Development of a gene cloning system in a fast-growing and moderately thermophilic Streptomyces species and heterologous expression of Streptomyces antibiotic biosynthetic gene clusters
© Chen and Qin; licensee BioMed Central Ltd. 2011
Received: 13 April 2011
Accepted: 28 October 2011
Published: 28 October 2011
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.
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.
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.
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 . 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 . 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: ), has been used as a standard host for gene cloning and expression for several decades . 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 ). 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 . 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 ; 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 . Growth of thermophilic Streptomyces strains is rapid at high temperature ; for example, S. thermoviolaceus has a doubling time of 1 h at 50°C . Thermophilic Streptomyces species produce thermostable enzymes and antibiotics , such as xylanase , alpha-amylase , granaticin  and anthramycin . 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
Strains used in this study
Genotype or description
Source or reference
S. coelicolor M145
S. lividans 1326
S. lividans ZX7
pro-2 str-6 rec-46 dnd SLP2- SLP3-
S. venezuelae ISP5230
A jadomycin B producer
22 strains (4F, 2C, T1-1, T1-2, T5A-1, T6-5, T6-1-4, T1A, T6A-2, T6A-3, T6B-1, T6B-2, T6B-5, T6B-6, T6B-8, T6C-1, T6C-5, T6D-1, T6D-2, T6E-1, T6E-2, T6F-2)
Isolated from garden soil at the Shanghai Institute of Plant Physiology and Ecology
11 strains (G101, G103, G303, G102, G507, G302, 403, 202G, G304, 301, 005)
Isolated from weed manure in Hunan province
8 strains (A1, Z2-1, X1-3, X1-6, X2-1, X3-3, X4-3, X5-5)
Isolated from swine manure in Hunan, Hubei and Fujian province
F- deoR recA1 endA1 hsdR17(rk- mk+) phoA supE44 λ- thi-1 gyrA96 relA1
Life Technologies, Inc
dam dcm hsdM cm kan
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
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): ). Eight ORFs (open reading frame) were predicted by "FramePlot 3.0 beta" ; 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  and pSNA1 .
Development of a gene cloning system in strains 2C and 4F
Plasmids used in this study
Genotype or description
Source or reference
A 6996-bp plasmid of strain X4-3
A 7.5-kb plasmid of strain X3-3
A 50-kb plasmid of strain T6-1-4
A 16-kb linear plasmid of strain T6-1-4
Life Technologies, Inc
pBluescript II SK
amp colEI-ori lacZ
A 2.6-kb BclI-fragment of melC/tsr cloned in pSP72 (BglII)
A 7-kb KpnI fragment of pTSC1 cloned in pQC156
A 7-kb KpnI fragment of pTSC1 cloned in pBluescript II SK
melC tsr pIJ101 origin
A 8.9-kb Sau3A1-fragment of pFP11 origin cloned in pQC156
A 4.1-kb Sau3A1-fragment of pFP1 origin cloned in pQC156
Two fragments (PCR) of SLP1 rep/imp cloned in pQC156
A 2.2-kb HindIII fragment of pFRL2 origin cloned into pQC156
A 2.9-kb fragment of SAP1 origin cloned in pQC156
Zhang and Qin, unpublished data
A 2-kb fragment of SCP2 origin cloned in pQC156
Yang and Qin, unpublished data
A 4.1-kb EcoRI/BglII fragment of pSHK1 origin cloned in pQC156
Streptomyces phage φC31-derived integration vector, apr r
amp colEI-ori cos melC tsr
pHAQ31 (BamHI) containing c. 33 kb sequence (5510413-5543521 bp) from S. coelicolor A3(2)
A 2.6-kb XbaI/NheI fragment containing the phiC31 integrase gene cloned in a pHAQ31-derived cosmid containing the actinorhodin biosynthetic gene cluster
The anthramycin biosynthetic gene cluster cloned into a cosmid CAO2
Transformation by plasmids of the moderately thermophilic Streptomyces 2C and 4F
Transformation frequency (transformants/μg DNA)
S. lividans ZX7
1.3 × 106
3 × 102
2.9 × 106
8 × 101
1.4 × 105
1.2 × 105
E. coli DH5α
1.3 × 103
2 × 101
E. coli DH5α
8.2 × 103
1 × 101
E. coli DH5α
1 × 102
2 × 101
E. coli DH5α
2 × 102
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  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 . 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 . 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
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.
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. . Streptomyces culture, plasmid isolation and preparation of protoplasts and transformation of Streptomyces lividans ZX7 followed Kieser et al. . Plasmid trans-conjugation from E. coli ET12567/pUZ8002 into thermophilic Streptomyces strains followed Bierman et al. . 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://0-www.ncbi.nlm.nih.gov.brum.beds.ac.uk/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)  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 . 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  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  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 , pFP1 and pFP11, linear plasmids SAP1 , pFRL2  and pSHK1 , and integrating plasmid SLP1 , 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  contained an E.coli replication origin and two cos sites of Supercos1  and Streptomyces selection markers melC/tsr genes . 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 , 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 .
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 . Detection of anthramycin production followed Hu et al. . 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.
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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