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1 Department of Biological Engineering, Inha University, Incheon 402-751, Korea
2 Life Sciences Institute and Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109-2216, USA
Correspondence
Eung-Soo Kim
eungsoo{at}inha.ac.kr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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-ketoacyl reductase; KS, -ketoacyl synthase; mAT, malonyl-specific AT; mmAT, methylmalonyl-specific AT; PKS, polyketide synthase; TE, thioesterase; TMC, tautomycetinThe GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is DQ983361.
A multiple sequence alignment using CLUSTALW of the substrate-specificity motifs of the AT domains of 10 modules from tmcA and tmcB, and sequence alignments between the key motifs of deduced TMC gene products and the conserved motifs of several PKS domains, are available as supplementary data with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Hopwood, 1987; Strauch et al., 1991; Myles, 2003; Hranueli et al., 2005). A secondary metabolite produced by Streptomyces sp. CK4412, originally isolated in Jeju Island, Korea, has been identified as an activated T cell-specific immunosuppressive compound with a novel mode of pharmacological action, both in vivo and in vitro, and whose chemical structure has been shown to be identical to that of a previously reported antifungal compound, tautomycetin (TMC), produced by Streptomyces griseochromogenes. (Cheng et al., 1989; Fig. 1). Inhibition of T cell proliferation with TMC has been observed at concentrations 100-fold lower than those needed to achieve maximal inhibition with cyclosporin A (CsA) (Shim et al., 2002). TMC is believed to specifically block tyrosine phosphorylation of intracellular signal mediators downstream of Src tyrosine kinases in a T cell-specific manner, leading to apoptosis due to cleavage of Bcl-2, caspase-9, caspase-3 and poly(ADP-ribose) polymerase, but not caspase-1 (Shim et al., 2002). Thus, it has been proposed that TMC, whose mechanism of action is different from that of CsA or FK506, is a novel, potent, T cell-specific immunosuppressive agent (Shim et al., 2002).
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). The genes, enzymes and mechanism for type I polyketide biosynthesis have been thoroughly reviewed (Katz & Donadio, 1993; Rawlings, 2001; Floss, 2006), and this has highlighted the enzyme complexes known as modular polyketide synthases (PKSs). The catalytic sites of type I PKSs are encoded by a series of catalytic domains within large multifunctional proteins. A simple carboxylic acid starter unit including acetyl-CoA or propionyl-CoA is typically transferred to the cysteine active site of a -ketoacyl synthase (KS) in the PKS complex. A specific extender unit determined by a unique acyl transferase (AT) domain in the PKS complex, such as a malonyl-CoA, methylmalonyl-CoA or ethylmalonyl-CoA, is transferred to the thiol group of the phosphopantetheine arm of an adjacent acyl carrier protein (ACP). These subunits are joined by a decarboxylative condensation catalysed by KS and remain covalently attached to ACP, followed by a partial or full series of keto group processing reactions, including -ketoacyl reductase (KR), dehydratase (DH) and enoyl reductase (ER). The growing chain is transferred from the ACP to a downstream KS, and further rounds of elongation and processing occur to achieve the full-length polyketide chain. The completed chain is typically released from the PKS by a terminal thioesterase (TE) to form a macrocyclic lactone ring or a linear product.
In many modular PKS systems, the arrangement of specific catalytic domains encoded in the PKS gene cluster is usually well correlated to the final structure of the corresponding polyketide compound. This collinearity of DNA sequence information from the PKS gene is usually sufficient to predict the final structure of the corresponding polyketide metabolite. However, despite the power of bioinformatic analysis in secondary metabolism, the isolation and characterization of key biosynthetic genes and enzymes is essential to understand the details of natural product assembly and tailoring. This information also provides opportunities to develop new natural product derivatives through combinatorial biosynthesis, chemoenzymic synthesis or heterologous expression approaches (Floss, 2006; Grunewald & Marahiel, 2006; Wenzel & Muller, 2005). The unique chemical structure of TMC, which includes an ester bond linkage between a cyclic C8 dialkylmaleic anhydride at one terminus, and a linear polyketide chain bearing a terminal alkene at the other, indicates that the corresponding biosynthetic pathway features a number of unique biochemical steps with significant potential for generating novel TMC derivatives (Li et al., 2006). Specifically, the genes and enzymes that specify biosynthesis of the cyclic C8 dialkylmaleic anhydride and subsequent linkage with the linear polyketide have not yet been investigated. Moreover, unlike most macrolactonization reactions catalysed by PKS TE domains, the TMC polyketide moiety is presumably released as a linear chain following a putative decarboxylative dehydration resulting in a terminal alkene residue.
