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Development of a gene cloning system in a fast-growing and moderately thermophilic Streptomyces species and heterologous expression of Streptomyces antibiotic biosynthetic gene clusters

BMC Microbiology volume 11, Article number: 243 (2011) Cite this article

Abstract

Background

Streptomyces species are a major source of antibiotics. They usually grow slowly at their optimal temperature and fermentation of industrial strains in a large scale often takes a long time, consuming more energy and materials than some other bacterial industrial strains (e.g., E. coli and Bacillus). Most thermophilic Streptomyces species grow fast, but no gene cloning systems have been developed in such strains.

Results

We report here the isolation of 41 fast-growing (about twice the rate of S. coelicolor), moderately thermophilic (growing at both 30°C and 50°C) Streptomyces strains, detection of one linear and three circular plasmids in them, and sequencing of a 6996-bp plasmid, pTSC1, from one of them. pTSC1-derived pCWH1 could replicate in both thermophilic and mesophilic Streptomyces strains. On the other hand, several Streptomyces replicons function in thermophilic Streptomyces species. By examining ten well-sporulating strains, we found two promising cloning hosts, 2C and 4F. A gene cloning system was established by using the two strains. The actinorhodin and anthramycin biosynthetic gene clusters from mesophilic S. coelicolor A3(2) and thermophilic S. refuineus were heterologously expressed in one of the hosts.

Conclusions

We have developed a gene cloning and expression system in a fast-growing and moderately thermophilic Streptomyces species. Although just a few plasmids and one antibiotic biosynthetic gene cluster from mesophilic Streptomyces were successfully expressed in thermophilic Streptomyces species, we expect that by utilizing thermophilic Streptomyces-specific promoters, more genes and especially antibiotic genes clusters of mesophilic Streptomyces should be heterologously expressed.

Background

Streptomyces species are high G+C, Gram-positive bacteria that are a major source of natural products, producing about half of all known microbial antibiotics [1]. Members of this genus also have a complex life cycle, in which uni-genomic spores geminate to produce a multi-genomic substrate mycelium of branching hyphae which gives rise to aerial hyphae and ultimately to spores [2]. Streptomyces coelicolor A3(2) is the genetically most studied Streptomyces species from the in vivo through in vitro to in silico phases and is an excellent model for studying antibiotic production and differentiation [3, 4]. Mainly because of a strong restriction barrier to introduction of foreign double-stranded DNA by transformation from Escherichia coli into A3(2), the closely related S. lividans, with no such barrier and cured of indigenous plasmids (SLP2 and SLP3: [5]), has been used as a standard host for gene cloning and expression for several decades [6]. However, compared with E. coli and Bacillus subtilis, S. coelicolor and S. lividans (also other species from the genus Streptomyces) grow slowly at their optimal temperature (e.g., S. coelicolor M145 - a plasmid-free derivative of A3(2) - grows exponentially with a doubling time of about 2.2 h on SMM medium at 28°C, see ref [6]). It takes about 2-3 weeks for Streptomyces strains to produce and accumulate antibiotics at a good yield on an industrial scale.

Fast-growing, thermophilic Streptomyces strains have been studied for a long time. Some earlier described thermophilic Streptomyces species (e.g., S. thermophilis and S. thermofuscus: [7, 8]) were not classified as thermophilic streptomycetes [9, 10]. Numerical classification of thermophilic streptomycetes showed three major, five minor and two single-member clusters [10]. Analysis of the 16S rRNA genes and morphological and chemical properties indicate their classification within the genus Streptomyces [11, 12]. Most thermophilic Streptomyces species have growth temperature ranges from 28 to 55°C and so are only moderately thermophilic [11, 12]. However, some thermophilic Streptomyces species can grow up to 68°C [13]; the optimum growth temperature of S. thermoautotrophicus is 65°C and no growth is observed below 40°C, so it is a truly thermophilic strain [14]. Growth of thermophilic Streptomyces strains is rapid at high temperature [15]; for example, S. thermoviolaceus has a doubling time of 1 h at 50°C [16]. Thermophilic Streptomyces species produce thermostable enzymes and antibiotics [15], such as xylanase [17], alpha-amylase [18], granaticin [16] and anthramycin [19]. Since thermophilic Streptomyces strains lack a genetic manipulation system, mesophilic strains (e.g. S. lividans) have been employed for expression of some genes or antibiotic biosynthetic gene clusters from thermophilic Streptomyces species [20–22].

We report here the development of a gene cloning system in a fast-growing (about twice the rate of S. coelicolor) and moderately thermophilic (growing at both 30°C and 50°C) Streptomyces strain, and successful heterologous expression of antibiotic biosynthetic gene clusters from both thermophilic and mesophilic Streptomyces species.

Results and Discussion

Isolation and identification of thermophilic Streptomyces strains from various soil samples

To isolate thermophilic Streptomyces strains, various soil samples from China were collected (see Methods). As summarized in Table 1, 22, 11 and eight strains were isolated from samples of garden soil, weed compost and swine manure, respectively. Thermophilic Streptomyces species have been isolated from composts, soil and sewage [23], as well as lakes and hot-springs [13]. Our results reinforce the idea of a widespread occurrence of these organisms.

