Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK
Correspondence
Stephen F. Haydock
stephen.haydock{at}addenbrookes.nhs.uk
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is DQ149987.
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INTRODUCTION |
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METHODS |
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Culture conditions and molecular genetic procedures.
E. coli strains were grown on 2x TY medium supplemented with appropriate antibiotics. S. neyagawaensis strains were maintained on tap water medium (Kieser et al., 2000). Liquid S. neyagawaensis cultures for isolation of genomic DNA were grown in tryptone soya broth (Difco). Cultures of S. neyagawaensis to be used in the analysis of concanamycin A production were grown in an aqueous medium containing 1 % glucose, 3 % soluble starch, 0·5 % peptone, 1 % soybean flour, 0·5 % yeast extract and 0·2 % calcium carbonate. Standard genetic techniques for E. coli and for in vitro DNA manipulations were as described by Sambrook et al. (1989)
. Total DNA was isolated from S. neyagawaensis by procedure B of Kieser et al. (2000)
. For the generation of the cosmid library, total DNA was partially digested with Sau3A, dephosphorylated with shrimp alkaline phosphatase, ligated directly into pSuperCos and packaged with Gigapack Gold packaging extract (Stratagene) without size fractionation. All procedures were in accordance with the manufacturer's recommendations. Cosmid DNA for complete sequencing was prepared using Qiagen midi-prep DNA purification kits from 50 ml liquid cultures in 2x LB medium containing ampicillin and kanamycin. The complete sequence of each cosmid was obtained from overlapping Sau3A fragments obtained after a partial digestion of the parent cosmid. The 25 kbp fraction of this digest was eluted from an agarose gel using the Gene Clean kit (Bio 101, Bio-Rad) and subcloned into pSG397 (Hashimoto-Gotoh et al., 1995
). Colony hybridization was performed with a digoxigenin-labelled probe in accordance with the manufacturer's recommendations (Boehringer Mannheim). For gene disruption by insertional activation, plasmid pOJconE was constructed by cloning a BamHI fragment of Cos1B6 internal to the conE gene (part of the putative concanamycin modular PKS), into the unique BamHI site of pOJ260. This plasmid was transferred by conjugation from E. coli into S. neyagawaensis by a previously described protocol (Kieser et al., 2000
). Conjugation took place on agar plates containing A medium (Kieser et al., 2000
) incubated overnight at 30 °C and overlaid after 12 h with nalidixic acid and apramycin. Potential exconjugants were transferred to fresh apramycin and nalidixic acid plates after a further 5 days' growth. Apramycin-resistant colonies were isolated and shown by Southern hybridization (data not shown) to contain the pOJconE plasmid integrated into the predicted genomic location by single crossover recombination within the conE ORF.
DNA sequencing analysis.
Automated DNA sequencing was carried out on double-stranded DNA templates by the dideoxynucleotide chain-termination method in the University of Cambridge Department of Biochemistry DNA sequencing facility. An Applied Biosystems 800 molecular biology CATALYST robot was used to apply Taq dideoxy terminator sequencing reactions (Big Dye Terminator kit; ABI) to an ABI 373A sequencer, according to the manufacturer's protocols. Cosmid DNA for initial end sequencing was prepared from overnight growth of 1 ml cultures in 96-well plates in 2x LB medium containing kanamycin and ampicillin. The complete sequence of the cosmid was obtained from analysis of overlapping Sau3A fragments obtained via a partial digestion of the parent cosmid. The 25 kbp size fraction of this digest was eluted from an agarose gel using the Gene Clean kit (Bio 101, Bio-Rad) and subcloned into the BamHI site of dephosphorylated pSG397 (Hashimoto-Gotoh et al., 1995). These random subclones were sequenced using template DNA obtained from 1 ml cultures grown overnight at 37 °C in 96-well plates, using the universal forward and reverse sequencing primers. SeqEd v 1.03 was used for sequence editing. Database searches used the BLAST algorithm. Sequence assembly employed GAP (Genome Assembly Program) version 4.2. (Bonfield et al., 1995
). To fill remaining contig gaps and resolve any remaining ambiguities, custom-designed oligonucleotide primers were used to sequence from selected subclones or from the parent cosmid. Both strands were completely sequenced with a mean eightfold coverage per base.
