2O]−, were clearly discerned, which also indirectly demonstrated the existence of a malonyl ester moiety in 4 (Figure S4). Compared to the 1H NMR spectrum of 2,22 the H-2 proton signal at δH 5.97 was absent, replaced in compound 4 by a pair of doublets of doublets at δH 2.93 and 2.75. The H-3 proton signal at δH 7.02 was shifted upfield to δH 5.26, whereas the H2-4 proton signals were shifted upfield to δH 2.22 and 1.95 from δH 2.45 and 2.55, respectively. All of the other proton signals were consistent with the signals from 2. Therefore, the main proton signal changes focused attention on the C-2, C-3, and C-4 positions and showed that a saturated lactone with an oxygenated C-3 had been generated in compound 4. The protons of the malonate-CH 2 were initially observed at δH 3.22 but disappeared rapidly, showing that they are relatively acidic protons that can readily undergo deuterium exchange in CD3OD. Compared with the previously established structure of compound 7, the major change was the signal of H-9, which 525

dx.doi.org/10.1021/np300667r | J. Nat. Prod. 2013, 76, 524−529

Journal of Natural Products

Article

Table 1. 1H NMR (400 MHz) Spectroscopic Data for Compounds 4 and 5 in CD3OD (ppm) position 1 2 3 4 5 6 7 8 CH3 (8) 9 10 11 12 13 14 15 16 17 18 2′

4

5

δH (J in Hz)

δH (J in Hz)

2.93, 2.75, 5.26, 2.22, 5.19, 5.86, 5.97,

dd (16.1, dd (16.1, m 1.95, m m dd (15.5, dd (15.5,

4.6) 4.6)

1.28, 4.23, 1.47, 4.85, 5.49, 6.45, 6.19, 5.75, 6.55, 5.97, 1.80, 3.20,

s m m m t (9.6) t (11.5) t (11.4) t (11.1) dd (14.0, 12.0) dd (14.0, 7.0) d (6.8) AB q (14.4)

6.9) 8.2)

2.91, 2.74, 5.26, 2.26, 5.20, 5.80, 5.90,

dd (16.1, dd (16.1, m 1.99, m m dd (15.5, dd (15.5,

1.28, 4.23, 1.48, 4.85, 5.52, 6.50, 6.35, 6.09, 6.78, 5.98, 4.15, 3.22,

s m 1.66, m m t (9.6) t (11.5) t (11.4) t (11.1) dd (14.0, 12.0) dd (14.7, 7.0) d (5.1) AB q (14.4)