Table 1 Strains used in this study
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To determine if these strains should be classified in the genus Streptomyces, 16S rRNA genes were amplified, cloned and sequenced. The sequences displayed high homology (97-99%) to those of known mesophilic Streptomyces and/or thermophilic Streptomyces species. A phylogenetic tree was drawn by using a neighbor-joining method [24]. The chosen 11 newly isolated strains have distinct phenotypes when cultured on R2YE and MS media, and the reference strains utilized for comparison are well-classified Streptomyces species. As shown in Figure 1, all 11 newly isolated strains (4F, T6C-1, T1A, T6E-2. X4-3, T6-1-4, X3-3, 2C, T5A-1, T6A-2 and T6A-3) resembled known thermophilic Streptomyces species (e.g., S. thermocarboxydus, S. thermoviolaceus and S. glaucescens). Moderately thermophilic Streptomyces species form at least two distinct clades [12, 23, 25], containing strains related to S. megasporus and S. thermodiastaticus, respectively. The phylogenetic tree of the 11 newly isolated strains reveals more clades (e.g., T5A-1 and T6E-2; see Figure 1). These results indicate that moderately thermophilic Streptomyces species are diverse in natural habitats.

Figure 1
figure 1

Identification of thermophilic Streptomyces strains. Phylogenetic tree for 11 newly identified strains and some known mesophilic and thermophilic Streptomyces species (Genbank numbers in parentheses). The tree is drawn to scale using the neighbor-joining method, with branch lengths in the same units as those of the evolutionary distances. Numbers next to the branches are the percentage of replicate trees (the bootstrap test is 500 replicates).

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Like typical Streptomyces species, these newly isolated strains produced spores on R2YE and MS media. Scanning electron microscopy showed that strains 4F and 2C formed long chains of smooth-surfaced spores after growth on MS medium at 42°C for 2 d (data not shown). Thus strains 4F and 2C were classified in the genus Streptomyces.

Characterization of the fast-growing and moderately-thermophilic Streptomyces strains 4F and 2C

As shown in Figure 2, strains 4F and 2C were able to grow from 30 to 50°C, while two mesophilic Streptomyces strains (S. coelicolor M145 and S. venezuelae ISP5230) grew at 30°C and 37°C. 4F and 2C grew well at 45°C and 50°C but poorly at 55°C, while M145 and ISP5230 could not grow at 45°C and 50°C (data not shown). Thus, 4F and 2C were concluded to be moderately thermophilic Streptomyces strains.

Figure 2
figure 2

Growth of strains 4F, 2C, M145 and ISP5230 on MS medium at different temperatures in a time-course. A series of 10× dilutions of spore suspensions were inoculated onto MS medium and incubated at 30, 37, 45 and 50°C in a time-course at 20, 30, 40 and 60 h. The numbers of spores of the four strains inoculated on plates are shown. 4F and 2C were cultured at 30, 37, 45 and 50°C, while M145 and ISP5230 were grown at 30 37 and 45°C.

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Strains 4F and 2C grew on MS medium at 37°C and 45°C faster than the mesophilic Streptomyces strains at 30°C and 37°C (Figure 2). To measure the growth rates of 4F and M145, equal numbers of spores were inoculated into TSB liquid medium, and three mycelial samples were harvested at various points during the time course. Each sample was weighed, and the three values were averaged for a particular time point. As shown in Figure 3, 4F rapidly accumulated biomass to a maximum at 45°C or 37°C within 16 h, then the growth curve fluctuated, and the final biomass of strain 4F is higher for M145 (especially at 45°C). The oscillations shown at 37 and 45°C resembling the "death/growth process" of S. coelicolor A3(2) in liquid medium with a diluted inoculum [26]. The doubling times of growth for 4F at 30, 37, 45 and 50°C and M145 at 30°C and 37°C in each logarithmic phase (14-20, 6-12, 8-14 and 12-18 h for 4F at 30, 37, 45 and 50°C, and 16-22 for M145 at 30 and 37°C) were 2.3, 1.4, 1.1 2.3, 2.2 and 2.4 h, respectively. Thus strain 4F grew at 45°C twice and at 37°C 1.6 times as fast as M145 at 30°C in TSB medium.

Figure 3
figure 3

Growth curves of 4F and M145 in liquid culture at four temperatures. The curves are based on the average of three weighings at each time point, and standard deviations are indicated.

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Figure 4
figure 4

Quantitation of actinorhodin production by M145 and by 4F containing the cloned actinorhodin gene cluster in liquid medium. About 1 × 106 spores of M145 and of 4F containing pCWH74 were inoculated into 50 ml R2YE liquid medium (lacking KH2PO4 and CaCl2) at 30 and 37°C. Samples of 1 ml culture were harvested in a time-course and treated with KOH; absorption at OD640 indicated actinorhodin production.

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Identification of one linear and three circular plasmids among 41 strains, and sequencing of pTSC1

We detected three circular plasmids, 7-kb pTSC1, from X4-3, 7.5-kb pTSC2 from X3-3, and 40-kb pTSC3 as well as 16-kb linear pTSL1 from T6-1-4. The complete nucleotide sequence of the circular pTSC1 consisted of 6996 bp (GenBank accession number GU271942), with 72% G+C, resembling that of a typical Streptomyces genome (e.g., 72.1% for S. coelicolor A3(2): [27]). Eight ORFs (open reading frame) were predicted by "FramePlot 3.0 beta" [28]; seven of them resembled Streptomyces or Mycobacterium genes (Additional file 1, Table S1). Notably, three genes resembled the transfer and spread genes (tra and spd) of Streptomyces plasmids pIJ101 [29] and pSNA1 [30].

