CN103865946A - Terramycin high-yielding method based on metabolism regulation - Google Patents
Terramycin high-yielding method based on metabolism regulation Download PDFInfo
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- CN103865946A CN103865946A CN201210550528.7A CN201210550528A CN103865946A CN 103865946 A CN103865946 A CN 103865946A CN 201210550528 A CN201210550528 A CN 201210550528A CN 103865946 A CN103865946 A CN 103865946A
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Abstract
本发明涉及基于代谢调控高产土霉素的方法。本发明公开了一种在龟裂链霉菌中通过调控土霉素合成途径提高土霉素产量的方法，通过增加一个或多个土霉素生物合成途径的调控因子的拷贝数，从而提高土霉素合成途径中相关基因的表达水平，进而提高土霉素的产量。The invention relates to a method for regulating high-yield oxytetracycline based on metabolism. The invention discloses a method for increasing the yield of oxytetracycline by regulating the synthetic pathway of oxytetracycline in Streptomyces fissa. The expression levels of related genes in the synthetic pathway of oxytetracycline can be increased, thereby increasing the production of oxytetracycline.
Description
Technical field
The invention belongs to streptomycete metabolic engineering technical field, more specifically, the present invention relates to a kind of novel process based on metabolic regulation high yield terramycin.
Background technology
Streptomycete is antibiotic main production bacterial strain, the genome of at present a lot of streptomycetes has completed order-checking, by the sequence analysis of gene order, a lot of functional genes (regulatory factor) are found, and identify in vitro and in vivo, a lot of regulatory factors are synthetic relevant to microbiotic, by the expression amount of the genes involved in regulation and control microbiotic route of synthesis, thereby improve antibiotic output.This obtains microbiotic enhanced variant and provides convenience for further utilizing these regulatory factors to carry out genetic modification as target spot.
Streptomyces rimosus (Streptomyces rimosus) is the production bacterial strain for the production of tetracycline antibiotics as industry, is also the main production bacterial strain of terramycin.Terramycin (Oxytetracycline, OTC) is a kind of Broad spectrum antibiotics of widespread use clinically, by the people such as Finlay in nineteen fifty reported first in Streptomycesrimosus, find.The OTC of report produces bacterium in addition at present: Streptomyces capuensis, Streptomyces henetus and Streptomyces platensis etc.OTC is to Gram-positive, negative bacteria, spirobacteria, and rickettsia and some virus have germ resistance, by being reversibly attached on 30S ribosomal subunit, stop the formation of aminoacyl-tRNA rrna complex body, thereby reach antibacterial effect.But in order to improve the output of terramycin, this area is also necessary that further research improves the method for terramycin output.
Be different from the method for traditional strain improvement, gene level operation has fast, effectively obtains the advantage of rite-directed mutagenesis strain.Although improving the method that the copy number of microbiotic route of synthesis genes involved in genome improves antibiotic output is employed in the prior art, but in raising route of synthesis, to have effect be uncertain to the copy of which or which gene, and the selection difficulty of gene is very large.And a lot of existing studies show that, the copy number that improves gene can not guarantee that the expression level of gene increases along with the increase of copy number, because bacterial strain itself exists self-control effect, can not change gene expression dose even improve gene copy number in a lot of situations, can not further improve antibiotic synthetic.Whether have effect also depend on the characteristic of gene itself, a lot of genes can not increase expression by improving copy number if improving gene copy number.Therefore, in order to improve the output of terramycin, need to screen genes involved in terramycin route of synthesis, critical to navigate to, can effectively realize the gene of regulation and control.
Summary of the invention
The object of the present invention is to provide a kind of novel process based on metabolic regulation high yield terramycin.
In a first aspect of the present invention, a kind of method of the terramycin output that increases streptomyces rimosus is provided, described method comprises: the rgC gene that increases 1-8 copy (as 2,3,4,5,6,7 copies) in streptomyces rimosus genome.
In a preference, described method also comprises: the otrC that increases 1-8 copy (as 2,3,4,5,6,7 copies) in streptomyces rimosus genome.
In another preference, described method comprises:
(1) provide expression vector, in described expression vector, comprise the sequence of rgC gene;
(2) expression vector is transformed to streptomyces rimosus, in Select gene group, be integrated with the restructuring streptomyces rimosus of the rgC gene order of external source.
In another preference, in the step of described method (1), in described expression vector, also comprise otrC gene; (2), in, in Select gene group, be integrated with the rgC gene order of external source, the restructuring streptomyces rimosus of otrC gene order.