Extraction, isolation and characterization of concanamycin A.
Cultures of wild-type S. neyagawaensis were grown in the aqueous medium described previously. A 100 ml seed culture of wild-type S. neyagawaensis was grown in a 500 ml Erlenmeyer flask for 4 days at 30 °C and 250 r.p.m. in an orbital shaker. The seed cultures were used to provide a 3 % inoculum in 2 l Erlenmeyer flasks that contained 300 ml of the same medium. The cultures were grown for a further 4 days under the same conditions and the cells were then harvested by centrifugation.
Cells of S. neyagawaensis collected from 3·6 l of culture were extracted in acetone (2x500 ml) and the acetone extracts were concentrated by evaporation to a volume of 400 ml. The acetone solution was then extracted with an equal volume of dichloromethane. The organic extract was dried with ammonium sulphate and then evaporated to afford a brown oil. The crude extract was analysed by LC-MS and LC-MS/MS. Concanamycin A was detected in the extracts from wild-type S. neyagawaensis, a component with a retention time of 14 min and a molecular ion of mass 888·7 (M+Na+). These results were identical to those obtained for an authentic sample of concanamycin A (Sigma) analysed in the same manner. The fragmentation pattern obtained for the selected 888·7 ion in LC-MS/MS analysis was also identical in the crude extract and standard concanamycin A samples. A mutant of S. neyagawaensis obtained by chromosomal integration of a pOJ260-derived construct into the putative concanamycin A locus (described above) was grown under identical conditions and the culture was harvested. The harvested cells from 3·6 l of culture were extracted and analysed using conditions identical to those applied to the cells of wild-type S. neyagawaensis. Concanamycin A was not detected by LC-MS in extracts from cells of the S. neyagawaensis mutant.
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RESULTS AND DISCUSSION |
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A total of 480 cosmid-bearing colonies, from a cosmid library of S. neyagawaensis DNA, housed in the vector pSuperCos, were therefore grown up in 2x LB medium in 96-well plates. Replica deeps were prepared in further 96-well plates by adding 100 µl of each culture to 100 µl 40 % glycerol. The remaining cells were used to prepare DNA sequencing template which were each end-sequenced using T3 and T7 primers. BLAST searches were performed on the sequence data and nine cosmids were identified that encoded either modular PKS genes or genes previously identified as components of biosynthetic gene clusters for complex macrolides. Large-scale cosmid DNA preparations were made from the glycerol deeps of these clones and partial sequence data were obtained for each of the nine clones by end sequencing of BamHI restriction fragments. Three clones (1B6, 2C7 and 4A8) between them were found to span a large portion of the same PKS locus whose organization appeared consistent with a role in the biosynthesis of concanamycin A, and this was confirmed by their subsequent complete sequencing. The other six clones initially identified by random sequencing did not belong to this locus. S. neyagawaensis appears to harbour genes for at least two other complex polyketides (S. F. Haydock, unpublished data). This provides further evidence of the unexpected biosynthetic potential of the streptomycetes (Omura et al., 2001; Haydock et al., 2004
).
Further cosmids spanning the remainder of the putative concanamycin cluster were obtained by hybridization of the cosmid library with digoxigenin-labelled BamHI fragments from the end of the cosmid clones 1B6, 2C7 and 4A8. The complete sequencing of these overlapping cosmid clones led to the determination of the sequence of the entire concanamycin biosynthetic gene cluster and the deduced gene organization was found to be as shown in Fig. 2. In addition to the six large genes required for the synthesis of the macrolactone core, the sequenced gene cluster encoded 22 other ORFs. The inferred protein products can be subdivided into (a) sugar biosynthesis and attachment; (b) regulatory genes and genes of unknown function; (c) provision of extender units or modification of the PKS; and (d) indeterminate (Table 1
).