Development of a gene cloning system in strains 2C and 4F

Followed the standard protocols of preparation and transformation of Streptomyces protoplasts with slight modifications (see Methods), pTSC1-derived pCWH1 (see Methods and Table 2) was introduced by transformation into ten well-sporulating thermophilic Streptomyces strains. Thiostrepton-resistant colonies were obtained for strains 2C and 4F at frequencies of 1.3 × 103 and 2 × 101 per μg DNA, but no transformants arose for the other eight strains. Many Streptomyces selection markers (e.g., tsr, apr, spec, hyg, erm and kan) could be used in strains 2C and 4F. No antibacterial activity (e.g., against Bacillus subtilis, Escherichia coli or Staphyloccocus aureus) was detected in the two strains (unpublished data). Thus, we found two promising cloning hosts, 2C and 4F.

Table 2 Plasmids used in this study
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Since 2C and 4F were classified in the genus Streptomyces, several mesophilic Streptomyces vectors were employed for transformation experiments. As shown in Table 3, pIJ702 (a pIJ101 derivative, [31]), pZR51 (pFRL2, [32]), pZR115 (pFP1, [33]) and pZR10 (pFP11, [33]) were able to transform both 2C and 4F. No transformants were obtained for SCP2 [34], SLP1[35], SAP1 [36] and pSHK1 [32] derivatives (pYQ40, pZR205, pHAQ61, and pGP9, respectively). pCWH1 could also transform S. lividans ZX7 [37] at high frequency (104/μg DNA). A Streptomyces integrating plasmid, pSET152 [38], could be introduced by conjugation from E. coli into many thermophilic Streptomyces strains (14 of 22 strains). Thus, pTSC1-derived pCWH1 can replicate in both thermophilic and mesophilic Streptomyces strains. On the other hand, several Streptomyces replicons, including circular plasmids pIJ101, pFP1 and pFP11 and linear plasmid pFRL2, were able to propagate in the thermophilic Streptomyces strains 2C and 4F, but no transformants were obtained for circular plasmids SCP2 and SLP1 and linear plasmids SAP1 and pSHK1.

Table 3 Transformation by plasmids of the moderately thermophilic Streptomyces 2C and 4F
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Comparing the transformation frequencies of pIJ702 from different hosts in 2C and 4F, as shown in Table 3, similar high frequencies of transformation (2.9 × 106 and 1.3 × 106) were obtained in 2C with pIJ702 from both 2C itself and the largely restriction-free S. lividans ZX7. Low frequencies of transformation (8 × 101 and 3 × 102) were obtained in 4F with pIJ702 from 2C and ZX7, although a high frequency (1.2 × 105) was obtained with plasmid DNA from the strain itself. These results indicated that strain 2C showed essentially no restriction barrier to the introduction of foreign double-stranded DNA from other Streptomyces species, whereas strain 4F had a strong restriction barrier. The evaluation of restriction barriers needs much more experimental data to be supported.

Heterologous expression of the actinorhodin biosynthetic gene cluster of S. coelicolor A3(2) in strain 4F

Since several mesophilic Streptomyces plasmids functioned in thermophilic Streptomyces, we chose a phage phiC31-derived integrating plasmid pSET152 [38] which is inherited stably in other hosts to perform experiment on heterologous expression of antibiotic biosynthetic genes in thermophilic Streptomyces strains. By using PCR with eight primers from the actinorhodin biosynthetic genes (sco5085-5092), we found that no bands for strains 4F and 2C were detected on agarose gel after electrophoresis of the PCR products, indicating no such genes in the strains. We cloned the complete actinorhodin biosynthetic gene cluster from S. coelicolor A3(2) in an integrating plasmid (see Methods), and the resulting plasmid, pCWH74, was introduced by conjugation into eight newly isolated strains, including 4F and 2C. PCR amplification experiments with eight paired primers from SCO5085 to SCO5092 confirmed the presence of the actinorhodin genes in the clones of 4F and 2C. Blue pigment was observed for strain 4F on both R2YE and MS media at 30 and 37°C after growth for 1 d, but no blue pigment was seen at 45°C. 2C with the actinorhodin gene cluster did not produce visible blue pigment on R2YE or MS media. To confirm that the blue pigment was actinorhodin, 4F containing pCWH74 was cultured in R2YE liquid medium lacking KH2PO4 and CaCl2 and the supernatant was treated with KOH and scanned at 640 nm [39]. The same pattern of absorption peaks was detected for 4F as for S. coelicolor A3(2) (data not shown). Thus the actinorhodin biosynthetic gene cluster from the mesophilic S. coelicolor A3(2) was heterologously expressed in strain 4F at low temperature (30 and 37°C), but not at high temperature (45°C).

To quantitate the productivity of actinorhodin, equal amounts of spores of M145 and 4F containing pCWH74 were inoculated into R2YE liquid medium lacking KH2PO4 and CaCl2, and 1 ml culture was harvested in a time-course. As shown in Figure 4, actinorhodin was produced in 4F at both 30 and 37°C, earlier than in M145 at 30°C. At 100 h, productivity of actinorhodin in 4F at 30°C was ~2.8 times higher than in M145 at 30°C. Strains M145 and 4F grew better in TSB than in R2YE liquid media (data no shown), but no actinorhodin was detected when cultured in TSB medium at 30 and 37°C. Growth curves of the two strains in R2 lacking KH2PO4 and CaCl2 at 30°C showed that their biomass values were similar from 20 to 120 hours (data not shown). Thus, better growth of M145 and 4F in TSB medium (Figure 3) did not correlate with delayed and less production of actinorhodin in R2YE medium (Figure 4).

