Bio-X Life Science Research Center, Shanghai Jiaotong University, Shanghai 200030, China1
Huazhong Agricultural University, Wuhan 430070, China2
Jiangxi Agricultural University, Nanchang 330045, China3
John Innes Centre, Colney, Norwich NR4 7UH, UK4
Author for correspondence: Zixin Deng. Tel: +86 21 62933404. Fax: +86 21 62933404. e-mail: zxdeng{at}mail.sjtu.edu.cn
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ABSTRACT |
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Keywords: antibiotic biosynthetic genes, dianemycin, avermectin, polyketide synthase, gene replacement in Streptomyces
Abbreviations: PKS, polyketide synthase; DEBS, 6-deoxyerythronolide B synthase; ACP, acyl carrier protein; KS, ketosynthase; KR, ketoreductase; AT, acyltransferase; TE, thioesterase
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INTRODUCTION |
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Many gene clusters encoding the enzymes of polyketide biosynthesis have been cloned and characterized (see Schwecke et al., 1995 ; Xue et al., 1998
; Ikeda et al., 1999
, for examples). Modular type I PKSs consisting of several large multifunctional proteins catalyse the biosynthesis of complex or reduced polyketides, the founding example being the erythromycin PKS (Cortes et al., 1990
; Donadio et al., 1991
). Combinatorial biosynthesis has been especially successful with many actinomycete PKSs following extensive structural and functional studies, and has led to the production of many unnatural natural products (Cane et al., 1998
; Hopwood, 1997
; Hutchinson, 1998
; Katz & McDaniel, 1999
).
Streptomyces nanchangensis was isolated from the soil in Nanchang, China (Ouyang et al., 1984 ). Str. nanchangensis produces at least two kinds of insecticidal antibiotics (Ouyang et al., 1993
). The polyether nanchangmycin (Fig. 1
) structurally and biologically resembles dianemycin (Czerwinski & Steinrauf, 1971
), which is used in poultry farming. The 16-membered macrolide meilingmycin (Fig. 2a
) resembles milbemycin
11 (Takahashi et al., 1993
) and has a similar aglycone and antiparasitic activity as avermectin (Fig. 2b
; Burg et al., 1979
). In addition, Str. nanchangensis produces at least two other antibiotics of unknown structure but with clearly different biological activities. Both meilingmycin and nanchangmycin are very active against a broad spectrum of harmful nematodes and insects, and are non-toxic for mammals and plants (Ouyang et al., 1993
). Their chemical structures (Figs 1
and 2
) indicate that they are both synthesized by modular type I PKSs.
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METHODS |
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Culture techniques, transformation and conjugation.
Str. nanchangensis NS3226 and its derivatives were grown on GS medium (2% soluble starch, 0·1% KNO3, 0·05% K2HPO4, 0·05% MgSO4.7H2O, 0·05% NaCl, 0·001% FeSO4, 2% agar, pH 7·5) or YEME liquid medium (Kieser et al., 2000 ) at 28 °C for growth of mycelium and isolation of total DNA, R2YE agar medium for transformation and protoplast regeneration (Kieser et al., 2000
), YD medium (0·4% Difco yeast extract, 1% maltose extract, 0·4% glucose, 0·2% MgCl2, 0·15% CaCl2, 2% agar, pH 7·5) for conjugation and B2-1 liquid medium (3% corn powder, 1% corn starch, 1% soy bean powder, 0·2% KH2PO4, 0·05% MgSO4, 0·05% NaCl, 0·2% (NH4)2SO4, 1% CaCO3, pH 7·5) for fermentation. Protoplast preparation was according to Hopwood et al. (1985)
.
E. coli strains were cultured according to Sambrook et al. (1989) . Cosmid clones were selected after infection of E. coli LE392 on L agar containing 100 µg ampicillin ml-1 or 10 µg apramycin ml-1. For Streptomyces, apramycin and thiostrepton were both used at 10 µg ml-1 in GS agar medium and at 5 µg ml-1 in liquid media.
Cloning techniques.