Many polyketide natural products contain unsaturated double bonds in their structures23 that are generated by ketoreductase-dehydratase (KR-DH) domains within the appropriate modules.24,25 A bioinformatics analysis of the terminal module (module 8) in the FST modular PKS12 suggested that it might generate a 3-hydroxy intermediate due to the lack of a cognate DH domain in the FST biosynthesis gene cluster. Such DH domains are also absent in the terminal modules for the biosynthesis of leptomycin26 and phoslactomycin,27 related natural products with unsaturated lactone moieties. Palaniappan et al.21 have reported that PlmT2 is responsible for the efficient formation of unsaturated lactones in the biosynthesis of phoslactomycins and proposed that FosM (named ORF4 in their study) might be the crucial enzyme involved in formation of the unsaturated lactone in FST biosynthesis, although experimental evidence was not available for the FST producer S. pulveraceus. To elucidate the specific role of FosM in FST biosynthesis, we inactivated the fosM gene in S. pulveraceus using the REDIRECT Technology20 to produce the ΔfosM mutant strain STQ0709 (Table S1). Its genotype was confirmed by PCR amplification using specific primers (Table S2 and Figure S3). When culturing the mutant STQ0709 side-by-side with the S. pulveraceus wild-type strain as a control, mutant STQ0709 accumulated one major new metabolite (5) and a small amount of FST, but no detectable 3 (Figure 2A-f, B). Compound 5 was then identified through HRMS, LC-MS/MS, and 1H NMR spectroscopic analysis (Table 1). A high-resolution MS measurement for compound 5 was consistent with a formula of C22H30O13P for m/z 533.1441 ([M − H]−). Compared with FST, the increase of 104 mass units in the molecular weight demonstrated that a malonyl ester moiety must also exist in 5, which finding was also confirmed by the LC-MS/MS (Figure S5) and 1H NMR analyses (Table 1). The same chemical shift changes of the protons at the C-2, C-3, and C-4 position were observed as in compound 4. The unique change for compound 5 compared with compound 4 was that there was an H2-18 methylene signal at δH 4.15 compared to an H3-18 methyl signal at δH 1.80 in 4, indicating that hydroxylation occurred at the C-18 position. Compound 5 also has very low stability, which is the most likely reason for the low-level production of FST in the fermentation of STQ0709 (Figure 2A-f, Figure S9). A complementation experiment was carried out in the STQ0709 strain using the expression plasmid for the fosM gene (pSET153M-tsr). When fermented for more than 2 days, the STQ0710 ( fosM) strain completely restored FST production in a complementation experiment. This result confirmed that FosM is critical for catalyzing an elimination of the malonate moiety to generate the unsaturated lactone in FST biosynthesis and can accelerate the production of FST (Figure 2A-g). According to prior PKS precedents,28 the C-3 site of the sixmembered ring of the FST polyketide should bear a hydroxy group when the polyketide is liberated by a thioesterase (TE) due to the lack of a DH domain in the terminal module. However, FST biosynthesis intermediates in which C-3 lacks a malonyl ester were not detected in the fermentation cultures of any of the post-PKS gene (fosJ, H, K, M) mutants we generated or from the wild-type strain, indicating that all of the post-PKS modification steps such as the C-8 hydroxylation, phosphorylation of the C-9 hydroxy group, and C-18 hydroxylation occurred on the polyketide chain bearing a malonyl ester at the C-3 position. Intermediates 4, accumulated from the ΔfosK

4.6) 4.6)

6.9) 8.2)

was shifted downfield to δH 4.23 from δH 3.69, indicating that a phosphoryl group could be linked to the hydroxy group at the C-8 position. Although compound 4 was not subjected to 13C NMR analysis here due to the isolation difficulties and low stability, the HRMS, LC-MS/MS, and 1H NMR analysis data were sufficient for interpretation of the chemical structure for compound 4 in comparison to closely related metabolites. The configuration of C-3 in 4 could not be confirmed according to the present NMR data. However, according to the amino acid sequence analysis of the KR8 domain, which lacks the conserved characteristic Leu-Asp-Asp (LDD) motif and can be designated as the A-type KR domain, A-type KRs give 3S hydroxy groups in the PKS elongation. So we can propose that the configuration of C-3 in the six-membered lactone ring of 4 may be an S malonyl ester moiety, similar to the configuration of C3-OH in PLM biosynthesis proposed by Palaniappan et al.21 Further confirmation work needs to be done by NOE analysis. Compound 2 was identified as a minor product in the fermentation broth of STQ0707, likely because 4 was comparatively unstable and underwent an elimination reaction to yield the observed 2 (Figure 2A-d, Figure S8). The structure of compound 4 (lacking the C-18 hydroxy group) upon inactivation of the fosK gene in S. pulveraceus provided direct evidence that FosK catalyzed the C-18 hydroxylation of FST (Figure 2B). Genetic complementation experiments were performed with expression of the intact genes (fosG and fosK) in mutant strains STQ0705 and STQ0707 using plasmids pSET153G-tsr and pSET153K-tsr, respectively. HPLC analyses (Figure 2A-c, A-e) showed a complete restoration of 3 production with fosG complementation in the STQ0706 strain and the restoration of FST production with fosK complementation in the STQ0708 strain. These data confirmed that fosGand fosK-encoded cytochrome P450 monooxygenases are responsible for the C-4 and C-18 hydroxylations, respectively. 526

