CN102277310B - Vector-host system for expressing antibiotic gene clusters and application thereof - Google Patents
Vector-host system for expressing antibiotic gene clusters and application thereof Download PDFInfo
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Abstract
The invention relates to a vector-host system for expressing antibiotic gene clusters and application thereof. According to the invention, high temperature streptomycete which has rapid growth and is moderately mesophile is found, and the streptomycete is able to carry out genetic transformation and can successfully express exogenous genes or gene clusters. The growth speed of the high temperature streptomycete is obviously greater than that of sky blue streptomycete, and the high temperature streptomycete is moderately mesophile, having a good production application value.
Description
Technical field
The present invention relates to genetically engineered or microbiology field, be specifically related to express carrier host system and the application thereof of antibiotic resistance gene bunch.
Background technology
Streptomycete is a class high GC content gram positive bacterium, and half known microbial antibiotic derives from this monoid.Poky normal temperature streptomycete is numerous important antibiotic sources bacterial strains, but the thermostreptomyces resource of Fast Growth is but excavated seldom.Because streptomycete type strain streptomyces coelicolor has very strong restriction modification for deriving from the conversion of intestinal bacteria double-stranded DNA, so be used as in decades, the heterologous host of gene clone and expression with the very near muta lead mycillin bacterial strain that does not have strong restriction modification system of having lost endogenous plasmid of streptomyces coelicolor taxonomy.Yet, compare normal temperature streptomycete poor growth (use the SMM substratum under 28 ℃ of conditions, M145 doubling time exponential phase of growth is 2.2 hours) with genus bacillus with intestinal bacteria.This is just so that need the time in 2-3 week to produce and accumulate the microbiotic product on the general industry.
The thermostreptomyces of Fast Growth has been found long time.The thermostreptomyces bacterial strain that some are described the earliest is not included into thermostreptomyces and belongs to.The thermostreptomyces numerical classification studies show that three main splits, five branches and two single strain branches.They belong to streptomyces to 16s rRNA gene and morphology and chemical property hint.Most of thermostreptomyces growth temperature is at 28-55 ℃, and they are that moderate is had a liking for thermostreptomyces.Yet some thermostreptomyces can be grown up to 68 ℃, and the growth temperature that S.thermoautotrophicus is suitable is 65 ℃ and is lower than 40 ℃ and just can not grows.Thermostreptomyces is grown under hot conditions fast, and for example the doubling time of S.thermoviolaceus needs only one hour at 50 ℃.Thermostreptomyces can produce heat-staple industrial enzyme such as zytase (xylanase), α-amylase (alpha-amylase) and microbiotic litmomycin (granaticin) and anthramycin (anthramycin).
Because thermostreptomyces is not also set up genetic operating system, the normal temperature streptomycete is used to express the gene that comes from thermostreptomyces source and antibiotic resistance gene bunch.The Host Strains of growing in the industrial production fast will shorten fermentation period and enhance productivity.Therefore, this area is in the urgent need to setting up the thermostreptomyces genetic operating system, and best this system's fast growth, and production efficiency is high.
Summary of the invention
The object of the present invention is to provide carrier host system and the application thereof of expressing antibiotic resistance gene bunch, described system fast growth, production efficiency is high.
In a first aspect of the present invention, a kind of streptomycete is provided, and this streptomycete is the streptomycete that moderate is had a liking for temperature, can grow at 30-50 ℃ (preferably 37-45 ℃), its at 45 ℃ growth Dai Shiwei less than 2 hours (preferably being less than 1.5 hours, more preferably is 1.1 hours).
In a preference, described streptomycete at 50 ℃ growth Dai Shiwei less than 2.5 hours; Better is less than 2.4 hours; It more preferably is 2.3 hours.
In another preference, the 16s rRNA sequence of described streptomycete is shown in SEQ ID NO:1; Or the chromosome duplication initiator of described streptomycete (ori, one section sequence between gene dnaA and dnaN) sequence is shown in SEQ ID NO:3.
In another preference, the preserving number of described streptomycete at Chinese Typical Representative culture collection center is CCTCC M 2010092 (4F).
In another preference, the 16s rRNA sequence of described streptomycete is shown in SEQ ID NO:2; Or the chromosome duplication initiator of described streptomycete (ori, one section sequence between gene dnaA and dnaN) ori sequence is shown in SEQ ID NO:4.
In another preference, the preserving number of described streptomycete at Chinese Typical Representative culture collection center is CCTCC M 2010091 (2C).
In another aspect of this invention, provide the purposes of described streptomycete, be used for as host system expression alien gene or gene cluster.
In another aspect of this invention, provide a kind of host system of restructuring, described host system comprises described streptomycete, and is contained in foreign gene or gene cluster in the described streptomycete.
In another preference, described foreign gene or gene cluster are integrated on the genome of described streptomycete; Perhaps described foreign gene or gene cluster are contained in the recombinant vectors, and described recombinant vectors is contained in the described streptomycete.
In another preference, described foreign gene or gene cluster are normal temperature or high temperature source.
In another preference, described foreign gene bunch is antibiotic resistance gene bunch.
In another preference, described microbiotic is the microbiotic that is produced by streptomycete or actinomycetes.
In another preference, described microbiotic is selected from: anthramycin, actinorhodin, validamycin, lincomycin, erythromycin, terramycin, duomycin, tsiklomitsin, Magnamycin A, romicil, mydecamycin, tetracenomycin, nystatin, amphotericin, bleomycin, rapamycin, teicoplanin, vancomycin, cicloxin, polymyxin B, daptomycin, tobramycin, Streptomycin sulphate, netilmicin, amikacin, gentamicin, kantlex, Streptomycin sulphate, pristinamycin, doractin, Avrmectin.
In another aspect of this invention, provide the method for a kind of expression alien gene or gene cluster, comprising: cultivate the host system of described restructuring, make it expression alien gene or gene cluster.
Other side of the present invention is because the disclosure of this paper is apparent to those skilled in the art.
Description of drawings
Fig. 1 has shown an evolutionary tree based on adjacent method (neighbor-joining) model that obtains 16S rRNA structure with separating.
Fig. 2 has shown scanning electron microscopic observation at 2 days 4F of MS substratum growth and the growing state of 2C bacterial strain sample, can see having formed long smooth spore chain.
Fig. 3 has shown that the spore suspension of the bacterial strain that each separation is obtained carries out being coated on the MS substratum behind 10 times of serial dilutions, respectively at 30,37, cultivates for 45 ℃, and carries out Taking Pictures recording at different time points and observe.
