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  <cit> .
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  <cit> .
streptomyces coelicolor a <dig> 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  <cit> .
mainly because of a strong restriction barrier to introduction of foreign double-stranded dna by transformation from escherichia coli into a <dig>  the closely related s. lividans, with no such barrier and cured of indigenous plasmids , has been used as a standard host for gene cloning and expression for several decades  <cit> .
however, compared with e. coli and bacillus subtilis, s. coelicolor and s. lividans  grow slowly at their optimal temperature  - grows exponentially with a doubling time of about  <dig>  h on smm medium at 28°c, see ref  <cit> ).
it takes about 2- <dig> 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  were not classified as thermophilic streptomycetes  <cit> .
numerical classification of thermophilic streptomycetes showed three major, five minor and two single-member clusters  <cit> .
analysis of the 16s rrna genes and morphological and chemical properties indicate their classification within the genus streptomyces  <cit> .
most thermophilic streptomyces species have growth temperature ranges from  <dig> to 55°c and so are only moderately thermophilic  <cit> .
however, some thermophilic streptomyces species can grow up to 68°c  <cit> ; 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  <cit> .
growth of thermophilic streptomyces strains is rapid at high temperature  <cit> ; for example, s. thermoviolaceus has a doubling time of  <dig> h at 50°c  <cit> .
thermophilic streptomyces species produce thermostable enzymes and antibiotics  <cit> , such as xylanase  <cit> , alpha-amylase  <cit> , granaticin  <cit>  and anthramycin  <cit> .
since thermophilic streptomyces strains lack a genetic manipulation system, mesophilic strains  have been employed for expression of some genes or antibiotic biosynthetic gene clusters from thermophilic streptomyces species  <cit> .
we report here the development of a gene cloning system in a fast-growing  and moderately thermophilic  streptomyces strain, and successful heterologous expression of antibiotic biosynthetic gene clusters from both thermophilic and mesophilic streptomyces species.
RESULTS
isolation and identification of thermophilic streptomyces strains from various soil samples
to isolate thermophilic streptomyces strains, various soil samples from china were collected .
as summarized in table  <dig>   <dig>   <dig> 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  <cit> , as well as lakes and hot-springs  <cit> .
our results reinforce the idea of a widespread occurrence of these organisms.
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  to those of known mesophilic streptomyces and/or thermophilic streptomyces species.
a phylogenetic tree was drawn by using a neighbor-joining method  <cit> .
the chosen  <dig> 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  <dig>  all  <dig> newly isolated strains  resembled known thermophilic streptomyces species .
moderately thermophilic streptomyces species form at least two distinct clades  <cit> , containing strains related to s. megasporus and s. thermodiastaticus, respectively.
the phylogenetic tree of the  <dig> newly isolated strains reveals more clades .
these results indicate that moderately thermophilic streptomyces species are diverse in natural habitats.
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  <dig> d .
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  <dig>  strains 4f and 2c were able to grow from  <dig> to 50°c, while two mesophilic streptomyces strains  grew at 30°c and 37°c.
4f and 2c grew well at 45°c and 50°c but poorly at 55°c, while m <dig> and isp <dig> could not grow at 45°c and 50°c .
thus, 4f and 2c were concluded to be moderately thermophilic streptomyces strains.
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 .
to measure the growth rates of 4f and m <dig>  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  <dig>  4f rapidly accumulated biomass to a maximum at 45°c or 37°c within  <dig> h, then the growth curve fluctuated, and the final biomass of strain 4f is higher for m <dig> .
the oscillations shown at  <dig> and 45°c resembling the "death/growth process" of s. coelicolor a <dig> in liquid medium with a diluted inoculum  <cit> .
the doubling times of growth for 4f at  <dig>   <dig>   <dig> and 50°c and m <dig> at 30°c and 37°c in each logarithmic phase  were  <dig> ,  <dig> ,  <dig>   <dig> ,  <dig>  and  <dig>  h, respectively.
thus strain 4f grew at 45°c twice and at 37°c  <dig>  times as fast as m <dig> at 30°c in tsb medium.