dx.doi.org/10.1021/np300667r | J. Nat. Prod. 2013, 76, 524−529

Journal of Natural Products

Article

mutant, and 6 and 7, from the ΔfosJ and ΔfosH mutants previously reported,12 respectively, all contain malonyl esters in their structures and are the major products in the fermentations of those mutants (Figure 4). The C-3 malonyl ester moiety in

as shown in Figure 3, similar to the process proposed by Palaniappan et al.21 Previously, we demonstrated that C-8 hydroxylation by FosJ occurs first and is followed by C-9 phosphorylation by FosH in the post-PKS modification reactions leading to FST.12 In this work, the accumulations of 4 in the ΔfosK mutant and 5 in the ΔfosM mutant support our previous hypothesis of the C-18 hydroxylation as the penultimate step and formation of the C2−C3 double bond as the final step in FST biosynthesis. Finally, the FosG mediates the hydroxylation of FST at C-4 to produce 3 (Figure 4). During the fermentation processes of the ΔfosH, ΔfosK, and ΔfosM mutants, all of the malonylated compounds, 4, 5 and 7, were accumulated as the major products with small amounts of nonmalonylated unsaturated six-membered lactone compounds, such as 2 and 8, gradually increasing at the end of the culture, as determined by HPLC analysis (data not shown). Furthermore, 5 was detected in neither the wild-type strain nor the ΔfosG mutant strain, which indirectly indicated that the elimination of the malonyl ester by FosM is instantaneous rather than slow and that compound 5 cannot be generated from modification of FST, even when FosM is present. To our regret, compounds 4 and 5 failed to restore FST formation when fed to the ΔfosH mutant STQ0703.12 It is possible that the malonylated compounds are unable to enter the host cell intact for use as substrates in enzymatic reactions. However, HPLC analysis showed a complete restoration of FST production with fosH, fosK, and fosM complementation in the corresponding gene deletion mutant strains. Although there is a lack of direct proof for

Figure 3. Proposed mechanism for the malonate elimination by FosM to generate the Δ2,3 double bonds of FST (1).

FST analogues could conceivably serve as a recognition element for post-PKS functionalization by FosJ, FosH, and FosK. In the last step of forstriecin biosynthesis, the binding site of FosM may be basic enough to induce a concerted decarboxylative elimination to generate the Δ2,3 double bonds

Figure 4. Proposed post-PKS modification of FST by tailoring enzymes FosJ, FosH, FosK, FosM, and FosG. Tailoring steps involving FosJ, FosH, FosK, FosM, and FosG and their ordering are assigned on the basis of metabolites 6, accumulated by the ΔfosJ mutant strain STQ0701, 7, accumulated by the ΔfosH mutant strain STQ0703, 4, accumulated by the ΔfosK mutant strain STQ0707, 5, accumulated by the ΔfosM mutant strain STQ0709, and FST (1), accumulated by the ΔfosG mutant strain STQ0705. 2 and 8 can be obtained from degradation in the ΔfosK and ΔfosH mutant strains, respectively. 527