Plasmid and total DNA was isolated from Streptomyces strains using procedure 3 of Hopwood et al. (1985) . For the generation of cosmid libraries, total DNA samples were partially digested with MboI, dephosphorylated with calf intestinal alkaline phosphatase and size fractionated by sucrose gradient centrifugation (Hopwood et al., 1985
). The DNA fragments were mixed at a 1:1 molar ratio with BamHI-digested cosmid vectors and ligated at
200 µg DNA ml-1. Packaging was done with
packaging mixes prepared according to Sambrook et al. (1989)
.
Antibiotic assay.
Production of nanchangmycin was detected using a bioassay and HPLC. The strains were grown at 28 °C for 57 d on GS agar medium. Agar plugs were transferred to L agar containing Bacillus cereus 1126, which is sensitive to nanchangmycin. Inhibition zones were visible after 12 h incubation at 37 °C. For HPLC analyses, the strains were cultured in 40 ml B2-1 fermentation medium in 250 ml baffled flasks at 28 °C and shaken at 220 r.p.m. for 7 d. After the mycelia were harvested by centrifugation at 600 g for 10 min, the products were extracted with an equal volume of methanol. The extract was directly applied to HPLC on a Waters Xterra RP18 5 µm (3·9x150 mm) column. The mobile phase was acetonitrile/water (85:15) and the flow rate was 1 ml min-1 at room temperature. The effluent was monitored at 243 nm with a Waters 996 photodiode array detector. The data were processed with a Waters Millennium Chromatography Manager.
Sequence analysis.
Regions with spontaneous mutations in the pIJ101-derived vector pHZ1351 were sequenced using pBluescript II SK(+) or SK(-) as vectors. Sequencing reactions were done using the Amersham Thermosequenase sequencing kit containing fluorescent dye terminators. M13 forward and reverse primers (24-mers) and the recommended standard PCR conditions (30 cycles; 1 min 96 °C, 2 min 50 °C and 4 min 60 °C) were used.
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RESULTS |
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Ordering of the cosmids was achieved by comparing PvuII (and BamHI) digests of the cosmids. Cosmids producing similar bands were grouped and fractionated again on agarose gels until the most likely order of the cosmids was found (Figs 3 and 4
). The order of the cosmids and restriction maps were further confirmed by Southern hybridization at high stringency (0·2xSSC, 65 °C) to prove the same origins of similar bands, using stepwise probes along the putative contigs (Figs 3
and 4
). Seventy-five out of the 90 cosmids were thus grouped into eight independent clusters. Cluster A (Fig. 3
) consists of 17 cosmid clones that hybridized to the ery PKS probe and spans
133 kb of continuous DNA. An
89 kb PKS-homologous region was located in the central part of the cluster; 28 kb flanking the left side, and at least 14 kb to the right of the PKS-homologous regions did not hybridize to the ery probe even at low stringency (6xSSC, 65 °C). Cluster B (Fig. 4
) has an
87 kb PKS-homologous region in a
132 kb contig (17 cosmids) and cluster C (Fig. 4
) has an
86 kb PKS-homologous region in a
104 kb contig (7 cosmids). PKS-homologous regions in other clusters (D,
174 kb; E,
122 kb; F,
54 kb; G,
37 kb; H,
59 kb; Fig. 4
), as detected using the 3·2 kb ery probe only containing KR4, ACP6, KS6 and AT7, are either not concentrated in one or two contiguous regions (e.g. clusters D and E in Fig. 4
) or not as large as in clusters AC (
10 kb in cluster F,
11 kb in cluster G and
33 kb in cluster H) along each contig.
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Localization of contigs to five different Str. nanchangensis AseI fragments separated by PFGE
AseI digestion of Str. nanchangensis NS3226 DNA generates 19 macro-restriction fragments (Fig. 5
) that can be separated by PFGE. The precise number of bands is not certain because several bands are clearly multiple, but
17 bands can be detected in a single gel. These were transferred to nylon membrane and hybridized at high stringency (0·2xSSC, 65 °C) against representative cosmids from each cluster as probes. Clearly, 32P-labelled 18 kb, 3·7 kb and 3·6 kb PvuII fragments internal to the insert in cosmid 20H7 from cluster A, 19 kb and 5·9 kb PvuII fragments in cosmid 8D2 from cluster B, 7·7 kb and 2·3 kb PvuII fragments in cosmid 12B11 from cluster C, 6·7 kb PvuII fragment in cosmid 7C5 from cluster F, and 3·9 kb and 3·8 kb PvuII fragments in cosmid 17G12 from cluster G hybridized to different AseI fragments (Fig. 5
). This supported the hypothesis of distinct PKS gene clusters because none of the inserts in cosmid contigs contain AseI sites. Thus, clusters A, B, C, F and G seemed to be fully contained within AseI fragments of 500, 290, 620, 940 and 800 kb, respectively. However, 3·6 kb and 2·6 kb PvuII fragments in cosmid 14G12 in cluster D, 3·8 kb PvuII fragment in cosmid 6D8 from cluster E and 7 kb PvuII fragment in cosmid 2E4 from cluster H all hybridized to the 620 kb AseI fragments, together with cluster C, which were not differentiated further by digestions with DraI and SspI because both enzymes resulted in smearing and poor resolution of the chromosomal fragments on PFGE.