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Formation of the sugar moiety of concanamycin A requires the elimination of the 2'-hydroxyl from the glucose ring. Such an elimination is seen in the formation of L-mycarose in erythromycin A biosynthesis (Summers et al., 1997). This process requires the action of two enzymes, namely a 2,3-dehydratase followed by a 2,3-enoyl reductase. A homologue of the gene for the latter enzyme is not seen in the concanamycin cluster. An alternative possibility is suggested by the presence of the two putative ketoreductases. We propose that the 2,3-dehydratase catalyses the formation of the 2-deoxy derivative as in erythromycin A. This intermediate is in equilibrium with the 2,4-diketo derivative. The 4- and 3-ketoreductases then directly reduce each keto group at C3 and C4 to give the 2-deoxy derivative with appropriate stereochemistry at the C3 and C4 positions. ORFs 8*, 9* and 10* again appear closely coupled together and may form part of the same transcription unit.
The pendant sugar of concanamycin A is unusual in that it is modified by the addition of a carbamoyl unit. Carbamoylation has been observed in other secondary metabolite natural products such as novobiocin (Steffensky et al., 2000) and cephamycin (Liras & Rodríguez-García, 2000
). ORF7* shows high end-to-end similarity to numerous authentic carbamoyltransferases. The best matches are to the carbamoyltransferase gene required for sugar O-carbamoylation in the synthesis of nodulation factors in many bacterial species. This homology might reflect the recruitment of a gene from such a nodulation cluster.
The final gene of this group, ORF11*, appears closely coupled to the downstream gene ORF10*. This gene product shows high end-to-end similarity to authentic glycosyltransferases. The best match is to the glycosyltransferase required for the addition of deoxyallose to tylosin, reflecting the close structural similarity in the sugar substrates of these two enzymes. The proposed pathway for the synthesis and attachment of the sugar moiety of concanamycin A is shown in Fig. 3.
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ORF1 shows end-to-end homology with discrete malonyltransferase enzymes acting on malonyl-CoA. The function of this enzyme in the biosynthesis of concanamycin A is uncertain. One other potential small ORF can be identified within the gene cluster. A potential 114 aa ORF, designated ORF4B*, lies between ORF4* and ORF5*, transcribed in the same direction as ORF5*. This ORF has no match in any published databases and its identity remains speculative.
The concanamycin gene cluster contains two pseudogenic remnants' within the gene cluster. These remnants flank the carbamoyltransferase activity. Downstream of this gene and transcribed in the same direction is the start of a gene that encodes the first 33 aa of deoxyallosyltransferase (the complete gene is located upstream and separate from it) and upstream of the carbamoyltransferase is a stretch of DNA that encodes 30 aa from an insertion sequence. We speculate that these remnants show evidence of recent evolution of the cluster characterized by the incorporation of a carbamoylation activity that modifies the attached sugar. Such gene remnants are not an unusual finding in macrolide biosynthetic clusters but are rarely commented upon.
The margins of the biosynthetic gene cluster can only be accurately determined by systematic gene disruption and analysis of the resulting phenotype. However, the upstream region of the cluster has been extended for a further 15 kbp and has been shown to encode a second cluster encoding the synthesis of a member of the anguicyclinone group of antibiotics. Downstream of the last two genes are the -hydroxybutyryl-CoA dehydrogenase- and crotonyl-CoA reductase-encoding genes as found flanking the oligomycin cluster (Omura et al., 2001
). Transcribed towards these ORFs is a gene with very high end-to-end similarity to ribonuclease and these two ORFs are therefore likely to mark the margin of the cluster on this side.