dx.doi.org/10.1021/np300667r | J. Nat. Prod. 2013, 76, 524−529

Journal of Natural Products

Article

soybean (MS) agar medium for sporulation and in yeast extract peptone dextrose (YPD) liquid medium for DNA isolation.35 DNA and Plasmid Manipulations. Plasmid extraction and DNA purification were carried out using commercial kits (TaKaRa), and genomic DNAs were isolated as described previously.35 Construction of ΔfosG, ΔfosK, and ΔfosM Mutant Strains. All target genes were inactivated using the REDIRECT Technology as described previously.34 An apramycin (Apr) resistance marker and oriT cassette was used to replace an internal region of each target gene. The replacement was accomplished in cosmids Cfos-F3 and Cfos-F4 (Table S1). Subsequently, these mutant cosmids were introduced into S. pulveraceus by conjugation from E. coli ET12567/pUZ8002 according to established methodologies with the following modifications.34,35 S. pulveraceus spores were suspended in 2 × YT medium and heat-shocked at 50 °C for 10 min. Manipulated spores (as conjugation recipients) were mixed with E. coli ET12567/pUZ8002 carrying mutant cosmids (as conjugation donors) and spread onto modified MS plates freshly supplemented with 10 mM MgCl2 and 5% Gly. After incubation at 28 °C for 17 h, each plate was covered with 1 mL of sterilized water containing Apr at a final concentration of 20 μg/ mL and nalidixic acid at a final concentration of 25 μg/mL. Incubation continued at 28 °C until exconjugants appeared. The desired doublecrossover mutants were selected as being apramycin-resistant and kanamycin-sensitive and named STQ0705 (ΔfosG), STQ0707 (ΔfosK), and STQ0709 (ΔfosM), the genotypes of which were confirmed by PCR amplification and sequencing (Table S1, Figures S1, S2, and S3). Complementation Studies. Complementation plasmids were generated using the integrative plasmid pSET153 (a derivative of pSET15225 into which the ErmE* promoter and ribosomal binding site (RBS) was cloned). The ErmE* promoter was amplified from pWHM3-ErmE* using ERM-F: 5′-GCTCTAGAAGCCCGACCCGAGCACGC-3′ (underlined is an XbaI site) and ERM-R: 5′CCGGAATTCGGATCCACTAGTTCCTCCTACCAACCGGCACGA-3′ (underlined are EcoRI and SpeI sites, italicized is an RBS site). The purified PCR product was first cloned into the pMD18-T simple vector, then confirmed by sequencing, and digested with XbaI and EcoRI. The digested fragment was inserted into the XbaI and EcoRI sites of pSET152, yielding pSET153. To add the new complementation plasmids into the S. pulveraceus mutant strains that contained apramycin resistance markers, we cloned the thiostrepton (tsr) resistance marker using TSR-F: 5′-ACATGCATGCTGATCAAGGCGAATACTT-3′ and TSR-R: 5′-ACATGCATGCTTATCGGTTGGCCGCGAG-3′ (underlined are SphI sites), which was digested with SphI and inserted into the SphI site of pSET153, with the resulting plasmid named pSET153-tsr. All target genes were amplified and digested with SpeI and EcoRI and then cloned into the same sites of pSET153-tsr to yield pSET153G-tsr (for fosG expression), pSET153K-tsr (for fosK expression), and pSET153M-tsr (for fosM expression). These complementation plasmids were introduced into the corresponding mutant strains by conjugation. Tsr- and Apr-resistant colonies were selected for product analysis, yielding complemented strains STQ0706 (i.e., STQ0705/pSET153Gtsr), STQ0708 (i.e., STQ0707/pSET153K-tsr), and STQ0710 (i.e., STQ0709/pSET153M-tsr), respectively (Table S3). Fermentation and Purification of S. pulveraceus Wild-Type and Recombinant Strains. The fermentation procedure used to grow the S. pulveraceus wild-type and recombinant strains for production of FST and its analogues was previously described.12 Thus, seed medium (50 mL YPD in a 250 mL flask) was inoculated with spores and incubated at 28 °C and 220 rpm for 24 h. This seed culture was then transferred into fermentation medium5 (5% glycerol, 0.4% baker’s yeast, 0.5% meat extract, 0.1% NaCl, 0.25% CaCO3, 0.25% K2HPO4, pH 7.0, 50 mL in a 250 mL flask with 2% inoculation amount; 40 flasks) and incubated at 28 °C and 220 rpm for another 3 days for production. The fermentation mixtures were centrifuged and filtered to pellet the mycelia. The resulting supernatants were loaded onto a HP20SS column equilibrated with 0.05 M phosphate buffer (pH 6.8). The column was eluted with 100% MeOH, and the fractions containing