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Cluster A contains genes for nanchangmycin biosynthesis
Cosmid 11A8, with a 37 kb insert in the middle of cluster A (Fig. 3), contains the tsr gene suitable for selection in Streptomyces. Its ColE1 origin of replication is non-functional in strain NS3226. The pIJ101 origin of replication and sti (second-strand origin of replication) (Deng et al., 1988
) are functional but result in extremely unstable replication in strain NS3226. For targeted gene replacement, 26 kb of the strain NS3226 DNA insert in cosmid 11A8 was replaced by a 1·4 kb apramycin resistance (AprR) determinant [aac3(IV)]. This was achieved first by complete digestion of 11A8 DNA by BamHI and religation to obtain pHZ1552 in E. coli. The internal 26 kb (marked N1 in Fig. 3
) of the 37 kb insert in 11A8 was found to be deleted and was further replaced by a 1·4 kb BamHI fragment carrying aac3(IV) inserted between 4·8 kb (proximal to bla) and 5·8 kb (proximal to oriT) fragments flanking both sides of the deleted region, giving pHZ1553 (Fig. 7a
).
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DISCUSSION |
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Our data presented here imply that micro-organisms may encode many more secondary metabolites than are readily detectable in standard fermentation. While the overwhelming study of polyketide biosynthetic gene clusters in micro-organisms, especially Streptomyces, is experiencing many exciting breakthroughs, the genetic, and thus their encoded metabolite, diversities might still be far from thorough understanding. The identification of six to eight PKS gene clusters in a single Sorangium cellulosum strain, which is known to produce only epothilone, had also been reported by Santi et al. (2000) . We suspect that type I PKS-like pathways could be a common theme among many different bacteria, apart from most commonly studied actinomycetes. Using conserved or even heterologous PKS probes for identifying unknown PKS gene clusters (or potential secondary metabolite pathways), especially in micro-organisms other than, for example, Streptomyces or Myxobacterium, for discovering the new polyketide compounds may have significant implications.
The identification and isolation of multiple PKS genes in a single strain could provide an opportunity for isolating Str. nanchangensis derivatives that produce only one of the antibiotics but in increased quantities, as the biosynthesis of compounds derived from similar precursors in the same cell might be competitive. Thus an increase in production, or selective antibiotic production, could be achieved by selective cluster-specific mutagenesis by gene replacement, as we have demonstrated. Indeed, the deletion of a large DNA fragment by gene replacement (not shown) in cluster C (Fig. 4) resulted in at least a threefold increase of the nanchangmycin production as detected by bioassay, but no obvious increase for meilingmycin production was detected in the disrupted strains for clusters AC (data not shown). An example of successful selective production of avermectin compounds, obviating the need for separation of avermectin and oligomycin by knocking out oligomycin production in an avermectin producer by Tn4560 mutagenesis had been reported by Ikeda & Omura (1995)
. The unknown or unwanted clusters could be deleted in a stepwise manner. Such targeted deletions should have no danger of introducing deleterious mutations elsewhere in the chromosome, which would otherwise affect, for example, cell growth.