Here we report the isolation and initial characterization of the TMC biosynthetic gene cluster. Using a type I PKS methylmalonyl-CoA AT-specific PCR screening strategy, three overlapping cosmids were isolated as a contiguous 110 kb sequence from a genomic DNA library of the TMC-producing strain Streptomyces sp. CK4412. Complete sequencing of an approximately 70 kb DNA region and subsequent bioinformatic analysis revealed two putative type I PKSs and 12 additional gene products, presumably involved in TMC biosynthesis. Most of the deduced functions of TMC PKS domains correlate well with their expected roles in TMC polyketide backbone biosynthesis. In addition, disruption of a putative TMC acyl-CoA transferase gene, located just upstream of the PKS gene in the tmc cluster, completely abolished natural product biosynthesis. Taken together, these results demonstrate that the cloned gene cluster identified in this study is responsible for TMC biosynthesis in Streptomyces sp. CK4412.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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), was kindly provided by ForHumanTech Ltd and used as the source of DNA for the construction of the genomic DNA library. The strain was cultivated at 28 °C in either R2YE or YEME liquid medium (Kieser et al., 2000). Escherichia coli DH5
strain was used for DNA cloning and plasmid propagation. E. coli XL-1 Blue MR strain was used for cosmid library construction. E. coli ET12567/pUZ8002 (dam–, dcm–, hrdM) was used as the transient host for E. coli–Streptomyces conjugation. All E. coli strains were cultured at 37 °C in Luria broth or on Luria agar, supplemented with the appropriate antibiotics when needed (Kieser et al., 2000).
Cloning and sequence analysis of the TMC gene cluster.
Total genomic DNA of Streptomyces sp. CK4412 grown on YEME medium was prepared by lysozyme digestion, phenol/chloroform/isoamyl alcohol extraction (25 : 24 : 1) and spooling from ethanol, as described by Kieser et al. (2000). A cosmid library was prepared using Streptomyces sp. CK4412 genomic DNA partially digested with Sau3AI and a commercially available Supercos-1 cosmid system (Stratagene), according to the manufacturer's protocol. The cosmid library was then screened by PCR using a type I PKS-specific primer pair. The PCR primer pair [forward primer 5'-TS(C/G)AAGTCS(C/G)AACATCGGB(C/G/T)CA-3' and reverse primer 5'-CGCAGGTTS(C/G)CS(C/G)GTACCAGTA-3'] was designed based on the conserved sequences found in a KS domain and a methylmalonyl AT domain of type I PKS genes (Ayuso-Sacido & Genilloud, 2005). PCR was performed in a final volume of 20 µl containing 0.4 µM each primer, 0.25 mM of each of the four dNTPs (Roche), 1 µl extracted DNA, 1 U Ex Taq polymerase (TaKaRa) with its recommended reaction buffer, and 10 % DMSO. Subsequent amplifications were then performed in a Rapid Cycler (Idaho Technology), according to the following profile: 30 cycles of 30 s at 95 °C, 30 s at 48 °C and 40 s at 68 °C. Amplification products were analysed by electrophoresis in 1 % (w/v) agarose gels and verified by sequencing using T7 promoter primer/T3 primer in a pSupercos-1 vector. Complete sequencing of the three positive cosmid clones was performed at Genotech. DNA sequences were assembled using BLAST searches on the National Center for Biotechnology Information (NCBI) server, and also analysed using the web-based program Frameplot 2.3.2 (http://www.nih.go.jp/
jun/cgi-bin/frameplot.pl). In addition, PKS amino acid sequence domain data were analysed using the Modular Polyketide Synthase Database (http://linux1.nii.res.in/pksdb/DBASE/page.html).
Inactivation of a putative TMC biosynthetic gene.
The tmcD biosynthetic gene encoding a putative acyl-CoA transferase located upstream of the PKS tmcA gene was inactivated using a PCR-targeted gene-disruption system (Gust et al., 2003). An apramycin-resistance gene/oriT cassette for the replacement of tmcD was amplified using the following primers: TFredF (5'-tcgtcggtgctcatgcctcggtctctcctgtgaattcctcaATTCCGGGGATCCGTCGACC-3') and TFredR (5'-ttcggtcacgcccagaacgacgtctcgctgctgtggaaggtgTGTAGGCTGGAGCTGCTTC-3'). Lower-case type represents 39 nt homologous extensions to the DNA regions inside tmcD. This cassette was introduced into E. coli BW25113/pIJ790 containing pTMC2982. The gene replacement in tmcD was confirmed by restriction analysis of the mutated pTMC2982 (pTMC2982
tmcD). pTMC2982tmcD was introduced into Streptomyces sp. CK4412 by conjugation from E. coli ET12567/pUZ8002. After incubation at 28 °C for 16 h, each plate was overlaid with 1 ml sterile water containing apramycin at a final concentration of 50 µg ml–1 and nalidixic acid at a final concentration of 25 µg ml–1. Incubation continued at 28 °C until conjugants appeared. The double-crossover recombinants were first selected by PCR and then confirmed by Southern blot hybridization of Streptomyces sp. CK4412 genomic DNA.