designating these compounds as intermediates, these present findings indirectly indicate that the malonylated compounds 4, 5, and 7 are likely to be intermediates of FST biosynthesis and not shunt products. Due to the high instability of the intermediates, compounds 4, 5, and 7 may have been degraded to 2, 1, and 8, respectively, to some degree via an elimination process that forms a cis double bond, which was reflected in the normal biosynthesis pathway and carried out by FosM. According to the structures of the isolated compounds (4, 5, 6, and 7) (Figure 4), we believe that the elimination process catalyzed by FosM to generate the Δ2,3 alkene of FST occurrs only after the hydroxylation at C-18 by FosK. The modifications to C-8 and the C-18 hydroxylation in FST biosynthesis are not influenced by the existence of the malonyl ester moiety. This study combined with our previous inferences supports the hypothesis that malonylation of the C-3 hydroxy residue is an efficient and normal process in FST biosynthesis and its occurrence is most likely triggered by the modular PKS, and FosM mediates the formation of the unsaturated lactone.12 Malonyl esters generated by modular PKSs have been found for some polyketide natural products having an unsaturated lactone such as azalomycin F,29 malolactomycin A,30 and the phoslactomycins.21 In contrast, hydroxy groups at the C-3 position are associated with saturated lactone compounds produced by modular PKSs.31 In summary, we have been able to correlate the inactivation of the fosG, fosK, and fosM genes with specific structural modifications of FST and to refine the scheme for the post-PKS modification steps in FST biosynthesis. These data confirm the proposed functions of FosG and FosK as two hydroxylases for the C-4 and C-18 hydroxylations, respectively, and FosM catalyzes the formation of the unsaturated lactone. All of the post-PKS modification steps in FST biosynthesis can occur with the polyketide chain bearing a malonyl ester at the C-3 position. Our results confirm that FosM is responsible for the formal elimination of malonate to generate the unsaturated lactone in FST biosynthesis only after the C-18 hydroxylation catalyzed by FosK is completed. The results reported here set an excellent stage for the future understanding of the mechanisms of FST biosynthesis and for generating structurally unique analogues of FST using combinatorial biosynthesis methods.

■

EXPERIMENTAL SECTION

General Experimental Procedures. 1H NMR spectra were recorded on a Bruker Avance II400 spectrometer in CD3OD with TMS as internal standard operating at 400 MHz. J values are expressed in Hz. The molecular mass was determined with Q-TOF-HRMS (Waters, Micromass). LC-MS/MS analysis was measured with Q-TOF (Waters, Micromass). Reversed-phase HPLC was performed on a Agilent 1200 liquid chromatograph equipped with a DAD detector and either a semipreparative (ZORBAX SB, 5 μm, 250 mm × 9.4 mm) or analytical (ZORBAX SB, 5 μm, 150 mm × 4.6 mm) C18 HPLC column. Bacterial Strains, Plasmids, and Culture Conditions. Bacterial strains and plasmids used in this study are listed in Table S4, Supporting Information. Escherichia coli DH5α was used as the host for general subcloning.32 E. coli ET12567/pUZ800233 was the nonmethylating plasmid donor strain for intergeneric conjugation with Streptomyces. E. coli BW25113/pIJ790 was the host for PCRtargeted mutagenesis.34 E. coli strains carrying plasmids were grown in Luria−Bertani (LB) medium supplemented with ampicillin (100 μg/ mL), apramycin (50 μg/mL), kanamycin (50 μg/mL), or chloramphenicol (25 μg/mL) when necessary.32 The FST producer S. pulveraceus was grown at 28 °C on ISP2 agar medium or mannitol− 528