In another preference, described rgC exists with the form of connecting with the sequence of otrC gene.
In another preference, in described expression vector, 5 ' end of rgC sequence, comprises ermEp1 promotor.
In another preference, described expression vector is pSET152 carrier.
In another aspect of this invention, provide a kind of streptomyces rimosus of restructuring, in its genome, be integrated with the rgC gene of 1-8 copy of external source.
In a preference, in the streptomyces rimosus of described restructuring, also contain the otrC gene of 1-8 copy of the external source of 1-8 copy.
In another preference, the streptomyces rimosus of described restructuring is to prepare by arbitrary described method above.
In another aspect of this invention, provide a kind of method of producing terramycin, described method comprises: cultivate the streptomyces rimosus of aforesaid restructuring, thereby produce terramycin.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Accompanying drawing explanation
Fig. 1, improve the schema of its terramycin output by increasing the copy number of rgC gene in streptomyces rimosus.
The structure of Fig. 2, integrated plasmid pSET152, it comprises Phi C 31 integrase gene order, phage attachment site attP sequence, and selection markers A Pula (Aprmycine) resistant gene sequence.
Fig. 3, recombinant plasmid pSET152-ermEp1-rgC (pSERC), wherein ermEp1 is an erythromycin strong promoter sequence, is positioned at the upstream of rgC gene in recombinant plasmid.
The terramycin of Fig. 4, mensuration recombinant bacterium SRI05/pSERC and starting strain SRI05 is tired.
Fig. 5, recombinant plasmid pSET152-ermep1-rgC-OtrC (pSETERO12), wherein ermEp1 is an erythromycin strong promoter sequence, is positioned at the upstream of rgC gene and the otrC gene of series connection in recombinant plasmid.
The terramycin of Fig. 6, mensuration recombinant bacterium SRI05/pSETERO12 and starting strain SRI05 is tired.
Embodiment
Transform difficult problem in order to overcome current terramycin industrial producing strain, the inventor is through deep research, develop a kind of method that improves terramycin output, by increasing the copy number of regulatory factor of one or more Oxytetracycline biosynthesis approach, thereby improve the expression level of genes involved in terramycin route of synthesis, and then improve the output of terramycin.
The present invention is by improving the copy number of a factor rgC of global regulation on streptomyces rimosus karyomit(e), thereby the expression amount of three initial structure genes in terramycin route of synthesis in raising streptomyces rimosus, thereby improve terramycin route of synthesis precursor---tsiklomitsin carbon skeleton, improve the flux of terramycin route of synthesis, thereby improve the output of terramycin.Described method can improve terramycin output and exceed 40%.
Described " streptomyces rimosus (Streptomyces rimosus) " is a kind of streptomycete that produces terramycin, and it can separate and obtain from soil.Its form is: fibrillae of spores volution 2～3 circles, closely, occasionally have the spacious spiral of pine.Spore cylindricality, 0.8 × 1.6～1 × 1.7 microns.
Described " terramycin " chemical name is: 6-methyl-4-dimethylamino)-3,5,6,10,12,12a-hexahydroxy--1,11-dioxo-Isosorbide-5-Nitrae, 4a, 5,5a, 6,11,12 α-octahydro-2-tetracene methane amide.Molecular formula: C
22h
24n
2o
9.It is tetracycline antibiotics, is broad-spectrum antibacterial agent.
Described " promotor " refers to a kind of nucleotide sequence, and the upstream (5 ' end) that it is present in goal gene encoding sequence conventionally, can be transcribed into mRNA by guiding nucleus acid sequence.Usually, promotor or promoter region provide RNA polymerase and correct initial recognition site of transcribing necessary other factors.
As used herein, " external source " or " allos " refers to from the relation between two or more pieces nucleic acid or the protein sequence of different sources.For example, if the combination of promotor and goal gene sequence is not naturally occurring conventionally, promotor is external source for this goal gene.Cell or the organism that particular sequence inserts for it is " external source ".
As used herein, " rgC " or " otrC " gene refers to the gene in Oxytetracycline biosynthesis approach, also be included under stringent condition with the molecule of described gene order hybridization or with the family gene molecule of above-mentioned numberator height homology, the expression of described gene has significant promoter action to the terramycin output of streptomyces rimosus.In this definition, be also included under stringent condition with the molecule of " rgC " or " otrC " hybridization or with the family gene molecule of above-mentioned numberator height homology.