HPLC quantification and antifungal bioassay for TMC.
For HPLC analysis, culture broth supernatants were extracted with equal volumes of chloroform. The extracts were dried using a rotoevaporator and then resuspended in methanol. Extracts were fractionated by HPLC using isocratic conditions of methanol : water : buffer (1 % diethylamine/formic acid, pH 7.3) (75 : 15 : 10) on a Genesis C18 4 µm column with UV detection at 273 nm. TMC production was also evaluated by biological assay against Aspergillus niger as an indicator using the agar-plug diffusion method (Isaacson & Kirschbaum, 1986). The agar plug from the 7-day-old Streptomyces solid agar culture was placed on top of A. niger that had been incubated on GY medium for 6 h at 30 °C, and this was followed by the measurement of the inhibition zone after overnight incubation at 30 °C.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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, Fig. 2). Although the 70 kb contiguous DNA cluster appears to contain most of the genes involved in TMC biosynthesis, we cannot rule out the possibility that additional genes are necessary for TMC biosynthesis.
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). All active-site motifs within individual catalytic domains were highly conserved in each of the 10 TMC PKS modules (Zirkle et al., 2004). TE was found to be the final catalytic domain encoded by tmcB. TmcB TE contains the highly conserved sequence GxSxG and GdH motifs (Zirkle et al., 2004), and is presumed to play a key role in the formation of the terminal alkene residue of TMC. Interestingly, several marine cyanobacterial natural products have been characterized that contain terminal alkene residues, including kalkitoxin (Wu et al., 2000), somocystinamide (Nogle & Gerwick, 2002) and the linear polyketide curacin A (William et al., 1994). Although there is limited amino acid sequence similarity between the terminal module of the curacin A biosynthetic pathway (CurM) and TmcB module 10, it is possible that the terminal decarboxylative dehydration of the linear TMC polyketide operates in a manner analogous to that recently proposed for the curacin A produced by Lyngbya majuscula (Chang et al., 2004). In the unique polyketide termination catalysed by TmcB module 10, the TMC TE domain is presumed to catalyse hydrolysis of the final chain-elongation intermediate to the corresponding carboxylic acid. Whether TMC TE is responsible for the additional steps resulting in terminal alkene formation, or whether a separate decarboxylase and dehydratase are involved, remains unclear. Detailed biochemical studies will be required to dissect the formation of this unusual terminal functional group.
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; Haydock et al., 1995; Del Vecchio et al., 2003). Based on the analysis of conserved sequence patterns, all 10 AT domains in TMC PKS genes were classified: AT domains in modules 1, 3, 5, 8 and 10 as malonyl extenders; AT domains in modules 2, 4, 6 and 7 as methylmalonyl extenders; and the AT domain in module 9 as an ethylmalonyl extender unit (Supplementary Fig. S1A). The order of AT domains in the TMC PKS was exactly matched with the structure of the expected extender unit for the corresponding TMC polyketide (Supplementary Fig. S1B). Although the AT domain in module 9 showed significant overall homology with mmATs, it is believed to load an ethylmalonyl-CoA, because the active-site recognition sequences (SVATH) are unique in comparison to those of mmATs (DYASH) (Supplementary Fig. S1A). Moreover, most mmATs closely resemble one another (Wu et al., 2000).
Organization of DH/ER/KR domains in TMC PKS genes
Within 10 TMC PKS modules, seven DH domains, three ER domains and nine KR domains were identified. All the TMC DH domains exhibited 57–76 % amino acid homology to each other. Unlike a typical DH active-site motif LXXHXXXGXXXXP (Zirkle et al., 2004), however, the two DH domains in TMC PKS modules were predicted to be inactive due to aberrant active-site sequences, including LXXPXXGXXXXP in TmcA DH1 and LXXYXXXGXXXXP in TmcA DH2 (Supplementary Fig. S2A). Although TmcA DH3 in module 5 seems to be functional, based on active-site-motif sequence alignment, this domain might also be non-functional, based on the absence of a corresponding double bond in the final TMC structure. A similar discrepancy regarding sequence-based structure prediction has also been reported in other DH domains in PKS systems (Aparicio et al., 1996).