dx.doi.org/10.1021/np300667r | J. Nat. Prod. 2013, 76, 524−529

Journal of Natural Products

Article

(6) De Jong, R. S.; De Vries, E. G.; Mulder, N. H. Anti-cancer Drugs 1997, 8, 413−418. (7) Boritzki, T. J.; Wolfard, T. S.; Besserer, J. A.; Jackson, R. C.; Fry, D. W. Biochem. Pharmacol. 1988, 37, 4063−4068. (8) Walsh, A. H.; Cheng, A.; Honkanen, R. E. FEBS Lett. 1997, 416, 230−234. (9) Cheng, A.; Balczon, R.; Zuo, Z.; Koons, J. S.; Walsh, A. H.; Honkanen, R. E. Cancer Res. 1998, 58, 3611−3619. (10) Roberge, M.; Tudan, C.; Hung, S. M. F.; Harder, K. W.; Jirik, F. R.; Anderson, H. Cancer Res. 1994, 54, 6115−6121. (11) Lewy, D. S.; Soenen, D. R.; Boger, D. L. Curr. Med. Chem. 2002, 9, 2005−2032. (12) Kong, R.; Liu, X.; Su, C.; Ma, C.; Qiu, R.; Tang, L. Chem. Biol. 2013, 20, 45−54. (13) Merson-Davies, L. A.; Cundiiffe, E. Mol. Microbiol. 1994, 13, 349−355. (14) Betlach, M. C.; Kealey, J. T.; Ashley, G. W.; McDaniel, R. Biochemistry 1998, 37, 14937−14942. (15) Xue, Y.; Wilson, D.; Zhao, L.; Liu, H.; Sherman, D. H. Chem. Biol. 1998, 5, 661−667. (16) Andersen, J. F.; Tatsuta, K.; Gunji, H.; Ishiyama, T.; Hutchinson, C. R. Biochemistry. 1993, 32, 1905−1913. (17) Ghatge, M. S.; Reynolds, K. A. J. Bacteriol. 2005, 187, 7970− 7976. (18) Byrne, B.; Carmody, M.; Gibson, E.; Rawlings, B.; Caffrey, P. Chem. Biol. 2003, 10, 1215−1224. (19) Carmody, M.; Murphy, B.; Byrne, B.; Power, P.; Rai, D.; Rawlings, B.; Caffrey, P. J. Biol. Chem. 2005, 280, 34420−34426. (20) Gust, B.; Kieser, T.; Chater, K., REDIRECT Technology: PCRTargeting System in Streptomyces coelicolor; The John Innes Centre: Norwich, UK, 2002. (21) Palaniappan, N.; Alhamadsheh, M. M.; Reynolds, K. A. J. Am. Chem. Soc. 2008, 130, 12236−12237. (22) Hokanson, G. C.; French, J. C. J. Org. Chem. 1985, 50, 462− 466. (23) Wu, J.; Zaleski, T. J.; Valenzano, C.; Khosla, C.; Cane, D. E. J. Am. Chem. Soc. 2005, 127, 17393−17404. (24) Reid, R.; Piagentini, M.; Rodriguez, E.; Ashley, G.; Viswanathan, N.; Carney, J.; Santi, D. V.; Hutchinson, C. R.; McDaniel, R. Biochemistry 2003, 42, 72−79. (25) Caffrey, P. ChemBioChem 2003, 4, 654−657. (26) Hu, Z.; Reid, R.; Gramajo, H. J. Antibiot. 2005, 58, 625−633. (27) Palaniappan, N.; Kim, B. S.; Sekiyama, Y.; Osada, H.; Reynolds, K. A. J. Biol. Chem. 2003, 278, 35552−35557. (28) Khosla, C.; Gokhale, R. S.; Jacobsen, J. R.; Cane, D. E. Annu. Rev. Biochem. 1999, 68, 219−253. (29) Chandra, A.; Nair, M. G. J. Antibiot. 1995, 48, 896−898. (30) Koshino, H.; Kobinata, K.; Uzawa, J.; Uramoto, M.; Isono, K.; Osada, H. Tetrahedron 1993, 49, 8827−8836. (31) Kim, B. S.; Cropp, T. A.; Florova, G.; Lindsay, Y.; Sherman, D. H.; Reynolds, K. A. Biochemistry 2002, 41, 10827−10833. (32) Maniatis, T. Molecular Cloning: a Laboratory Manual; Sambrook, J., Fritsch, E. F., Maniatis, T., Eds.; Cold Spring Harbor Laboratory Press: New York, 1989. (33) Paget, M. S. B.; Chamberlin, L.; Atrih, A.; Foster, S. J.; Buttner, M. J. J. Bacteriol. 1999, 181, 204−211. (34) Gust, B.; Challis, G. L.; Fowler, K.; Kieser, T.; Chater, K. F. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 1541−1546. (35) Kieser, T.; Bibb, M. J.; Buttner, M. J.; Chater, K. F.; Hopwood, D. A. Practical Streptomyces Genetics; The John Innes Foundation: Norwich, UK, 2000.