identification of one linear and three circular plasmids among  <dig> strains, and sequencing of ptsc1
we detected three circular plasmids, 7-kb ptsc <dig>  from x4- <dig>   <dig> -kb ptsc <dig> from x3- <dig>  and 40-kb ptsc <dig> as well as 16-kb linear ptsl <dig> from t6-1- <dig>  the complete nucleotide sequence of the circular ptsc <dig> consisted of  <dig> bp , with 72% g+c, resembling that of a typical streptomyces genome :  <cit> ).
eight orfs  were predicted by "frameplot  <dig>  beta"  <cit> ; seven of them resembled streptomyces or mycobacterium genes .
notably, three genes resembled the transfer and spread genes  of streptomyces plasmids pij <dig>  <cit>  and psna <dig>  <cit> .
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 , ptsc1-derived pcwh <dig>  was introduced by transformation into ten well-sporulating thermophilic streptomyces strains.
thiostrepton-resistant colonies were obtained for strains 2c and 4f at frequencies of  <dig>  ×  <dig> and  <dig> ×  <dig> per μg dna, but no transformants arose for the other eight strains.
many streptomyces selection markers  could be used in strains 2c and 4f.
no antibacterial activity  was detected in the two strains .
thus, we found two promising cloning hosts, 2c and 4f.
since 2c and 4f were classified in the genus streptomyces, several mesophilic streptomyces vectors were employed for transformation experiments.
as shown in table  <dig>  pij <dig> , pzr <dig> , pzr <dig>  and pzr <dig>  were able to transform both 2c and 4f.
no transformants were obtained for scp <dig>  <cit> , slp <dig> <cit> , sap <dig>  <cit>  and pshk <dig>  <cit>  derivatives .
pcwh <dig> could also transform s. lividans zx <dig>  <cit>  at high frequency .
a streptomyces integrating plasmid, pset <dig>  <cit> , could be introduced by conjugation from e. coli into many thermophilic streptomyces strains .
thus, ptsc1-derived pcwh <dig> can replicate in both thermophilic and mesophilic streptomyces strains.
on the other hand, several streptomyces replicons, including circular plasmids pij <dig>  pfp <dig> and pfp <dig> and linear plasmid pfrl <dig>  were able to propagate in the thermophilic streptomyces strains 2c and 4f, but no transformants were obtained for circular plasmids scp <dig> and slp <dig> and linear plasmids sap <dig> and pshk <dig>
comparing the transformation frequencies of pij <dig> from different hosts in 2c and 4f, as shown in table  <dig>  similar high frequencies of transformation  were obtained in 2c with pij <dig> from both 2c itself and the largely restriction-free s. lividans zx <dig>  low frequencies of transformation  were obtained in 4f with pij <dig> from 2c and zx <dig>  although a high frequency  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 a <dig> in strain 4f
since several mesophilic streptomyces plasmids functioned in thermophilic streptomyces, we chose a phage phic31-derived integrating plasmid pset <dig>  <cit>  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 , 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 a <dig> in an integrating plasmid , and the resulting plasmid, pcwh <dig>  was introduced by conjugation into eight newly isolated strains, including 4f and 2c.
pcr amplification experiments with eight paired primers from sco <dig> to sco <dig> 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  <dig> and 37°c after growth for  <dig> 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 pcwh <dig> was cultured in r2ye liquid medium lacking kh2po <dig> and cacl <dig> and the supernatant was treated with koh and scanned at  <dig> nm  <cit> .
the same pattern of absorption peaks was detected for 4f as for s. coelicolor a <dig> .
thus the actinorhodin biosynthetic gene cluster from the mesophilic s. coelicolor a <dig> was heterologously expressed in strain 4f at low temperature , but not at high temperature .
to quantitate the productivity of actinorhodin, equal amounts of spores of m <dig> and 4f containing pcwh <dig> were inoculated into r2ye liquid medium lacking kh2po <dig> and cacl <dig>  and  <dig> ml culture was harvested in a time-course.
as shown in figure  <dig>  actinorhodin was produced in 4f at both  <dig> and 37°c, earlier than in m <dig> at 30°c.
at  <dig> h, productivity of actinorhodin in 4f at 30°c was ~ <dig>  times higher than in m <dig> at 30°c.
strains m <dig> and 4f grew better in tsb than in r2ye liquid media , but no actinorhodin was detected when cultured in tsb medium at  <dig> and 37°c.
growth curves of the two strains in r <dig> lacking kh2po <dig> and cacl <dig> at 30°c showed that their biomass values were similar from  <dig> to  <dig> hours .
thus, better growth of m <dig> and 4f in tsb medium  did not correlate with delayed and less production of actinorhodin in r2ye medium .
like in 4f, m <dig> 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  <cit> .