Provision of extender units and modification of the PKS
The aglycone core of complex polyketides is derived from the repeated condensation of acyl-CoA building blocks, most commonly acetate units derived from malonyl-CoA and propionate units derived from methylmalonyl-CoA. In addition, less commonly, butyrate extender units are found, derived from ethylmalonyl-CoA and methoxyacetate units, derived from methoxymalonyl-ACP. Condensation 10 of the biosynthesis of the concanamycin aglycone would appear to require ethylmalonyl-CoA. The examination of the AT domain for this extension cycle is consistent with a preference for ethylmalonyl-CoA over methylmalonyl-CoA (Reeves et al., 2001) (see also below). ORFs 18* and 19* appear to encode a crotonyl-CoA reductase and
-hydroxybutyryl-CoA reductase, respectively, which could both play a role in furnishing butyrate extender units. Crotonyl-CoA reductase is commonly found in polyketide biosynthetic gene clusters where an ethylmalonyl extender unit is required, as in tylosin (Butler et al., 2001
), elaiophylin (Haydock et al., 2004
) and oligomycin (Omura et al., 2001
) biosynthesis.
-Hydroxybutyryl-CoA reductase is less common but is also found in the oligomycin biosynthetic cluster (Omura et al., 2001
). ORFs 18* and 19* are therefore likely to be involved in the synthesis of the butyrate extender unit for module 10. The extension units for modules 6 and 14 could be derived from malonyl-CoA, followed by subsequent hydroxylation and methylation. Alternatively, this unit could be derived from direct incorporation of methoxymalonyl-ACP as is seen in the biosynthetic clusters for ascomycin (Wu et al., 2000
; Reeves et al., 2002
), ansamitocin (Yu et al., 2002
) and geldanamycin (Rascher et al., 2003
). The sequenced gene cluster contains no P450 hydroxylase genes and only one O-methyltransferase. This latter gene, ORF12*, is most similar to FkbG of the ascomycin biosynthetic cluster and is one of several genes suggested to be involved in biosynthesis of methoxymalonyl-ACP. Homologues of the remaining genes required for this pathway are also present and clustered together immediately downstream of ORFF in the concanamycin cluster, but separated from the FkbG homologue. ORF1* is homologous to FkbB, ORF2* to FkbJ, ORF3* to FkbI and ORF4* to FkbH. The products of these ORFs are thought to convert a primary metabolite to methoxymalonyl-ACP by a series of reactions that have not been well characterized. ORFs 1*4* again may be transcriptionally coupled to each other and to the upstream PKS genes. It is therefore reasonable to conclude that modules 6 and 13 of the concanamycin PKS incorporate methoxymalonate extender units.
Organization of the concanamycin PKS
The biosynthesis of the polyketide backbone of concanamycin A is encoded by six large ORFs designated conA, conB, conC, conD, conE and conF which are adjacent and transcribed in the same direction and which encode, respectively, the multienzyme polypeptides CON16. Examination of the encoded AT domains of CON polypeptides for distinctive amino acid sequence motifs allows the chemical nature of the extender unit recruited by each AT domain (especially malonate versus methylmalonate or ethylmalonate) to be inferred (Haydock et al., 1995; Lau et al., 1999
; Reeves et al., 2001
; Del Vecchio et al., 2003
). A signature motif for AT domains that incorporate methoxymalonate has, because of the rarity of this extension unit, hitherto eluded definition (but see below). Examination of the reductive loop (Staunton & Weissman, 2001
) domains (KR, DH, ER) in each extension module allows the level of reduction to be deduced for that extension. In the case of hydroxyacyl extension units, even the stereochemical configuration of the hydroxy group can be predicted (Caffrey, 2003
). The predicted polyketide product of the concanamycin PKS locus from our analysis is entirely consistent with assembly of the polyketide chain of concanamycin A, the modules being encoded along the chromosome in the order they are used from conA to conF (Fig. 2
). CON1 (2841 aa, approx. 300 kDa) encodes the loading domain and module 1 of the polyketide chain extension; CON2 (4825 aa, approx. 500 kDa) encodes modules for extensions 24; CON3 (4970 aa, approx. 520 kDa) encodes modules for extensions 57; CON4 (4490 aa, approx. 430 kDa) encodes modules 89; CON5 (5599 aa, approx. 530 kDa) encodes modules 1013; CON6 (2056 aa, approx. 215 kDa) encodes the final module for condensation 14 and has a C-terminal integral thioesterase domain required for lactonization and release of the polyketide chain (Fig. 4
).