The three ER domains in TMC PKS modules showed 64–74 % amino acid homology to each other, and contained the conserved active motif GGVGXAAXQXA (Supplementary Fig. S2B). All nine KR domains in TMC PKS modules showed the conserved active site motifs GXGXXG(A)XXXA and LXS(G)RXG(T,A). The TmcA KR3 domain in module 4, which was significantly different from other KRs, seemed to be inactive due to a 16-amino-acid deletion in the catalytic domain (Keatinge-Clay & Stroud, 2006; Supplementary Fig. S2C).
TMC genes involved in post-PKS tailoring functions
There are seven ORFs organized as a transcriptional unit located upstream of tmcA. The deduced amino acid sequences showed significant similarity to enzymes that catalyse tailoring functions expected in TMC biosynthesis, including a carboxylesterase and citrate lyase, consistent with a proposed biosynthetic origin of the cyclic C8 dialkylmaleic anhydride moiety. The product of tmcC is believed to be responsible for the linking of the cyclic C8 dialkylmaleic anhydride moiety to the linear polyketide moiety (Gandolfi et al., 2001). Thus, tmcD, which exhibits significant homology to an acyl-CoA transferase/carnitine dehydratase, is believed to encode an enzyme that activates the cyclic C8 dialkylmaleic anhydride moiety (Engemann et al., 2005) as the corresponding CoA ester. Based on previous studies to determine the biosynthetic origin of TMC, it is evident that the cyclic anhydride is generated by condensation of one molecule of propionate with 2-oxoglutarate (Ubukata et al., 1995). Thus, the tmcE–tmcI-encoded gene products might be responsible for the aldol condensation, dehydration and subsequent hydration of the allylic position at C3' (Fig. 1). However, these biochemical steps remain to be explored in detail and confirmed. Prior to esterification, the polyketide moiety initially synthesized by TmcA and TmcB might be further modified by several enzymes, such as a putative decarboxylase encoded by tmcJ (and/or tmcK), or a dehydratase encoded by tmcM. The ketone group located close to the right-hand end of TMC in Fig. 1 is believed to be introduced after polyketide biosynthesis by a separate cytochrome P450 hydroxylase enzyme, whose gene has been identified outside the cloned TMC cluster (S.-S. Choi and others, unpublished data). Since tmcL, encoding a putative crotonyl-CoA reductase, and tmcM, encoding a putative L-carnitine dehydratase, are located downstream of tmcB and show significant homologies with the genes responsible for the biosynthesis of ethylmalonate, these two gene products might be responsible for the synthesis of ethylmalonyl-CoA, an extender unit for TMC PKS AT9 (Table 1; Wu et al., 2000). A 3 kb ORF (tmcN) located downstream of tmcB could be a pathway-specific regulatory gene due to its chromosomal location within the cluster as well as its homology to LuxR-family regulatory genes, often found in secondary-metabolite gene clusters of Gram-positive bacteria (Haydock et al., 2005). In addition, a rare UUA codon was found in tmcN, suggesting that this gene encodes a pathway-specific regulatory protein whose expression is regulated by the bldA gene that encodes UUA-specific tRNA (Haydock et al., 2005). The current proposed mechanism for TMC biosynthesis is shown in Fig. 3.
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tmcD that was introduced into Streptomyces sp. CK4412 by conjugative gene transfer (Fig. 4A). Construction of the tmcD mutant (Streptomyces sp. CK4412-001) generated by PCR-targeted disruption was confirmed by PCR analysis. The expected 1.3 kb PCR-amplified bands were observed in genomic DNA samples isolated from Streptomyces sp. CK4412 and pTMC2982 DNA, while the expected size (1.5 kb) PCR-amplified bands were observed in genomic DNA samples isolated from Streptomyces sp. CK4412-001 and pTMC2982tmcD (Fig. 4B, top). In addition, the expected size (0.5 kb) PCR-amplified fragment, using an alternative PCR primer pair designed to detect an apramycin-resistance gene/oriT cassette, was observed only in Streptomyces sp. CK4412-001 and pTMC2982tmcD (Fig. 4B, bottom), implying that tmcD was specifically disrupted, as expected. Both Streptomyces sp. CK4412 wild-type and Streptomyces sp. CK4412-001 strains were grown under conditions optimal for TMC production, followed by antifungal bioassay and HPLC quantification. Although a very weak antifungal activity was detected for the Streptomyces sp. CK4412-001 strain, probably due to the yet-to-be-confirmed linear polyketide precursor, the significantly reduced antifungal activity against Aspergillus niger (Fig. 5A), as well as the absence of TMC in extracts of the Streptomyces sp. CK4412-001 strain under the same culture conditions (Fig. 5B), provides strong evidence that tmcD (encoding a putative acyl-CoA transferase) plays an essential role in TMC biosynthesis.
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ACKNOWLEDGEMENTS |
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Edited by: M. S. Paget
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Received 6 October 2006;
revised 4 December 2006;
accepted 22 December 2006.
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