The spore inoculating that Fig. 4 has shown equal number to the TSB liquid nutrient medium (prescription: Oxoid tryptonesoya broth powder (CM129) 30 grams, adding distil water is to 1000ml; Or referring to T.Kieser., M.J.Bibb., M.J.Buttner., K.F.Chater., D.A.Hopwood, page412, Practical Streptomycesgenetics, The John Inns Foundation, Norwich, England.2000) cultivate, check and accept the bacterium sampling in the different time, observe 4F and M145 in the growth velocity of differing temps.
Fig. 5 has shown the production of quantitative examination actinorhodin, and the M145 spore of equivalent amount and 4F zygote are not adding KH
2PO
4And CaCl
2The R2YE solution culture fermentation cultivate, the 1ml nutrient solution is in different point in time measurement OD values.
Fig. 6 has shown that the tunning of 4F carries out LC-MS and analyzes, and observes the mass peak of anthramycin.
Embodiment
The inventor is through long-term research and screening, found a kind of moderate of Fast Growth to have a liking for the thermostreptomyces of temperature, and this streptomycete can carry out genetic transformation, gene or gene cluster that can the successful expression external source.The speed of growth of described streptomycete approximately is 2 times of streptomyces coelicolor, and is the thermostreptomyces that moderate is had a liking for temperature, has good production application and is worth.
As used herein, described " gene cluster " refers to the assortment of genes that a plurality of sequences in the gene (such as series connection) form, and these a plurality of genes are closely related on function, and a common gene cluster contains 1-100 gene.
As used herein, described " moderate is had a liking for the streptomycete of temperature " referring to can be at 30-50 ℃ (better 37-50 ℃; More preferably 37-45 ℃) growth streptomycete.
As used herein, described " for time " refers to streptomycete in logarithmic phase biomass multiplication needed mean time once, and the average length of time with this streptomyces cell replicative cycle on the numerical value is identical.Usually take hour as unit.
As used herein, described " microbiotic " is to have the microorganism growth of inhibition and breeding, even a kind of chemical substance of kill microorganisms function.Microbiotic is widely used, except be used for suppressing microorganism, but the also inhibition tumor cell, the enzyme that have, but or reducing cholesterol etc.Antibiotic source comprises: the microorganisms such as bacterium, actinomycetes, mould, fungi, plant, animal.In addition, also can produce microbiotic with the method for full chemistry or half chemistry.
As used herein, described " foreign gene (bunch) " is used interchangeably with " heterologous gene (bunch) ", all refer to the gene that non-streptomycete itself carries (bunch).Described foreign gene or gene cluster are normal temperature and high temperature source.
Streptomycete
Streptomycete is the main monoid that microbiotic produces in the microorganism, but normal temperature streptomycete poor growth usually often needs the fermentation period (2-3 week) grown, with the more energy of intestinal bacteria genus bacillus phase specific consumption and material in the Industrial processes.In view of the poky problem of existing normal temperature streptomycete, the inventor has carried out repeatedly studying and screening, thereby the moderate that has obtained Fast Growth (being two times faster than streptomyces coelicolor) is had a liking for warm thermostreptomyces.But utilize described streptomycete successful expression to come from the gene cluster of high temperature and normal temperature streptomycete.
Therefore, the invention provides a kind of streptomycete, this streptomycete is the streptomycete that moderate is had a liking for temperature, can be 30-50 ℃ of growth, its at 45 ℃ growth Dai Shiwei less than 2 hours; Preferably be less than 1.5 hours; It more preferably is 1.1 hours.
In another preference, described streptomycete at 50 ℃ growth Dai Shiwei less than 2.5 hours; Better is less than 2.4 hours; It more preferably is 2.3 hours.
The inventor has found 2 better thermostreptomyces 2C and 4F under study for action.Selective marker and the carrier of a lot of streptomycetes can use in these two bacterial strains.With protoplastis preparation regeneration and the Transformation Program of streptomycete standard, find that 2C has very high transformation efficiency.2C does not almost have restricted for the external source double-stranded DNA that derives from streptomycete, and 4F has strong restriction modification effect.
Therefore, as optimal way of the present invention, the preserving number of described streptomycete at Chinese Typical Representative culture collection center is CCTCC M 2010092 (4F).More preferably, the 16s rRNA sequence of described streptomycete is shown in SEQ ID NO:1; Or the chromosome duplication initiator sequence (ori sequence) of described streptomycete is shown in SEQID NO:3.
In a preference, described streptomycete 4F has following physiological characteristic: can grow in 30-50 ℃ temperature range, belong to the moderate streptomyces thermophilus.4F can just begin to produce spore at 20 hours on the MS substratum of 37 ℃ and 45 ℃, the speed of growth must be fast 30 ℃ and 37 ℃ of growths than middle temperature streptomycete on the MS solid medium.
As optimal way of the present invention, the preserving number of described streptomycete at Chinese Typical Representative culture collection center is 2C CCTCC M 2010091 (2C).More preferably, the 16s rRNA sequence of described streptomycete is shown in SEQID NO:2; Or the chromosome duplication initiator sequence (ori sequence) of described streptomycete is shown in SEQ IDNO:4.
Described streptomycete can be used for as host system, expression alien gene or gene cluster.The present invention has no particular limits for foreign gene, as long as it can be expressed by streptomycete.In addition, foreign gene also comprises its variant, and the albumen that this variant is expressed has identical function with the albumen of this exogenous gene expression.It carries out random or rite-directed mutagenesis etc. and obtains by inserting or the deletion base.
Plasmid
The present invention also comprises linear plasmid or the ring-like plasmid that separation obtains from described thermostreptomyces.
The inventor obtains having detected a plurality of linear plasmids or ring-like plasmid many plant height temperature streptomycetes from separation, through the known plasmid of these plasmids and normal temperature streptomycete that checks order higher homology is arranged.Pulse electrophoresis has detected the karyomit(e) band, shows that karyomit(e) is the line style configuration.Copying in showing 25 11 of genes involved amplification with conservative telomere is the telomere of guarding, and the situation of this ratio and normal temperature streptomycete is similar.The pTSC1 replicon can be in normal temperature streptomycete and thermostreptomyces normal replication, and come from the plasmid pIJ101 of normal temperature streptomycete, pFRL2, pFP1 and pFP11 also can be good at copying in thermostreptomyces.
Host system and the expression of restructuring
Based on new discovery of the present invention, the present invention also provides a kind of host system of restructuring, and described host system comprises streptomycete of the present invention, and is contained in foreign gene or gene cluster in the described streptomycete.Described foreign gene or gene cluster are integrated on the genome of described streptomycete; Perhaps described foreign gene or gene cluster are contained in the recombinant vectors, and described recombinant vectors is contained in the described streptomycete.