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We therefore sought to exploit the high mutual sequence homology between AT domains within the concanamycin PKS to identify residues that might hallmark AT domains specific for methoxymalonyl-ACP and differentiate them from AT domains that recruit methylmalonyl-CoA. We identified a short span of residues that distinguished these two AT groups and which lie immediately N-terminal of the previously described YASHS motif that distinguishes methylmalonyl- from malonyl-incorporating AT domains (Lau et al., 1999). All the concanamycin AT domains that are proposed to incorporate methylmalonate have the sequence RXRR(X)3YASHS (where X is any amino acid) whilst those proposed to incorporate methoxymalonate all have the sequence MXWR(X)3YASHS.
To confirm the significance of this motif we used the program BLAST to search the UNIPROT protein sequence databases with a 40 residue span of the peptide sequence of module 13 of the concanamycin PKS centred on the methionine and tryptophan residues identified above. Interestingly, we found very few streptomycete modular PKS AT domains with a tryptophan in the position found in the concanamycin AT domains. This position is highly conserved in methylmalonyl-specific AT domains and is usually occupied by an arginine (as in the methylmalonate-specific domains of the concanamycin PKS), or by a lysine or less commonly by serine or threonine. In addition, the position corresponding to the methionine in the motif is usually occupied by a positively charged residue such as arginine. We were able to identify only six streptomycete modular AT peptide sequences in the database in which the tryptophan was present at the specified position with a non-polar residue in the position corresponding to methionine. The alignment of these sequences is shown in Table 2.
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Confirmation of the identity of the concanamycin biosynthetic locus by PKS gene disruption
Although the organization of the gene cluster isolated and sequenced from S. neyagawaensis is entirely and exactly consistent with its encoding concanamycin A biosynthesis, confirmation of the identity of this locus was sought by gene disruption. To do this a 9 kbp BamHI fragment internal to the putative PKS gene conE was subcloned into the E. coli/Streptomyces shuttle vector pOJ260 (Bierman et al., 1992). The resulting plasmid was used to transform E. coli ET12567 (MacNeil et al., 1992
) containing the helper plasmid pUZ8002 (Paget et al., 1999
) and this recombinant strain was used for conjugation with S. neyagawaensis. Stable apramycin-resistant integrants were obtained via homologous recombination between the 9 kbp insert cloned into the suicide vector and the host genome. Both wild-type and recombinant strains were grown under conditions optimal for production of concanamycin A. Using liquid chromatography linked to mass spectroscopy, a peak was observed for the wild-type strain, whose exact mass corresponded to that predicted for concanamycin A. No compound of this mass was detected in the recombinant strain grown under the same conditions (see Methods), confirming the proposed role for this locus in concanamycin A biosynthesis.
Concluding remarks
The analysis of the concanamycin A biosynthetic locus from S. neyagawaensis has revealed a set of modular PKS genes, clustered with an extensive set of genes involved in provision of unusual precursor units. The uncovering of a specificity motif in certain of the AT domains of the PKS which reliably distinguishes those that are specific for methoxymalonate will not only aid in the identification of newly cloned PKS genes, but paves the way for AT engineering by mutagenesis of this motif. Finally, the provision of the sequence for this key plecomacrolide, together with methods for genetic manipulation of the producing strain, should allow the genetic modification of the cluster to derive novel structural variants of concanamycin with potential utility in a wide range of medical conditions.
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ACKNOWLEDGEMENTS |
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Received 12 May 2005;
accepted 29 July 2005.
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