Like in 4F, M145 produced more actinorhodin in R2YE medium at 30°C than at 37°C, suggesting that expression of the actinorhodin biosynthetic genes might be temperature-dependent. Temperature-dependent antibiotic gene clusters have been reported in Streptomyces, for example, much higher productivity of validamycin A produced by Streptomyces hygroscopicus was found at 37°C than at 30°C [40]. We infer that by replacement of thermophilic-specific promoters, many single genes and especially antibiotic genes clusters of mesophilic Streptomyces should be heterologously expressed in the fast-growing and thermophilic Streptomyces.

Heterologous expression of the anthramycin biosynthetic gene cluster of the thermophilic S. refuineus subsp. thermotolerans in strain 4F

Expression of the anthramycin biosynthetic genes of S. refuineus subsp. thermotolerans could be detected at high temperature (i.e. 47°C), but not at 30 or 37°C [22]. An integrating cosmid, 024COA-3, containing the whole anthramycin biosynthetic gene cluster was introduced by conjugation from E. coli into strain 4F. PCR amplification experiments confirmed the presence of the anthramycin genes in the clone of 4F. After culturing in AP1 medium at 30, 37 and 47°C for 24 h, mycelium was extracted, dried and re-dissolved in MeOH. Thin-layer chromatography, followed by a bio-assay by overlaying with LB agar containing as indicator strain a Bacillus sp., revealed a zone of growth inhibition on 4F at 47°C, but no inhibition zone was found at 30 and 37°C (data not shown). A spot on a TLC plate was further purified for HPLC-MS analysis. As shown in Figure 5, an anthramycin-specific peak (ES+ = 316 Dalton, see ref [41]) was detected. Thus the anthramycin biosynthetic gene cluster of the thermophilic S. refuineus subsp. thermotolerans was heterologously expressed in strain 4F. We introduced the same cosmid 024COA-3 containing the anthramycin gene cluster into strain 2C, but no transformants were obtained. To see if strain 2C might be a better host than 4F, more antibiotic biosynthetic gene clusters should be tested.

Figure 5
figure 5

Analysis of anthramycin production by HPLC/MS. After separating anthramycin on an HPLC column, mass spectrometry was performed using 6520 Agilent Accurate-Mass Q-TOF LC/MS.

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Conclusions

This study shows that by isolation of new strains and testing several plasmids, a host-vector system in a fast-growing and moderately thermophilic Streptomyces species could be developed. Two antibiotic biosynthetic gene clusters from mesophilic and thermophilic Streptomyces were heterlogously expressed in one strain. We expect that by utilizing thermophilic Streptomyces-specific promoters, more genes and especially antibiotic genes clusters of mesophilic Streptomyces should be heterologously expressed.

Methods

Bacterial strains, plasmids, and general methods

Strains used in this work are listed in Table 1. Plasmid isolation, transformation of E. coli DH5α and PCR amplification followed Sambrook et al. [42]. Streptomyces culture, plasmid isolation and preparation of protoplasts and transformation of Streptomyces lividans ZX7 followed Kieser et al. [6]. Plasmid trans-conjugation from E. coli ET12567/pUZ8002 into thermophilic Streptomyces strains followed Bierman et al. [38]. KpnI-treated pTSC1 was cloned in pBluescript II SK to obtain pCWH100 and was sequenced by primer-walking at Shanghai Invitrogen Inc. Sequence comparisons were done with software from the National Center for Biotechnology Information http://www.ncbi.nlm.nih.gov/BLAST. The complete nucleotide sequence of pTSC1 was deposited in the GenBank database under no. GU271942.

Isolation and identification of thermophilic Streptomyces strains

Samples of garden soil, weed compost and swine manure were collected from Shanghai city, Hunan, Hubei and Fujian provinces in the summers of 2005 and 2006. The samples were dried at 100°C for 1 h and cultivated on SC medium (starch 10 g, casein 0.3 g, KNO3 2 g, MgSO4.7H2O 0.05 g, FeSO4.7H2O 0.01 g, CaCO3 0.02 g, agar 18 g, H2O to 1000 ml, pH7.2) [43] at 50°C for 3-5 d. Thermophilic Streptomyces strains were cultured in TSB (Oxoid tryptone soya broth powder, 30 g, H2O to 1000 ml) liquid medium at 45°C for 1 d and genomic DNA was isolated followed the Kirby mix procedure [6]. 16S rRNA genes were amplified by PCR with primers (5'-AGAGTTTGATCCTGGCTCAG-3' and 5'-TCAGGCTACCTTGTTACGACTT-3'). PCR conditions were: template DNA denatured at 95°C for 5 min, then 95°C 30 s, 55°C 30 s, 72°C 2 min, for 35 cycles. PCR products were cloned in pBluescript II SK and sequenced with its T7 and T3 primers.

Strains were inoculated on MS (mannitol 20 g, soya flour 20 g, agar 20 g, H2O to 1000 ml, pH7) medium covered with cellophane disks. After 2 days incubation at 42°C, the cells were fixed with fresh 2% glutaraldehyde (pH7.2) and 1% osmium tetroxide. Spores were examined with a JSM-6360LV scanning electron microscopy (Jeol).