Preferably, the host system of described restructuring can be used for antibiotic production.Normally a kind of chemical substance of microbiotic (secondary metabolites, micromolecular compound), it can be produced by biological engineering method.Adopt the method production microbiotic of culturing micro-organisms generally to need microorganism cells to give expression to the relevant a series of enzymes of purpose microbiotic biosynthetic pathway, by the catalysis of enzyme, specific metabolic precursor thereof in the cell is synthesized the purpose microbiotic through corresponding microbiotic pathways metabolism.The inventor finds unexpectedly, streptomycete of the present invention is behind the microbiotic biological synthesis gene cluster that imports external source, can express well the synthetic needed biosynthetic enzyme of purpose microbiotic, because the characteristics of streptomycete fast growth of the present invention, can shorten significantly antibiotic fermentation period, thereby produce quickly, efficiently microbiotic.
Described microbiotic can be the various microbiotic of being produced by streptomycete or actinomycete fermentation.As optimal way of the present invention, described microbiotic is selected from but is not limited to: anthramycin, actinorhodin, validamycin, lincomycin, erythromycin, terramycin, duomycin, tsiklomitsin, Magnamycin A, romicil, mydecamycin, tetracenomycin, nystatin, amphotericin, bleomycin, rapamycin, teicoplanin, vancomycin, cicloxin, polymyxin B, daptomycin, tobramycin, Streptomycin sulphate, netilmicin, amikacin, gentamicin, kantlex, Streptomycin sulphate, pristinamycin, doractin, Avrmectin etc.
Should understand, although embodiment has enumerated the example that utilizes streptomycete of the present invention to produce anthramycin, actinorhodin, yet those skilled in the art know that different antibiotic formation mechanism and production method are similarly, and therefore streptomycete of the present invention also can be used for other antibiotic production obviously.
Expression alien gene or gene cluster
Cultivate recombinant host of the present invention system (restructuring streptomycete), the method that makes it expression alien gene or foreign gene bunch can be cultivated with reference to this area Streptomycin sulphate the ordinary method of (or fermentation), and the present invention has no particular limits.
As one embodiment of the present invention, provide a kind of production antibiotic method, comprising: the microbiotic synthetic gene bunch conversion in normal temperature and high temperature source is entered streptomycete of the present invention, and then obtain the streptomycete of conversion.The streptomycete of this conversion is cultivated in substratum, obtained the purpose microbiotic with the conversion streptomycete fermentation after cultivating again.This provides the Host Strains platform that can at high temperature grow for heterogenous expression.
The method that imports foreign gene or gene cluster in streptomycete also is that those skilled in the art understand, and the present invention has no particular limits.Preferably, a kind of method that is transformed into foreign gene or gene cluster in streptomycete is provided, comprise: the protoplastis of preparation streptomycete, the carrier that method by intestinal bacteria conjugal transfer will contain described foreign gene or gene cluster changes in the streptomycete, can stablize and copy and express gene or the gene cluster that changes over to.
Major advantage of the present invention is:
(1) found a kind of moderate of Fast Growth to have a liking for the thermostreptomyces of temperature, 2 times of its speeds of growth are faster than streptomyces coelicolor, but successful expression foreign gene or gene cluster; And, when being used for expression alien gene or gene cluster, can effectively shorten fermentation period.
(2) thermostreptomyces of the present invention can be produced microbiotic well, efficiently, and can at high temperature produce, and has great production application and is worth.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable among the present invention.The usefulness that better implementation method described in the literary composition and material only present a demonstration.
I. materials and methods
Bacterial strain, plasmid, experimental technique
Bacterial strain and the plasmid that the present invention relates to are listed in table 1.
Table 1
| PBluescript II SK | Amp colEI-ori lacZ | Stratagene, Inc |
| PQC156 | With the partially digested pIJ702 of BCLI, obtain the fragment that comprises melanoma gene and sulphur company silk rhzomorph resistant gene of from 2189 to 4805 base pairs, be cloned into the BglII site on the pSP72. | Reference 30 |
| PCWH1 | KpnI digested plasmid pTSC1, the dna fragmentation of acquisition 7kbp is inserted in the pQC156 that the KpnI enzyme is cut.PTSC1 sequence: GU271942.1 GI:284080577 | The pTSC1 sequence is seen GI:284080577 |
| PIJ702 | The replicon in melC tsr pIJ101 source | Reference 20 |
| PZR10 | The partially digested plasmid pFP11 of Sau3AI obtains the replication regions dna fragmentation of pFP1 from 20312 to 29249 base pairs, is inserted into pFP11 AY943952.1 GI:61661434 on the BamHI site of pQC156 | Reference 38 |
| PZR115 | With the partially digested plasmid pFP1 of Sau3AI, obtain the replication regions dna fragmentation of pFP1 from 14907 to 19074, be inserted on the BamHI site of pQC156 the pFP1 sequence and see: NC_006912.1 GI:62184588 | Reference 38 |
| PZR205 | Pcr amplification is incorporated into rep (sequence from 210917 to 213801) and the imp (sequence from 219836 to 221375) of the plasmid SLP1 on Streptomyces coelicolor A3 (2) karyomit(e), the two segment DNA fragment BamHI enzymes that obtain are cut connection, are inserted in the BamHI site of pQC156.Streptomyces coelicolor A3 (2) chromosome sequence is seen GenBank:AL939120.1, GI:24413871 | Reference 38 |
| PZR51 | The HindIII site pFRL2 sequence that 59-2282 base pair plasmid duplicate field dna fragmentation on the pFRL2 of pcr amplification is cloned into carrier pQC156 is seen DQ884964.1 GI:113367024 | Reference 39 |
| PHAQ61 | PstI fragment 30462-44234 base pair on the SAP1 (NC_004719.1 GI:29826443) is cloned into the PstI site of streptomycete replicon probe pQC156 | The research of streptomycete linear plasmid and ring-like plasmid and use in April, 2009 |
| Zhang Ran, Qin Chongjun | ||
| PQC578 | The MluI endonuclease bamhi from pSLA2 of 6kb contains the MluI site (in the melC gene) that ori/rlrA/rorA is cloned into pQC156, has obtained the pQC578pSLA2 sequence and has seen AB088224.2 GI:169994890 | |
| PYQ40 | The sulphur of 1 kilobase connects silk rhzomorph resistant gene tsr fragment with reclaiming approximately behind the BCLI complete degestion pIJ702 (reference 20), with klenow enzyme (New England Biolabs, Inc) fill, the EcoRV site that is connected among the pSP72 (Life Technologies, Inc) obtains subclone pYQ39; From 28683 to 30305bp sequence, be cloned into the EcoRI site of subclone pYQ39 on pcr amplification SCP2 (NC_003904.1 GI:21233964) plasmid, obtain pYQ40. | |
| PGP9 | The replication regions dna fragmentation of from 12595 to 16696 base pairs on the pcr amplification pSHK1 (EU372836.1 GI:166162348) is cloned in the pQC156 carrier with EcoRI and BglII double digestion | Reference 39 |
| PSET152 | The integrative plasmid carrier in Streptomyces Phage ф C31 source, Apramycin sulfate resistance (AJ414670.1 GI:17974209) | |
| PHAQ31 | Amp colEI-ori cos melC tsr | The building process document 34 that sees reference |
| PCWH74 | The gene library carrier that comprises the pHAQ31 source of actinorhodin biological synthesis gene cluster.Make up as follows: with pstI complete degestion pSET152 carrier, reclaim 2.4 kilobase to 2.6 kilobase to fragment, connect and obtain subclone pCWH71, reclaim 2.6 kilobase to fragment with XbaI and NheI double digestion pCWH71, this fragment is cloned in the XbaI site that enters the library subclone that is numbered N7-85 that comprises the actinorhodin gene cluster obtain heterogenous expression carrier pCWH74.The section of the streptomyces coelicolor genomic fragment that N7-85 comprises is 5510413 to 5543521 base pairs, and building the storehouse carrier is pHAQ31, and the library construction process sees reference 34 for details | The present invention's preparation |
| 024CAO-3 | Derive from the carrier that gene library support C AO-2 comprises the anthramycin biological synthesis | Reference | 15 |
Separate and evaluation thermostreptomyces bacterial strain
2005, summer in 2006, the inventor is respectively from Shanghai, the Hunan, and take a sample in the soil in Fujian, the weeds pig manure in Hubei.Sample was cultivated 3-5 days at the SC substratum after 1 hour 100 ℃ of dryings.Further purifying cultivation on the SC substratum of the clone who grows.The spore shape scanning electron microscopic observation.