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 , but not at  <dig> or 37°c  <cit> .
an integrating cosmid, 024coa- <dig>  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 ap <dig> medium at  <dig>   <dig> and 47°c for  <dig> 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  <dig> and 37°c .
a spot on a tlc plate was further purified for hplc-ms analysis.
as shown in figure  <dig>  an anthramycin-specific peak  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- <dig> 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.
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.

 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 a 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. mainly because of a strong restriction barrier to introduction of foreign double-stranded dna by transformation from escherichia coli into a the closely related s. lividans, with no such barrier and cured of indigenous plasmids, 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 grow slowly at their optimal temperature - grows exponentially with a doubling time of about h on smm medium at 28°c, see ref ). it takes about 2- 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 were not classified as thermophilic streptomycetes. 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. most thermophilic streptomyces species have growth temperature ranges from to 55°c and so are only moderately thermophilic. 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 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 have been employed for expression of some genes or antibiotic biosynthetic gene clusters from thermophilic streptomyces species. we report here the development of a gene cloning system in a fast-growing and moderately thermophilic streptomyces strain, and successful heterologous expression of antibiotic biosynthetic gene clusters from both thermophilic and mesophilic streptomyces species.  isolation and identification of thermophilic streptomyces strains from various soil samples to isolate thermophilic streptomyces strains, various soil samples from china were collected. as summarized in table 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, as well as lakes and hot-springs. our results reinforce the idea of a widespread occurrence of these organisms. 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 to those of known mesophilic streptomyces and/or thermophilic streptomyces species. a phylogenetic tree was drawn by using a neighbor-joining method. the chosen 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 all newly isolated strains resembled known thermophilic streptomyces species. moderately thermophilic streptomyces species form at least two distinct clades, containing strains related to s. megasporus and s. thermodiastaticus, respectively. the phylogenetic tree of the newly isolated strains reveals more clades. these results indicate that moderately thermophilic streptomyces species are diverse in natural habitats. 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 d. 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 strains 4f and 2c were able to grow from to 50°c, while two mesophilic streptomyces strains grew at 30°c and 37°c. 4f and 2c grew well at 45°c and 50°c but poorly at 55°c, while m and isp could not grow at 45°c and 50°c. thus, 4f and 2c were concluded to be moderately thermophilic streptomyces strains. 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. to measure the growth rates of 4f and m 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 4f rapidly accumulated biomass to a maximum at 45°c or 37°c within h, then the growth curve fluctuated, and the final biomass of strain 4f is higher for m. the oscillations shown at and 45°c resembling the "death/growth process" of s. coelicolor a in liquid medium with a diluted inoculum. the doubling times of growth for 4f at and 50°c and m at 30°c and 37°c in each logarithmic phase were,,, and h, respectively. thus strain 4f grew at 45°c twice and at 37°c times as fast as m at 30°c in tsb medium. identification of one linear and three circular plasmids among strains, and sequencing of ptsc1 we detected three circular plasmids, 7-kb ptsc from x4- -kb ptsc from x3- and 40-kb ptsc as well as 16-kb linear ptsl from t6-1- the complete nucleotide sequence of the circular ptsc consisted of bp, with 72% g+c, resembling that of a typical streptomyces genome: ). eight orfs were predicted by "frameplot beta"; seven of them resembled streptomyces or mycobacterium genes. notably, three genes resembled the transfer and spread genes of streptomyces plasmids pij and psna. 