A search of the isolated multiple PKS clusters for the ones that could be involved in nanchangmycin or meilingmycin biosynthesis led to the discovery of the cluster that is essential for the former. Several DNA fragments in clusters AC respectively were disrupted or replaced for targeted mutations to achieve this result, although none of the three clusters were found to encode meilingmycin production. To our knowledge, the identification of a large region involved in nanchangmycin production would constitute a first documented example of what seems to be a complete polyether antibiotic biosynthesis gene cluster. The coverage of 89 kb PKS-homologous DNA (Fig. 3) involved in the biosynthesis of nanchangmycin (Fig. 1
) strongly supports the hypothesis of a modular organization for polyether biosynthesis, as has been reported for macrolide and other antibiotics. We tend to think that the interruption of the 89 kb PKS-homologous region by a 12 kb PKS-non-homologous region is not a division of two independent PKS clusters, but all necessary for nanchangmycin production. Based on erythromycin and avermectin examples, a standard PKS module appears to be encoded by
5 kb of DNA (Bevitt et al., 1992
; Donadio & Katz, 1992
; MacNeil et al., 1992
). The DNA encoding the PKS for the nanchangmycin polyether production would require at least 70 kb of genetic information; enough for 14 modules. The 89 kb PKS-homologous region might be a maximal estimate because four PvuII end fragments flanking the two separate contigs (Fig. 3
) may only have a partial PKS-homologous region. The most interesting and possibly unique enzymes for polyether biosynthesis would be the cyclase(s) that catalyses the cascade of cyclizations to form the polyether structure, but such new information will have to wait until the gene cluster is sequenced: this work is now in progress. The genetic information required for such enzymes [or/and additional genes necessary for the side-group (Fig. 1
) biosynthesis] could either be located in the 12 kb DNA sandwiched between two PKS-homologous regions or flanking both sides of the PKS-homologous regions. The interrupted PKS regions in a single PKS pathway had also been reported in other clusters, including avermectin (MacNeil et al., 1992
; Ikeda et al., 1999
).
Str. nanchangensis NS3226 is refractory to transformation by plasmid DNA from E. coli or Str. lividans, possibly because of the poor frequency of protoplast regeneration, and/or strong restriction system(s). Such problems were circumvented effectively by the development of mobilized conjugation of StreptomycesE. coli shuttle cosmid vectors, also carrying oriT from RP4, from E. coli to Str. nanchangensis. Most significant is the construction and efficient utilization of replicative Streptomyces plasmids derived from pIJ101 for generating mutations by targeted gene disruption and replacement experiments, which were used in this study for the demonstration that a specific gene cluster was involved in antibiotic production. Different from the suicide plasmids or temperature-sensitive Streptomyces plasmid vectors frequently used so far for gene disruption and replacement, pHZ1351 and pHZ1358 (Fig. 6) are replicative but genetically very unstable in Str. nanchangensis. Fewer than 1 in 1000000 colonies lost thiostrepton resistance and thus still could carry plasmids after one round of non-selective growth. pHZ1351 (Fig. 6a
) has a polylinker cloning site and can be propagated both in Streptomyces and in E. coli. pHZ1358 (Fig. 6b
) has additionally the origin of transfer of the conjugative plasmid RP4 for efficient conjugative transfer from E. coli to Streptomyces, and a phage
cos site flanked by T3 and T7 promoters for the efficient generation of ordered cosmid libraries. It is not clear whether sti has anything to do with the structural stability of the plasmids as has been suggested by Zaman et al. (1993)
and Pigac et al. (1988)
because there are also structurally stable plasmids such as pIJ702 that lack this function, and a case of instability of pIJ702 and its use for gene disruption experiments had been reported in Sac. erythraea (Weber et al., 1990
). The extreme segregational instability of pHZ1351 (Fig. 6a
) and pHZ1358 (Fig. 6b
) is likely to be caused by a spontaneous deletion of the 693 bp fragment which we have characterized between rep and sti, a region suggested to be required for plasmid maintenance (Kieser et al., 1982
).
Successful gene replacement experiments using pHZ1351 or pHZ1358 were also performed for mutagenizing a peptide pathway gene in Streptomyces hygroscopicus 10-22 and a polyene pathway gene in Streptomyces sp. FR008 in our laboratory (unpublished results) and in other laboratories, for mutagenizing the milbemycin PKS of Streptomyces griseochromogenes (P. Dyson, personal communication) and the clavulanic acid pathway of Streptomyces clavuligerus, a goal that could not be achieved with many other vector plasmids (P. Liras, personal communication).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 15 May 2001;
revised 29 August 2001;
accepted 9 October 2001.