SC culture medium prescription wherein: starch 10g, casein 1g (difco company, lot number: 6117551), KH
2PO
40.5g, MgSO
4.7H
2O 0.5g, H
2O 1000ml, Agar 20g, nalidixic acid 25ug/ml, heavy cadmium acid potassium 500ug/ml; Or with reference to Nature.1964 May 30; 202:928-9 Selection of mediafor isolation of streptomycetes.KUESTER E, WILLIAMS ST.
Protoplastis preparation and conversion
The inventor prepares program with improved protoplastis and prepares protoplastis.
(a) preparation bacterial strain 2C protoplastis
The spore suspension of inoculation thermostreptomyces bacterial strain 2C adds 5mmol/L MgCl to 50ml
2, 0.5% glycine and sucrose concentration be in the 250ml shaking flask of 25% YEME substratum.Cultivated 9 hours for 42 ℃.Centrifugal collection mycelium (5000r/min, 10min).Abandoning supernatant, in the sucrose solution of 15ml 10.3%, centrifugal (5000r/min, 10min) repeats once with mycelium suspended.Mycelium is resuspended in the P damping fluid that 15ml contains the 1mg/ml N,O-Diacetylmuramidase, processes 20-30min for 30 ℃.Per 5 minutes jogs once therebetween.Add 30 ℃ of 10ml P damping fluids again and continue to process 15min, the test tube that rear usefulness is equipped with absorbent cotton filters, and filtrate changes in the centrifuge tube.Centrifugal (3000r/min, 10min) leniently precipitates protoplastis.Abandoning supernatant is suspended in protoplastis in the 1ml P damping fluid, and is stand-by.
Wherein, the YEME culture medium prescription is every liter and contains: yeast extract paste 3g; Peptone 5g; Malt extract 3g; Glucose 10g; Sucrose 340g; Or the document 20 that sees reference.
Wherein, P damping fluid compound method is: prepare first base soln: sucrose 103g, K
2SO
40.25g, MgCl
2.6H
2O 2.02g, trace element solution 2ml, adding distil water are distributed into the aliquot sample of 80ml, then autoclaving to 800ml.Before the use, add K in every bottle
2HPO
4(0.5%), 1mlCaCl
2.2H
2O (3.68%) 10ml TES damping fluid (5.73%, pH7.2) 10ml; Or the document 20 that sees reference.
(b) preparation bacterial strain 2C protoplastis
The spore suspension of inoculation thermostreptomyces bacterial strain 4F adds 5mmol/L MgCl to 50ml
2, 0.3% glycine sucrose concentration be in the 250ml shaking flask of 25% YEME substratum.Cultivated 9 hours for 42 ℃.Centrifugal collection mycelium (5000r/min, 10min).Abandoning supernatant, with mycelium suspended in the sucrose solution of 15ml 10.3%, centrifugal (5000r/min, 10min).Repeat once.Mycelium is resuspended in the P damping fluid that 15ml contains the 1mg/ml N,O-Diacetylmuramidase, processes 20-30min for 30 ℃.Per 5 minutes jogs once therebetween.Add 30 ℃ of 10ml P damping fluids again and continue to process 15min, the test tube that rear usefulness is equipped with absorbent cotton filters, and filtrate changes in the centrifuge tube.Centrifugal (3000r/min, 10min) leniently precipitates protoplastis.Abandoning supernatant is suspended in protoplastis in the 0.5ml P damping fluid, and is stand-by.
(c) protoplast transformation
Carry out protoplast transformation with the pCWH1 carrier, the method for transformation document 20 that sees reference.As a result, the transformation efficiency of 2C and 4F is respectively 1.3 * 10
3With 2 * 10
1Every μ g DNA.
Because 2C and 4F also belong to streptomyces, the inventor attempts the carrier of middle temperature streptomycete is used for doing transformation experiment.
Found that, pIJ702 (derivative vector of pIJ101), pZR10 (pFP11), pZR51 (pFRL2), pZR115 (pFP1) can both transform 2C and 4F bacterial strain, with SCP2, SLP1, the derivative vector pYQ40 that the replicon of SAP1 and pSHK1 makes up, pZR205, the Plasmid Transformation of pHAQ61 and pGP9 does not obtain transformant.Streptomycete integrative plasmid pSET152 also can belong to by enter many thermostreptomyces from intestinal bacteria conjugal transfer (referring to reference 20).Therefore, the carrier of normal temperature streptomycete also can be worked in 2C and 4F.
Relatively derive from the transformation efficiency of pIJ702 plasmid in 2C and 4F of different strains: the pIJ702 that derives from 2C and the low restricted bacterial strain ZX7 of modification has obtained high transformation efficiency, and these plasmids are very low by (8 * 10 to the transformation efficiency of 4F
1With 3 * 10
2), can obtain high transformation efficiency (1.2 * 10 although come from the plasmid of self
5).These presentation of results, the similar external source double-stranded DNA for deriving from other streptomycetes of 2C and ZX7 does not almost have restriction modification, and bacterial strain 4F has strong restriction modification.