Isolation of plasmids from thermophilic Streptomyces strains

Isolating plasmid from thermophilic Streptomyces strains followed the protocol of Kieser [44] with sight modification. Strains were cultured in TSB liquid medium at 42°C overnight and mycelium was harvested by spinning at 4000 rpm for 15 min. About 50 μl mycelium was suspended in 350 μl TES buffer (25 mM Tris-HCL pH8, 25 mM EDTA pH8, 0.3 M sucrose, 2 mg/ml lysozyme, 5 μg/ml pre-boiled RNase A) and incubated at 37°C for 30 min. 44 μl of 10% SDS was added and mixed immediately by rotating and then 4 μl of 10 mg/ml proteinase K was added, followed by incubation for 60 min. 225 μl of 0.3 N NaOH/2% SDS was added and mixed immediately by vortexing, incubated at 70°C for 15 min and then cooled. 200 μl acid phenol/chloroform was added and vortexed and centrifuged at 12000 rpm for 10 min. The supernatant was transferred to a new centrifuge tube containing 55 μl un-buffered sodium acetate and 500 μl isopropanol was added. After mixing and centrifugation at 12000 rpm for 10 min and all liquid was removed using a pipette. The pellet was washed twice with 1 ml 70% ethanol, air dried and dissolved in 50 μl TE buffer.

Growth curve of thermophilic Streptomyces strains in liquid culture

About 1.5 × 107 spores were inoculated into 50 ml TSB liquid medium supplemented with 0.01% antifoam289 (Sigma A 5551) and cultured at 30, 37, 45 and 50°C. 1 ml culture was harvested at each time-point and wet mycelium was harvested by centrifugation at 12000 rpm for 5 min. After drying for 10 min in a vacuum, the pellet was weighed with a fine balance (min. 10 mg). Growth curves were drawn with an average of three weighings at each time-point.

Protoplast preparation and transformation of thermophilic Streptomyces strains

Protoplast preparation, regeneration and transformation of the thermophilic Streptomyces strains 2C and 4F followed standard Streptomyces protocols [6, 45] with slight modifications. About 1 × 109 spores were inoculated into 50-ml YEME liquid medium (yeast extract powder 3 g, peptone 5 g, malt extract powder 3 g, glucose 10 g, with 25% sucrose, H2O to 1000 ml, pH7, supplemented with 0.5% glycine for 2C and 0.3% for 4F) at 45°C for ~7 h. Mycelium was harvested, washed once with 10.3% sucrose, and 1 mg/ml lysozyme solution in P buffer was added at 30°C (ca. 15 min for 2C and 30 min for 4F) to make protoplasts. After transformation, regeneration of protoplasts was achieved on R2YE medium at 45°C for ca. 9 h, to be selected by antibiotics.

Construction of plasmids for transformation of thermophilic Streptomyces strains

Plasmids used in this work are listed in Table 2. Sizes of circular plasmids pTSC1, pTSC2 and pTSC3 and linear plasmid pTSL1 from thermophilic Streptomyces strains were measured by electrophoresis with known DNA markers (i.e. 1-kb supercoiled ladder and sequenced circular/linear plasmids). pQC156 [46] containing Streptomyces selection markers melC/tsr was cloned in an E.coli plasmid pSP72. KpnI-treated pTSC1 was cloned in pQC156 to obtain pCWH1. The mesophilic Streptomyces replicons, including circular plasmids SCP2 [34], pFP1 and pFP11[33], linear plasmids SAP1 [36], pFRL2 [32] and pSHK1 [32], and integrating plasmid SLP1 [35], were cloned in pQC156 to yield pYQ40, pZR115, pZR10, pQC578, pHAQ61, pZR51, pGP9 and pZR205, respectively. These plasmids were introduced by protoplast transformation into thermophilic Streptomyces strains.

Cloning and heterologous expression of the actinorhodin gene cluster in thermophilic Streptomyces

pHAQ31 [47] contained an E.coli replication origin and two cos sites of Supercos1 [48] and Streptomyces selection markers melC/tsr genes [31]. pHAQ31-derived cosmid N7-85 contained the whole actinorhodin biosynthetic gene cluster (5510413-5543521 bp) from S. coelicolor A3(2). A 3.4-kb XbaI/NheI fragment containing the phage фC31 integrase gene of pSET152 was cloned in a XbaI site of N7-85. The resulting plasmid, pCWH74, was introduced by conjugation from E. coli into thermophilic Streptomyces strains [38], which were cultured on R2YE (sucrose 103 g, K2SO4 0.25 g, MgCl2.6H2O 10.12 g, glucose 10 g, Difco Casaminoacids 0.1 g, trace element solution 2 ml, Difco yeast extract 5 g, TES 5.73 g, agar 22 g, H2O to 1000 ml, after autoclave and add 0.5% KH2PO4 5 ml, 5 M CaCl2.2H2O 4 ml, 20% L-proline 15 ml, 1N NaOH 7 ml) and MS media at 30, 37 and 45°C to detect blue actinorhodin pigment. To quantitate the production of actinorhodin, about 1 × 106 spores of M145 and 4F containing pCWH74 were inoculated into 50 ml R2YE liquid medium (lacking KH2PO4 and CaCl2) at 30 and 37°C; 1 ml culture was harvested in a time-course and treated with KOH, whereupon absorption at OD640 indicated actinorhodin production [39].