FST or its analogues were collected and evaporated to produce an oily residue. The oily residue was dissolved in MeOH and diluted to 10% MeOH concentration with 0.1 M phosphate buffer (pH 6.8), then loaded onto a C18 chromatography column with the MeOH− phosphate buffer mixture as the mobile phase. A stepped gradient of MeOH−buffer mixtures (10%, 20%, 30%, 40%, 50%, 60% MeOH in buffer) was used to elute the column. All of the fractions containing FST or its analogues were concentrated in vacuo and exchanged into MeOH using an HP20SS column. The MeOH solvent was then evaporated under reduced pressure to produce an oily residue. The samples containing objective compounds were dissolved in 10% MeOH (5 mL) in buffer and subjected to final purifications using semipreparative chromatography (gradient of 10% to 40% MeOH in buffer over 50 min, 2 mL/min). Two pure fractions were collected: FOS K-1 (4) (5 mg) and FOS M-1 (5) (4 mg). All filtered supernatant samples of the fermentation broths were analyzed using an Agilent 1200 HPLC system connected to a DAD detector. For each sample, a linear gradient elution from 10% to 60% MeOH in 0.005 M phosphate buffer over 25 min was developed at a flow rate of 1 mL/min and monitored by UV detection at 267 nm. Purified compounds were characterized using HRMS, LC-MS/MS, and 1H NMR spectrometry. FOS K-1 (4): amorphous solid; 1H NMR (CD3OD, 400 MHz) see Table 1; LC-MS/MS m/z 518, 473, 413; HRESIMS m/z 517.1460 [M − H]− (calcd for C22H31O12P, 517.1475). FOS M-1 (5): amorphous solid; 1H NMR (CD3OD, 400 MHz) see Table 1; LC-MS/MS m/z 533, 489, 429; HRESIMS m/z 533.1441 [M − H]− (calcd for C22H30O13P, 533.1424)

■

ASSOCIATED CONTENT

S Supporting Information *

Full experimental details describing production and confirmation of the ΔfosG, ΔfosK, and ΔfosM mutant strains STQ0705, STQ0707, and STQ0709, expression constructs for complementation of the ΔfosG, ΔfosK, and ΔfosM mutants, and PCR confirmation of the genotypes of the mutant strains. HRMS, LC-MS/MS, and 1H NMR were used to analyze the FST intermediates 4 and 5. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Tel and Fax: +86-0411-84706750. E-mail: tangli63b@yahoo. com. Author Contributions §

X.-j. Liu and R.-x. Kong contributed equally to this work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This research was supported by National Natural Science Funds for Distinguished Young Scholar award 30688003 to L.T.

■

REFERENCES

(1) Katz, L. Chem. Rev. 1997, 97, 2557−2575. (2) Weissman, K. J.; Leadlay, P. F. Nat. Rev. Microbiol. 2005, 3, 925− 936. (3) Zhou, H.; Xie, X.; Tang, Y. Curr. Opin. Biotechnol. 2008, 19, 590− 596. (4) Stampwala, S. S.; Bunge, R. H.; Hurley, T. R.; Willmer, N. E.; Brankiewicz, A. J.; Steinman, C. E.; Smitka, T. A.; French, J. C. J. Antibiot. 1983, 36, 1601−1605. (5) Tunac, J. B.; Graham, B. D.; Dobson, W. E. J. Antibiot. 1983, 36, 1595−1600. 529

dx.doi.org/10.1021/np300667r | J. Nat. Prod. 2013, 76, 524−529