Heterogenous expression actinorhodin and anthramycin antibiotic resistance gene bunch in thermostreptomyces
PhiC31 integrase gene (GenBank accession number GI:17974212) is cloned into an XbalI site that comprises the cosmid carrier library of actinorhodin total length gene cluster obtains integrated expression vector pCWH74, by entering the thermostreptomyces of the present invention from intestinal bacteria conjugal transfer.Whether zygote has blue pigment to produce 30,37 and 45 ℃ of observations respectively on R2YE substratum (referring to reference 20) and MS substratum.In order to study the output of actinorhodin, positive zygote is cultivated in liquid R2YE, in each different time points sampling, 1ml nutrient solution 12000 is got supernatant after turning centrifugation in 3 minutes, measure 640 light absorption value after KOH processes, the measurement of actinorhodin is according to the method (reference 20) of Kieser etc.
An integrated plasmid 024CAO-3 who comprises complete anthramycin gene cluster is (referring to Hu Y, PhelanVV, Farnet CM, Zazopoulos E, Bachmann BO:Reassembly of anthramycinbiosynthetic gene cluster by using recombinogenic cassettes.Chembiochem 2008,9 (10): 1603-1608) be engaged transfer and import among the 4F.Zygote is 47 ℃ of cultivation 24h on the AP1 substratum, thalline extracts with butanols, heavy molten with methyl alcohol after the extraction liquid volatilization, (method is seen Hu Y to anthramycin with the LC-MC detection, Phelan VV, Farnet CM, Zazopoulos E, Bachmann BO:Reassembly ofanthramycin biosynthetic gene cluster by using recombinogenic cassettes.Chembiochem2008,9 (10): 1603-1608.
II. embodiment
The inventor summer from having gathered the soil in hot environment source all over China.Cultivate streptomycete and carry out separation and purification at 50 ℃ of selective enrichments with the SC substratum.The inventor is respectively at vegetable soil, separates obtaining 20,11 and 8 strain bacterium in weeds compost and the pig manure.The 16S rRNA of the bacterial strain of the separation and purification Cloning and sequencing that increases, sequence homology analysis shows, they and various normal thermostreptomyces have very high homology.For example, 16S rRNA (the Genbank X79322.1 GI:488843) similarity of the 16S rRNA of 2C (CCTCC M 2010091) (shown in SEQ ID NO:2) and S.glaucescens DSM40716 is 99%, with 16S rRNA (Genbank AB249926.1GI:109945079) similarity of S.thermocarboxydus NBRC 16323 be 98%.The 16S rRNA (shown in SEQ IDNO:1) of 4F (CCTCC M 2010092) is 99% with the 16S rRNA similarity of S.thermocarboxydus.With separation obtain that 16S rRNA made up based on the evolutionary tree of evolutionary tree in abutting connection with algorithm model.In Fig. 1, (4F, T6C-1, T1A, T6E-2.X4-3, T614 and X3-3) is similar to thermostreptomyces for most of bacterial strain, and also some bacterial strain (2C, T6A-2 and T6A-3) is more similar to the normal temperature streptomycete.
In addition, through order-checking, the chromosome duplication initiator ori sequence of thermostreptomyces bacterial strain 2C is shown in SEQ IDNO:4; The chromosome duplication initiator ori sequence of 4F is shown in SEQ ID NO:3.
The same with classical streptomycete, these bacterial strains that are separated to also produce spore on R2YE and MS substratum.Scanning electron microscopic observation can see that thermostreptomyces bacterial strain 4F and 2C have formed long smooth spore chain, such as Fig. 2 at 2 days sample of MS substratum growth.These bacterial strains of 4F and 2C are accredited as streptomyces, and the growth suitable temp is at 30-50 ℃.
The 4F of the moderate streptomyces thermophilus of embodiment 2, Fast Growth and 2C growth characteristics are identified
The inventor carries out the spore suspension of the thermostreptomyces bacterial strain that separation obtains to be coated on the MS substratum behind 10 times of serial dilutions, respectively at 30,37, cultivates for 45 ℃, and carries out Taking Pictures recording at different time points and observe.As showing among Fig. 3,4F grows in 30-50 ℃ temperature range respectively, and 3 strain normal temperature streptomycetes are grown in 30 ℃ and 37 ℃.4F grows very slowly at 55 ℃, M145, and ISP5230 and GLA 4-26 can not be 45 ℃ and 50 ℃ of growths.Thereby 4F belongs to the moderate streptomyces thermophilus.
4F can just begin to produce at 20 hours spore on the MS substratum of 37 ℃ and 45 ℃, and this moment M145, ISP5230 and GLA 4-26 almost also do not have obvious visible growth sign.Significantly better than 30 ℃ or 50 ℃, ISP5230 is approximately beginning to produce spore in the 30h to 4F at 37 ℃ or 45 ℃ of growing states, and this wants Zao than M145 and GLA 4-26.These presentation of results, under the condition of 37 ℃ and 45 ℃, the speed of growth must be fast 30 ℃ and 37 ℃ of growths than middle temperature streptomycete on the MS solid medium for 4F.
In order to measure the growth velocity of 4F and M145, the spore inoculating of equal number is cultivated to the TSB liquid nutrient medium, checks and accepts the bacterium sampling in the different time, and the experiment triplicate is averaged.As shown in Figure 4,4F grows fast at 45 ℃ or 37 ℃, just reached the peak value of biomass within 16 hours time, then the growth curve fluctuation presents and the similar dead process of growth of the growth curve situation of streptomyces coelicolor, and the unit volume biomass of 4F is larger than the M145 of 30 ℃ of cultivations.The same with the situation of solid culture, 4F will be faster than the M145 of 30 ℃ of cultivations 45 ℃ or 37 ℃ of growths, and 4F growth when 50 ℃ or 30 ℃ is little with the M145 speed of growth difference of 30 ℃ of cultivations, and 4F is 30,37,45 and 50 ℃ for the time be respectively 2.3,1.4,1.1,2.3, M145 30 ℃ for the time be 2.2h, in the TSB liquid nutrient medium, 4F is that M145 is fast 30 ℃ twice 45 ℃ growth velocity.Similarly, 2C also is that a moderate is had a liking for thermostreptomyces, and growth velocity is the same with 4F fast.