Heterologous expression of the anthramycin biosynthetic gene cluster in thermophilic Streptomyces

An integrating cosmid, 024COA-3, containing the whole anthramycin biosynthetic gene cluster (EU195114.1, 1-33150 bp) (kindly provided by Prof. Brian Bachmann) was introduced by conjugation from E. coli into strain 4F [38]. Detection of anthramycin production followed Hu et al. [41]. After culturing in AP1 (corn starch 10 g, 2% peptonized milk, yeast extract powder 30 g, H2O to 1000 ml, pH7) medium at 47°C for 24 h, mycelium was extracted, dried and re-dissolved in MeOH. Anthramycin was first isolated on a HPLC column (Zorbax eclips 1.8 μm XDB-C18) and then mass spectrometry was performed using 6520 Agilent Accurate-Mass Q-TOF LC/MS. Anthramycin was separated by using a Zorbax eclips 1.8 μm XDB-C18 with a linear water-acetonitrile gradient containing 10 mM ammonium acetate (0.2 ml/min). The electrospray needle of the mass spectrometer was at 4000 V, the voltage of the skimmer was set to 65 V, Oct RF Vpp750V, collision ev 45 V, nebulizer pressure at 45 psig, and drying gas N2 350°C 9 L/min.

References

  1. Bérdy J: Bioactive microbial metabolites. J Antibiot (Tokyo). 2005, 58: 1-26. 10.1038/ja.2005.1.

    Article  Google Scholar 

  2. Chater KF: Genetics of differentiation in Streptomyces. Annu Rev Microbiol. 1993, 47: 685-713. 10.1146/annurev.mi.47.100193.003345.

    Article  PubMed  CAS  Google Scholar 

  3. Hopwood DA: Forty years of genetics with Streptomyces: from in vivo through in vitro to in silico. Microbiology. 1999, 145 (Pt 9): 2183-2202.

    Article  PubMed  CAS  Google Scholar 

  4. Hopwood DA: Soil to genomics: the Streptomyces chromosome. Annu Rev Genet. 2006, 40: 1-23. 10.1146/annurev.genet.40.110405.090639.

    Article  PubMed  CAS  Google Scholar 

  5. Hopwood DA, Kieser T, Wright HM, Bibb MJ: Plasmids, recombination and chromosome mapping in Streptomyces lividans 66. J Gen Microbiol. 1983, 129: 2257-2269.

    PubMed  CAS  Google Scholar 

  6. Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA: Practical Streptomyces Genetics. 2000, The John Innes Institute, The John Innes Foundation Press

    Google Scholar 

  7. Gilbert R: Ueber Actinomyces thermophilus und andere Actinomyceten. Zeitschrift für Hygiene und Infektionskeiten. 1904, 47: 383-406. 10.1007/BF02284567.

    Article  Google Scholar 

  8. Waksman SA, Umbreit WW, Cordon TC: Thermophilic actinomycetes and fungi in soils and in composts. Soil Science. 1939, 47: 37-61. 10.1097/00010694-193901000-00005.

    Article  CAS  Google Scholar 

  9. Skerman VBD, McGowan V, Sneath PHA: Approved lists of bacterial names. Int J Syst Bacteriol. 1980, 30: 225-420. 10.1099/00207713-30-1-225.

    Article  Google Scholar 

  10. Goodfellow M, Lacey J, Todd C: Numerical classification of thermophilic streptomycetes. J Gen Microbiol. 1987, 133: 3135-3149.

    Google Scholar 

  11. Kim SB, Falconer C, Williams E, Goodfellow M: Streptomyces thermocarboxydovorans sp. nov. and Streptomyces thermocarboxydus sp. nov., two moderately thermophilic carboxydotrophic species from soil. Int J Syst Bacteriol. 1998, 48: 59-68. 10.1099/00207713-48-1-59.

    Article  PubMed  Google Scholar 

  12. Kim SB, Goodfellow M: Streptomyces thermospinisporus sp. nov., a moderately thermophilic carboxydotrophic streptomycete isolated from soil. Int J Syst Evol Microbiol. 2002, 52: 1225-1228. 10.1099/ijs.0.02003-0.

    PubMed  CAS  Google Scholar 

  13. Xu LH, Tiang YQ, Zhang YF, Zhao LX, Jiang CL: Streptomyces thermogriseus, a new species of the genus Streptomyces from soil, lake and hot-spring. Int J Syst Bacteriol. 1998, 48: 1089-1093. 10.1099/00207713-48-4-1089.

    Article  PubMed  Google Scholar 

  14. Gadkari D, Schricker K, Acker G, Kroppenstedt RM, Meyer O: Streptomyces thermoautotrophicus sp. nov., a thermophilic CO- and H(2)-oxidizing obligate chemolithoautotroph. Appl Environ Microbiol. 1990, 56: 3727-3734.

    PubMed  CAS  PubMed Central  Google Scholar 

  15. Edwards C: Isolation properties and potential applications of thermophilic actinomycetes. Appl Biochem Biotech. 1993, 42: 161-179. 10.1007/BF02788050.

    Article  CAS  Google Scholar 

  16. James PD, Edwards C: The effects of temperature on growth and production of the antibiotic granaticin by a thermotolerant streptomycete. J Gen Microbiol. 1989, 135: 1997-2003.

    PubMed  CAS  Google Scholar 

  17. Tsujibo H, Miyamoto K, Kuda T, Minami K, Sakamoto T, Hasegawa T, Inamori Y: Purification, properties, and partial amino acid sequences of thermostable xylanases from Streptomyces thermoviolaceus OPC-520. Appl Environ Microbiol. 1992, 58: 371-375.

    PubMed  CAS  PubMed Central  Google Scholar 

  18. Bahri SM, Ward JM: Sequence of the Streptomyces thermoviolaceus CUB74 alpha-amylase-encoding gene and its transcription analysis in Streptomyces lividans. Gene. 1993, 127: 133-137. 10.1016/0378-1119(93)90628-G.