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, ptsc1-derived pcwh was introduced by transformation into ten well-sporulating thermophilic streptomyces strains. thiostrepton-resistant colonies were obtained for strains 2c and 4f at frequencies of × and × per μg dna, but no transformants arose for the other eight strains. many streptomyces selection markers could be used in strains 2c and 4f. no antibacterial activity was detected in the two strains. thus, we found two promising cloning hosts, 2c and 4f. since 2c and 4f were classified in the genus streptomyces, several mesophilic streptomyces vectors were employed for transformation experiments. as shown in table pij, pzr, pzr and pzr were able to transform both 2c and 4f. no transformants were obtained for scp, slp, sap and pshk derivatives. pcwh could also transform s. lividans zx at high frequency. a streptomyces integrating plasmid, pset, could be introduced by conjugation from e. coli into many thermophilic streptomyces strains. thus, ptsc1-derived pcwh can replicate in both thermophilic and mesophilic streptomyces strains. on the other hand, several streptomyces replicons, including circular plasmids pij pfp and pfp and linear plasmid pfrl were able to propagate in the thermophilic streptomyces strains 2c and 4f, but no transformants were obtained for circular plasmids scp and slp and linear plasmids sap and pshk comparing the transformation frequencies of pij from different hosts in 2c and 4f, as shown in table similar high frequencies of transformation were obtained in 2c with pij from both 2c itself and the largely restriction-free s. lividans zx low frequencies of transformation were obtained in 4f with pij from 2c and zx although a high frequency 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 a in strain 4f since several mesophilic streptomyces plasmids functioned in thermophilic streptomyces, we chose a phage phic31-derived integrating plasmid pset 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, 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 a in an integrating plasmid, and the resulting plasmid, pcwh was introduced by conjugation into eight newly isolated strains, including 4f and 2c. pcr amplification experiments with eight paired primers from sco to sco 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 and 37°c after growth for 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 pcwh was cultured in r2ye liquid medium lacking kh2po and cacl and the supernatant was treated with koh and scanned at nm. the same pattern of absorption peaks was detected for 4f as for s. coelicolor a. thus the actinorhodin biosynthetic gene cluster from the mesophilic s. coelicolor a was heterologously expressed in strain 4f at low temperature, but not at high temperature. to quantitate the productivity of actinorhodin, equal amounts of spores of m and 4f containing pcwh were inoculated into r2ye liquid medium lacking kh2po and cacl and ml culture was harvested in a time-course. as shown in figure actinorhodin was produced in 4f at both and 37°c, earlier than in m at 30°c. at h, productivity of actinorhodin in 4f at 30°c was ~ times higher than in m at 30°c. strains m and 4f grew better in tsb than in r2ye liquid media, but no actinorhodin was detected when cultured in tsb medium at and 37°c. growth curves of the two strains in r lacking kh2po and cacl at 30°c showed that their biomass values were similar from to hours. thus, better growth of m and 4f in tsb medium did not correlate with delayed and less production of actinorhodin in r2ye medium. like in 4f, m 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 expression of the anthramycin biosynthetic genes of s. refuineus subsp. thermotolerans could be detected at high temperature, but not at or 37°c. an integrating cosmid, 024coa- 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 ap medium at and 47°c for 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 and 37°c. a spot on a tlc plate was further purified for hplc-ms analysis. as shown in figure an anthramycin-specific peak 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- 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.  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. 
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. 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. ptsc1-derived pcwh could replicate in both thermophilic and mesophilic streptomyces strains. streptomyces species are a major source of antibiotics. most thermophilic streptomyces species grow fast, but no gene cloning systems have been developed in such strains. on the other hand, several streptomyces replicons function in thermophilic streptomyces species. we have developed a gene cloning and expression system in a fast-growing and moderately thermophilic streptomyces species. the actinorhodin and anthramycin biosynthetic gene clusters from mesophilic s. coelicolor a and thermophilic s. refuineus were heterologously expressed in one of the hosts. we report here the isolation of fast-growing, moderately thermophilic streptomyces strains, detection of one linear and three circular plasmids in them, and sequencing of a 6996-bp plasmid, ptsc from one of them.
 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. most thermophilic streptomyces species grow fast, but no gene cloning systems have been developed in such strains.  we report here the isolation of fast-growing, moderately thermophilic streptomyces strains, detection of one linear and three circular plasmids in them, and sequencing of a 6996-bp plasmid, ptsc from one of them. ptsc1-derived pcwh 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 a 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. 