From the new streptomycete bacterial strain that separates, extract ring-like and linear plasmid with alkaline SDS method with neutral SDS method respectively.The inventor separates the ring-like plasmid pTSC1 that obtains 7-kb from X4-3, separate obtaining the ring-like plasmid pTSC2 of 7.5-kb from X3-3, separates pTSC3 and the linear plasmid 16-kb that obtains 40-kb from T614, and PFGE does not detect large linear plasmid band.
7 kilobase that obtain with KpnI digested plasmid pTSC1 are to the dna fragmentation of length, and it is inserted into kpnI site among the pBluescript II SK, and obtain full sequence 6996bp (sequence is seen GI:284080577) by the primer extension order-checking.Sequential analysis shows that GC content is 72%, similar to the composition of classical streptomyces gene group (for example S.coelicolor A3 (2) is 72.1%).Table 2 is that seven genes to streptomycete or mycobacterium in them are similar with eight ORFs of FramePlot 3.0beta analyses and prediction.And four genes are genes (tra and spd) relevant with diffusion with pSNA1 conjugal transfer with streptomycete plasmid pIJ101, do not find the gene very high with known replication protein homology at pTSC1, and new replicanism may be arranged.
Table 2
The gene clone system of embodiment 4, development 2C and 4F
PTSC1 is cloned among the escherichia coli plasmid pQC156 that comprises Streptomyces tsr resistant gene, obtains carrier pCWH1.The random ten plant height temperature streptomycetes of selecting of the inventor prepare program with aforesaid protoplastis and prepare protoplastis, then transform with pCWH1, and the transformation efficiency of 2C and 4F is respectively 1.3 * 10
3With 2 * 10
1Every μ g DNA, other 8 bacterial strains do not obtain transformant.
Because 2C and 4F also belong to streptomyces, the inventor attempts the carrier of middle temperature streptomycete is used for doing transformation experiment.PIJ702 (pIJ101 derivative vector), pZR51 (pFRL2), pZR115 (pFP1) can both transform 2C and 4F bacterial strain, SCP2, SLP1, SAP1 and pSHK1 derive (pYQ40, pZR205, pHAQ61 and pGP9) Plasmid Transformation do not obtain transformant.Streptomycete integrative plasmid pSET152 also can belong to (14 strains 22 strains) by enter many thermostreptomyces from intestinal bacteria conjugal transfer.The carrier of normal temperature streptomycete also can be worked in 2C and 4F.
Relatively derive from the transformation efficiency of pIJ702 plasmid in 2C and 4F of different strains: the pIJ702 that derives from 2C and the low restricted bacterial strain ZX7 of modification has obtained high transformation efficiency, and these plasmids are very low by (8 * 10 to the transformation efficiency of 4F
1With 3 * 10
2), can obtain high transformation efficiency (1.2 * 10 although come from the plasmid of self
5), these presentation of results, the similar external source double-stranded DNA for deriving from other streptomycetes of 2C and ZX7 does not almost have restriction modification, and bacterial strain 4F has strong restriction modification.
Whether also can express the microbiotic synthetic gene bunch that comes from middle temperature streptomycete in order to test thermostreptomyces, the inventor has cloned complete actinorhodin gene cluster from streptomyces coelicolor A3 (2), cloning process: with pstI complete degestion pSET152 carrier, reclaim 2.4 kilobase to 2.6 kilobase to fragment, connect and obtain subclone pCWH71, reclaim 2.6 kilobase to fragment with XbaI and NheI double digestion pCWH71, this fragment is cloned in the XbaI site that enters the library subclone that is numbered N7-85 that comprises the actinorhodin gene cluster obtain heterogenous expression carrier pCWH74.The section of the streptomyces coelicolor genomic fragment that N7-85 comprises is 5510413 to 5543521 base pairs, and building the storehouse carrier is pHAQ31, and the library construction process sees reference 34 for details.Gene cluster is transferred in 8 high temperature bacterial strains that newly separate with conjugal transfer between the streptomycete by intestinal bacteria, comprises 4F and 2C.
Thermostreptomyces bacterial strain 4F and the 2C of restructuring can produce spore at the MS substratum, cultivate at the R2YE liquid nutrient medium.
30 or 37 ℃ of growths are after one day, and the 4F zygote has produced blue pigment at R2YE and MS substratum, but 45 ℃ are not seen that blue pigment produces when cultivating.The zygote of other bacterial strains is not all observed the generation of blue pigment.
In order to determine whether blue pigment is exactly actinorhodin, the inventor cultivates the 4F zygote at the R2YE solution culture fermentation, method by reference 20 is carried out full wavelength scanner, and the wavelength absorption pattern is consistent with the fermented liquid of streptomyces coelicolor A3 (2).Bunch can be by successful expression in lower temperature (30 or 37 ℃) from the actinorhodin synthetic gene of normal temperature streptomycete.
The inventor's quantitative examination production of actinorhodin, the M145 spore of equivalent amount and 4F zygote are not adding KH
2PO
4And CaCl
2The R2YE solution culture fermentation cultivate, the 1ml nutrient solution is at different point in time measurement.As shown in Figure 5, the pigment formation time will be early than M145 at 30 ℃ under the culture condition of actinorhodin 30 ℃ and 37 ℃ in 4F.At 100 hours, actinorhodin output under 30 ℃ the culture condition of 4F was M145 30 ℃ 2.8 times.
Anthramycin synthetic gene bunch can only be under higher culture temperature could the synthetic ammonia Anthramycin, when lower culture temperature, just can not produce anthramycin.The inventor will comprise an integrative plasmid 024COA-3 of whole anthramycin biological synthesis gene cluster (referring to Hu Y, Phelan VV, Farnet CM, ZazopoulosE, Bachmann BO:Reassembly of anthramycin biosynthetic gene cluster by usingrecombinogenic cassettes.Chembiochem 2008,9 (10): 1603-1608) import among the 4F by the conjugal transfer from intestinal bacteria to the streptomycete.30,37, under 47 ℃ the temperature, with AP1 culture medium culturing zygote, thalline heavily is dissolved in the methyl alcohol after processing after 24 hours.Also demonstrated the growth-inhibiting point with the genus bacillus biological indicator with the thin plate chromatographic separation, 47 ℃ have antibacterial substance to produce, but 30 and 37 ℃ do not produce antibacterial substance.
Tunning is carried out LC-MS analyze, observed as shown in Figure 6 the mass peak of anthramycin.Above result shows that 4F can heterogenous expression comes from the anthramycin antibiotic resistance gene bunch of thermophilic S.refuineus subsp.Thermotolerans.