    Article  PubMed  CAS  Google Scholar 

  19. Leimgruber W, Stefanović V, Schenker F, Karr A, Berger J: Isolation and characterization of anthramycin, a new antitumor antibiotic. J Am Chem Soc. 1965, 87: 5791-5793. 10.1021/ja00952a050.

    Article  PubMed  CAS  Google Scholar 

  20. Mellouli L, Guerineau M, Bejar S, Virolle MJ: Regulation of the expression of amy TO1 encoding a thermostable alpha-amylase from Streptomyces sp. TO1, in its original host and in Streptomyces lividans TK24. FEMS Microbiol Lett. 1999, 181: 31-39.

    PubMed  CAS  Google Scholar 

  21. Park HJ, Kim ES: An inducible Streptomyces gene cluster involved in aromatic compound metabolism. FEMS Microbiol Lett. 2003, 226: 151-157. 10.1016/S0378-1097(03)00585-8.

    Article  PubMed  CAS  Google Scholar 

  22. Hu Y, Phelan VV, Farnet CM, Zazopoulos E, Bachmann BO: Reassembly of anthramycin biosynthetic gene cluster by using recombinogenic cassettes. Chembiochem. 2008, 9: 1603-1608. 10.1002/cbic.200800029.

    Article  PubMed  CAS  Google Scholar 

  23. O'Donnell AG, Falconer C, Goodfellow M, Ward AC, Williams E: Biosystematics and diversity amongst novel carboxydotrophic actinomycetes. Antonie Van Leeuwenhoek. 1993, 64: 325-340.

    Article  PubMed  Google Scholar 

  24. Saitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987, 4: 406-425.

    PubMed  CAS  Google Scholar 

  25. Kim D, Chun J, Sahin N, Hah Y, Goodfellow M: Analysis of thermophilic clades within the genus Streptomyces by 16S ribosomal DNA sequence comparisons. Int J Syst Bacteriol. 1996, 46: 581-587. 10.1099/00207713-46-2-581.

    Article  CAS  Google Scholar 

  26. Manteca A, Alvarez R, Salazar N, Yagüe P, Sanchez J: Mycelium differentiation and antibiotic production in submerged cultures of Streptomyces coelicolor. Appl Environ Microbiol. 2008, 74: 3877-3886. 10.1128/AEM.02715-07.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  27. Bentley SD, Chater KF, Cerdeno-Tarraga AM, Challis GL, Thomson NR, James KD, Harris DE, Quail MA, Kieser H, Harper D, Bateman A, Brown S, Chandra G, Chen CW, Collins M, Cronin A, Fraser A, Goble A, Hidalgo J, Hornsby T, Howarth S, Huang CH, Kieser T, Larke L, Murphy L, Oliver K, O'Neil S, Rabbinowitsch E, Rajandream MA, Rutherford K, Rutter S, Seeger K, Saunders S, Sharp D, Squares R, Squares S, Taylor K, Warren T, Wietzorrek A, Woodward J, Barrell BG, Parkhill J, Hopwood DA: Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature. 2002, 417: 141-147. 10.1038/417141a.

    Article  PubMed  Google Scholar 

  28. Ishikawa J, Hotta K: FramePlot: a new implementation of the frame analysis for predicting protein-coding regions in bacterial DNA with a high G + C content. FEMS Microbiol Lett. 1999, 174: 251-253. 10.1111/j.1574-6968.1999.tb13576.x.

    Article  PubMed  CAS  Google Scholar 

  29. Kieser T, Hopwood DA, Wright HM, Thompson CJ: pIJ101, a multi-copy broad host-range Streptomyces plasmid: functional analysis and development of DNA cloning vectors. Mol Gen Genet. 1982, 185 (2): 223-238. 10.1007/BF00330791.

    Article  PubMed  CAS  Google Scholar 

  30. Mendes MV, Aparicio JF, Martin JF: Complete nucleotide sequence and characterization of pSNA1 from pimaricin-producing Streptomyces natalensis that replicates by a rolling circle mechanism. Plasmid. 2000, 43 (2): 159-165. 10.1006/plas.1999.1446.

    Article  PubMed  CAS  Google Scholar 

  31. Katz E, Thompson CJ, Hopwood DA: Cloning and expression of the tyrosinase gene from Streptomyces antibioticus in Streptomyces lividans. J Gen Microbiol. 1983, 129: 2703-2714.

    PubMed  CAS  Google Scholar 

  32. Zhang R, Xia H, Guo P, Qin Z: Variation in the replication loci of Streptomyces linear plasmids. FEMS Microbiol Lett. 2009, 290: 209-216.

    Article  PubMed  CAS  Google Scholar 

  33. Zhang R, Zeng A, Fang P, Qin Z: Characterization of the replication and conjugation loci of Streptomyces circular plasmids pFP11 and pFP1 and their ability to propagate in linear mode with artificially attached telomeres. Appl Environ Microbiol. 2008, 74: 3368-3376. 10.1128/AEM.00402-08.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  34. Haug I, Weissenborn A, Brolle D, Bentley S, Kieser T, Altenbuchner J: Streptomyces coelicolor A3(2) plasmid SCP2*: deductions from the complete sequence. Microbiology. 2003, 149: 505-513. 10.1099/mic.0.25751-0.

    Article  PubMed  CAS  Google Scholar 

  35. Bibb MJ, Ward JM, Kieser T, Cohen SN, Hopwood DA: Excision of chromosomal DNA sequences from Streptomyces coelicolor forms a novel family of plasmids detectable in Streptomyces lividans. Mol Gen Genet. 1981, 184 (2): 230-240.