Culture presevation
All quote in this application as a reference at all documents that the present invention mentions, just as each piece document is quoted separately as a reference.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Reference
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Sequence table
＜110〉Shanghai Inst. of Life Science, CAS
＜120〉express carrier host system and the application thereof of antibiotic resistance gene bunch
<130>101418
<160>4
<170>PatentIn version 3.3
<210>1
<211>1377
<212>DNA
＜213〉streptomycete (Streptomyces)
<400>1
acttcggtgg ggattagtgg cgaacgggtg agtaacacgt gggcaatctg ccctgcactc 60
tgggacaagc cctggaaacg gggtctaata ccggatactg atccgcttgg gcatcttgga 120
tgatcgaaag ctccggcggt gcaggatgag cccgcggcct atcagctagt tggtgaggta 180
atggctcacc aaggcgacga cgggtagccg gcctgagagg gcgaccggcc acactgggac 240
tgagacacgg cccagactcc tacgggaggc agcagtgggg aatattgcac aatgggcgaa 300
agcctgatgc agcgacgccg cgtgagggat gacggccttc gggttgtaaa cctctttcag 360
cagggaagaa gcgaaagtga cggtacctgc agaagaagcg ccggctaact acgtgccagc 420
agccgcggta atacgtaggg cgcgagcgtt gtccggaatt attgggcgta aagagctcgt 480
aggcggcttg tcacgtcggt tgtgaaagcc cggggcttaa ccccgggtct gcagtcgata 540
cgggcaggct agagttcggt aggggagatc ggaattcctg gtgtagcggt gaaatgcgca 600
gatatcagga ggaacaccgg tggcgaaggc ggatctctgg gccgatactg acgctgagga 660
gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg taaacggtgg 720
gcactaggtg tgggcgacat tccacgtcgt ccgtgccgca gctaacgcat taagtgcccc 780
gcctggggag tacggccgca aggctaaaac tcaaaggaat tgacgggggc ccgcacaagc 840
ggcggagcat gtggcttaat tcgacgcaac gcgaagaacc ttaccaaggc ttgacataca 900
ccggaaagca tcagagatgg tgcccccctt gtggtcggtg tacaggtggt gcatggctgt 960
cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgtcccg 1020
tgttgccagc aggcccttgt ggtgctgggg actcacggga gaccgccggg gtcaactcgg 1080
aggaaggtgg ggacgacgtc aagtcatcat gccccttatg tcttgggctg cacacgtgct 1140
acaatggccg gtacaatgag ctgcgatacc gcgaggtgga gcgaatctca aaaagccggt 1200
ctcagttcgg attggggtct gcaactcgac cccatgaagt cggagtcgct agtaatcgca 1260
gatcagcatt gctgcggtga atacgttccc gggccttgta cacaccgccc gtcacgtcac 1320
gaaagtcggt aacacccgaa gccggtggcc caaccccttg tgggagggag cttcgaa 1377
<210>2
<211>1398
<212>DNA
＜213〉streptomycete (Streptomyces)
<400>2
tgcaagtcga acgatgaacc acttcggtgg ggattagtgg cgaacgggtg agtaacacgt 60
gggcaatctg ccctgcactc tgggacaagc cctggaaacg gggtctaata ccggatactg 120
attgtcttgg gcatccttga tgatcgaaag ctccggcggt gcaggatgag cccgcggcct 180
atcagcttgt tggtgaggta acggctcacc aaggcgacga cgggtagccg gcctgagagg 240
gcgaccggcc acactgggac tgagacacgg cccagactcc tacgggaggc agcagtgggg 300
aatattgcac aatgggcgca agcctgatgc agcgacgccg cgtgagggat gacggccttc 360
gggttgtaaa cctctttcag cagggaagaa gcgaaagtga cggtacctgc agaagaagcg 420
ccggctaact acgtgccagc agccgcggta atacgtaggg cgcaagcgtt gtccggaatt 480
attgggcgta aagagctcgt aggcggcttg tcgcgtcggt tgtgaaagcc cggggcttaa 540
ccccgggtct gcagtcgata cgggcaggct agagttcggt aggggagatc ggaattcctg 600
gtgtagcggt gaaatgcgca gatatcagga ggaacaccgg tggcgaaggc ggatctctgg 660
gccgatactg acgctgagga gcgaaagcgt ggggagcgaa caggattaga taccctggta 720
gtccacgccg taaacggtgg gcactaggtg tgggcaacat tccacgttgt ccgtgccgca 780
gctaacgcat taagtgcccc gcctggggag tacggccgca aggctaaaac tcaaaggaat 840
tgacgggggc ccgcacaagc ggcggagcat gtggcttaat tcgacgcaac gcgaagaacc 900
ttaccaaggc ttgacataca ccggaaacgt ccagagacag gcgccccctt gtggtcggtg 960
tacaggtggt gcatggctgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa 1020
cgagcgcaac ccttgtcccg tgttgccagc aggcccttgt ggtgctgggg actcacggga 1080
gaccgccggg gtcaactcgg aggaaggtgg ggacgacgtc aagtcatcat gccccttatg 1140
tcttgggctg cacacgtgct acaatggccg gtacaaagag ctgcgatacc gtgaggtgga 1200
gcgaatctca aaaagccggt ctcagttcgg attggggtct gcaactcgac cccatgaagt 1260
cggagtcgct agtaatcgca gatcagcatt gctgcggtga atacgttccc gggccttgta 1320
cacaccgccc gtcacgtcac gaaagtcggt aacacccgaa gccggtggcc caaccccttg 1380
tgggagggag cttcgaag 1398
<210>3
<211>1441
<212>DNA
＜213〉streptomycete (Streptomyces)
<400>3
gcggccgctc tagaactagt ggatcccccg ggctgcagga attcgatgcc atgtacctct 60
gccgcgagct gacggacctg tcgctgccga agatcggcgc gctgttcggc gggcgcgacc 120
acacgacggt gatgcacgcc gaccggaaga tccgcaatct gatggccgag cgccggtcca 180
tctacaacca ggtcaccgag ctgaccaacc gcatcaaggc cggctgacgg cccgcacgca 240
cccttgaggg cgcccctgga gagatccggg ggcgcccttc ttcatgcgtc cggagcccgc 300
cccggcctcc gaagcccgcc cccggccccg gaagcccacc ttcggctccg gaagcccacc 360
ttcgccctcc ggaagcctgc ctcggacctc tggaagcccg gtccgcgcgc ttccggtgcc 420
catcccatgc ctctccgagc cccgccccct gacaccggac cgagcgccct gttcgatttc 480
caccgcgggt tacggccttc ctccacagat tcggcgactt tttcccgtcc acacgctggg 540
gaccggaaag ttgtcccgac accgtccaca gccgcccctg gtgagaagct gtctgcccag 600
gtcacctgcc tgtggattcg tggacgaacg atctccacag actgtggacg acgagatgat 660
ccacagcctg tgcatcaagt tgtccacggg cttcccacaa gctgcggccg gttgtcccca 720
gtgatcggcc gcttctccac atggctgtcc actgttcggc aacgtgacgc gccttctcac 780
cggcccgagt gaaagccgtc acatcgagct gccggagggg gctgtgggaa aggagctcaa 840
acctggggac gcgtctgggg agaagtcggc ctcccctgtg cacgggatgt gcagaacttt 900
ccgttctcca cagtgaggcc gggttgtcca ccgcccccgc ccacaggccc cgtggacaaa 960
aaaccggctc tgacctgcgc aaacgaggtt atccacggta tccacaggcc ctactactac 1020
tcccgactag agagagcgag gaattcgttt cgaagagggt cctgtgcaca actcgctgtc 1080
tcggccccga ctgccactcg tcacgacttg accccgacgg gcacctagtg tcagtgcggt 1140
gcgtcagact ggaccccggt gtccttcccc tcagaggggc cgacgacacc gagtcagacg 1200
acgaagccag gcacagggcg agaagcgccg gcaacagcag gaggcggcaa cagtgaagat 1260
ccgggtggaa cgcgacgtac tcgcggaggc agtggcctgg gcggctcgca gcctcccggc 1320
ccgtccgccg gcgcctgtcc tcgtcggcct tgagcagcag gccggcgagg gccagctgag 1380
cctgtccagc ttcgactacg aggtctgggc gatcaagctt atcgataccg tcgacctcga 1440
g 1441
<210>4
<211>1571
<212>DNA
＜213〉streptomycete (Streptomyces)
<400>4
ggaacaaaag ctggagctcc accgcggtgg cggccgctct agaactagtg gatcccccgg 60
gctgcaggaa ttcgatcgcg gagacctcgt agtcgaagct ggacaggctc agctggccgt 120
cctcggcctt cagcagcagg ccggcgagga caggcgccgg cggacgggcc gggaggctgc 180
gggccgccca ggccactgcc tccgcgagta cgtcgcgttc cacccggatc ttcacggtaa 240
gccgcctcct gctgttgccg gcgcttgtcg ccctgcttcg ccttcgtcgt ctgtctcggt 300
gtcaccggcc ccggggaggg gaaggacacc gtggaccagt ctgacgcacc gcactgacag 360
taggtgcctg tcggggtcaa gtcgtgacga ggggcagccg gtcaaccgac agcgagttgt 420
gcacaggccc cgcttcgaaa cgaattccgg gctctctcta gtcgtcggta gtagtagggc 480
ctgtggatac cgtggataac ctcgtttgcc cagctcaggg cgcagatttt gtccacgggc 540
cctgtgggcg gagacggtgg acaactcacg gttcctgtgg agaacggaaa gttctgcaca 600
ccccgtgcac aggccggggt gagttctccc cagcggcgtc cccagtttta cccacgtttc 660
ccacagccca atccggcagc ttcgtgtgac ggctttcact cgacgcggtg agggggcgcg 720
tcggcttgcc gaacagtgga cagacatgtg gagaagccgc tcctcgctgg ggacaaccgg 780
ccccagcctg tgggttgccc gtggacaact ctttgcacag ggtgtggacc gccgagttgt 840
ccacaccctg tggatcttct tcgtccacgg atccacagcc acctgagctg ctgtgatggc 900
tttcccccag gctcccctgt ggacacgatc tggacaactt cgcagtcccc agcgtgtgga 960
cacgaaaaag tcgccgaatc tgtggagaga agccgtaacc cggcatcgat tcgaacaaga 1020
gatgacagat gtgcgacggc cgggccggcg gctccgggcg cccggggacg gctgaggggc 1080
cttcgcgagg ccctcggacg gctctggcgt ggcctgtgag gaggctctcg ggcggctccg 1140
gggtcggctg aggcggccga ggcagtctgc gggcggtgcc cgaaggggcc aggggacggc 1200
tgtggcgccg ctgtggggac gggcgtggca cgcctggcga cgcgcgtggc ggccctgagg 1260
ccggcgagcg gccgcgtgcc ccgatacggc gaaggcgccc ccggagagtc cggaggcgcc 1320
ctcgggtggc cgctgcggcg gtgtcagccg ttcttgatgc ggttggtcag ctcggtgacc 1380
tggttgtaga tggagcgccg ctcggccatc agattgcgga tcttgcggtc cgcgtgcatc 1440
accgtcgtgt ggtcccggcc gccgaacagc gcaccgatct tgggcagcga cagatccgtc 1500
agctcccggc acaggtacat ggcatcaagc ttatcgatac cgtcgacctc gagggggggc 1560
ccggtaccca a 1571
Claims (8)
1. a streptomycete is characterized in that, this streptomycete is the streptomycete that moderate is had a liking for temperature, can be 30-50 ℃ of growth, its at 45 ℃ growth Dai Shiwei less than 2 hours;
The preserving number of described streptomycete at Chinese Typical Representative culture collection center is CCTCC M 2010092.
2. streptomycete as claimed in claim 1 is characterized in that, the 16s rRNA sequence of described streptomycete is shown in SEQ ID NO:1; Or
The chromosome duplication initiator sequence of described streptomycete is shown in SEQ ID NO:3.
3. the purposes of the arbitrary described streptomycete of claim 1-2 is used for as host system expression alien gene or gene cluster.
4. the host system of a restructuring is characterized in that, described host system comprises the arbitrary described streptomycete of claim 1-2, and is contained in foreign gene or gene cluster in the described streptomycete.
5. the host system of restructuring as claimed in claim 4 is characterized in that, described foreign gene bunch is antibiotic resistance gene bunch.
6. the host system of restructuring as claimed in claim 5 is characterized in that, described microbiotic is the microbiotic that is produced by streptomycete or actinomycetes.
7. the host system of restructuring as claimed in claim 5 is characterized in that, described microbiotic is selected from: anthramycin, actinorhodin, validamycin, lincomycin, erythromycin, terramycin, duomycin, tsiklomitsin, Magnamycin A, romicil, mydecamycin, tetracenomycin, nystatin, amphotericin, bleomycin, rapamycin, teicoplanin, vancomycin, cicloxin, polymyxin B, daptomycin, tobramycin, Streptomycin sulphate, netilmicin, amikacin, gentamicin, kantlex, Streptomycin sulphate, pristinamycin, doractin, Avrmectin.
8. the method for an expression alien gene or gene cluster is characterized in that, comprising: cultivate the host system of the arbitrary described restructuring of claim 4-7, make it expression alien gene or gene cluster.
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