    PubMed  CAS  Google Scholar 

  36. Ikeda H, Ishikawa J, Hanamoto A, Shinose M, Kikuchi H, Shiba T, Sakaki Y, Hattori M, Omura S: Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis. Nat Biotechnol. 2003, 21 (5): 526-531. 10.1038/nbt820.

    Article  PubMed  Google Scholar 

  37. Zhou X, Deng Z, Firmin JL, Hopwood DA, Kieser T: Site-specific degradation of Streptomyces lividans DNA during electrophoresis in buffers contaminated with ferrous iron. Nucleic Acids Res. 1988, 16: 4341-4352. 10.1093/nar/16.10.4341.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  38. Bierman M, Logan R, Obrien K, Seno ET, Rao RN, Schoner BE: Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene. 1992, 116 (1): 43-49. 10.1016/0378-1119(92)90627-2.

    Article  PubMed  CAS  Google Scholar 

  39. Bystrykh LV, FernandezMoreno MA, Herrema JK, Malpartida F, Hopwood DA, Dijkhuizen L: Production of actinorhodin related "blue pigments" by Streptomyces coelicolor A3(2). J Bacteriol. 1996, 178 (8): 2238-2244.

    PubMed  CAS  PubMed Central  Google Scholar 

  40. Liao YQ, Wei ZH, Bai LQ, Deng ZX, Zhong JJ: Effect of fermentation temperature on validamycin A production by Streptomyces hygroscopicus 5008. J Biotechnol. 2009, 142: 271-274. 10.1016/j.jbiotec.2009.04.015.

    Article  PubMed  CAS  Google Scholar 

  41. Hu Y, Phelan V, Ntai I, Farnet CM, Zazopoulos E, Bachmann BO: Benzodiazepine biosynthesis in Streptomyces refuineus. Chem Biol. 2007, 14: 691-701. 10.1016/j.chembiol.2007.05.009.

    Article  PubMed  CAS  Google Scholar 

  42. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. 1989, Cold Spring Harbor, Cold Spring Harbor Laboratory Press

    Google Scholar 

  43. Mackay SJ: Improved enumeration of Streptomyces spp. on a starch casein salt medium. Appl Environ Microbiol. 1977, 33: 227-230.

    PubMed  CAS  PubMed Central  Google Scholar 

  44. Kieser T: Factors affecting the isolation of ccc DNA from Streptomyces lividans and Escherichia coli. Plasmid. 1984, 12: 19-36. 10.1016/0147-619X(84)90063-5.

    Article  PubMed  CAS  Google Scholar 

  45. Bibb MJ, Ward JM, Hopwood DA: Transformation of plasmid DNA into Streptomyces at high frequency. Nature. 1978, 274: 398-400. 10.1038/274398a0.

    Article  PubMed  CAS  Google Scholar 

  46. Qin Z, Shen M, Cohen SN: Identification and characterization of a pSLA2 plasmid locus required for linear DNA replication and circular plasmid stable inheritance in Streptomyces lividans. J Bacteriol. 2003, 185: 6575-6582. 10.1128/JB.185.22.6575-6582.2003.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  47. Xia H, Huang J, Hu M, Shen M, Xie P, Zhang L, Wang H, Qin Z: Construction of an ordered cosmid library of S. avermitilis for genetic modification of the industrial strains. Chin J antibiot. 2009, 34: 340-343.

    Google Scholar 

  48. Evans GA, Lewis K, Rothenberg BE: High efficiency vectors for cosmid microcloning and genomic analysis. Gene. 1989, 79 (1): 9-20. 10.1016/0378-1119(89)90088-7.

    Article  PubMed  CAS  Google Scholar 

  49. Yang K, Han L, He J, Wang L, Vining LC: A repressor-response regulator gene pair controlling jadomycin B production in Streptomyces venezuelae ISP5230. Gene. 2001, 279: 165-173. 10.1016/S0378-1119(01)00723-5.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

We are very grateful to Sir David Hopwood for critical reading of and useful suggestions and corrections on the manuscript. We thank Brian Bachmann for kindly providing a cosmid containing the anthramycin biosynthetic gene cluster, Keqian Yang for S. venezuelae ISP5230, and Yiguang Wang for S. glaucescens GLA 4-26. These investigations were supported by grants from the National Nature Science Foundation of China (30770045, 31121001), National "973" project (2011CBA00801, 2012CB721104) and the Chinese Academy of Sciences project (KSCX2-EW-G-13) to Z. Qin.

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  1. Key Laboratory of Synthetic Biology, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, 300 Fenglin Road, Shanghai, 20032, People's Republic of China

    Weihua Chen & Zhongjun Qin

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  1. Weihua Chen
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WHC designed and performed all the experiments. ZJQ was involved in project design, and prepared the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest.

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12866_2011_1521_MOESM1_ESM.DOC

Additional file 1: Predicted ORFs of plasmid pTSC1. Detailed information and possible functions of the eight ORFs of pTSC1. (DOC 36 KB)

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Chen, W., Qin, Z. Development of a gene cloning system in a fast-growing and moderately thermophilic Streptomyces species and heterologous expression of Streptomyces antibiotic biosynthetic gene clusters. BMC Microbiol 11, 243 (2011). https://doi.org/10.1186/1471-2180-11-243

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BMC Microbiology

ISSN: 1471-2180

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