As used herein, term " stringent condition " refers to: (1) at the hybridization compared with under low ionic strength and comparatively high temps and wash-out, as 0.2 × SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only the homogeny between two sequences at least 50%, preferably more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85% or more than 90%, be more preferably 95% and just hybridize when above.
NCBI discloses the gene order of " rgC " or " otrC ", and therefore, those skilled in the art are easy to obtain and prepare these genes.As optimal way of the present invention, described rgC gene has the nucleotide sequence shown in SEQ ID NO:1; Shown nucleotide sequence; Described otrC has the nucleotide sequence shown in SEQ ID NO:2.
Invention also relates to the polynucleotide varient of rgC or otrC, the polypeptide (enzyme) that its coding is identical with the aminoacid sequence of wild type gene coding.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural occurs.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing in fact the function of polypeptide of its coding.
The Nucleotide full length sequence of rgC of the present invention or otrC or its fragment can obtain by the method for pcr amplification method, recombination method or synthetic conventionally.For pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.
In order to increase the terramycin output of streptomyces rimosus, the inventor has done research widely, has found the gene rgC or the otrC that are suitable for improveing, and has built corresponding construction.
Therefore, the invention provides a kind of construction, described construction comprises: the expression cassette of rgC, otrC.Described expression cassette possesses the required all elements of genetic expression (comprising promotor, coding DNA and terminator etc.), thereby can intactly give expression to corresponding albumen.
As optimal way of the present invention, described rgC is present in same expression cassette with the form of connecting with otrC.In addition, selectable, described rgC can be present in respectively in different expression cassettes and (possess expression original paper separately, comprise promotor, terminator etc.) from otrC.
As optimal way of the present invention, rgC is connected with otrC, and drive and express with ermEp1.
Conventionally, described construction is positioned on expression vector.Therefore, the present invention also comprises a kind of carrier, and it contains described construction.Described expression vector also contains replication orgin and/or marker gene etc. conventionally.Method well-known to those having ordinary skill in the art can be used for building expression vector required for the present invention.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described DNA sequence dna can be effectively connected in the suitable promotor in expression vector, to instruct mRNA synthetic.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
Described rgC and otrC can be present in the same expression cassette of same expression vector or in different expression cassette; Also can be present in different expression vectors.As long as should be understood that and can realize the expression that increases rgC, otrC in streptomyces rimosus, the mode of any recombinant expressed rgC, otrC is all available.
In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of the host cell transforming, as eukaryotic cell is cultivated Tetrahydrofolate dehydrogenase, the neomycin resistance of use.Preferably, the selected marker on expression vector of the present invention is Apramycin sulfate (Apramycin) resistant gene.
When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe insert enhancer sequence in carrier time.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs, act on promotor transcribing with enhancing gene conventionally.Persons skilled in the art are all known the suitable carrier of How to choose, promotor, enhanser and host cell.
The carrier that comprises above-mentioned suitable polynucleotide sequence and suitable promotor or control sequence, can be for transforming suitable host.In the method for the invention, described host is streptomyces rimosus.
Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell, for example calcium phosphate precipitation, conventional mechanical method is as microinjection, electroporation, liposome packing etc.As the preferred mode of one, the method can electricity consumption transforming is carried out.
As optimal way of the present invention, provide a kind of method flow that improves terramycin output in streptomyces rimosus as shown in Figure 1.First, extract the genomic dna of streptomyces rimosus, design and synthesize primer, for the factor rgC of the global regulation gene that increases.Second step, is cloned in shuttle vectors, obtains recombinant vectors.The 3rd step, the recombinant vectors that second step is obtained turns by electricity or the method that engages is transferred in parent strain.The 4th step, adopts resistance screening to obtain recombinant vectors and is incorporated into the mutant strain in parental set of chromosome.The 5th step, the mutant strain that adopts method validation the 4th step of pcr amplification to obtain.The 6th step, the terramycin throughput of the mutant strain that mensuration the 5th step obtains.
Present method by testing in type strain and industrial superior strain, result shows, the genetic background clearly industrial superior strain of type strain and genetic background complexity can be utilized present method transformation, obtains the mutant strain of high yield terramycin, simple.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, writes molecular cloning experiment guide, the third edition, Science Press, the condition described in 2002, or the condition of advising according to manufacturer conventionally as J. Pehanorm Brooker etc. according to normal condition.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
The copy number of embodiment 1,1 factor rgC of global regulation gene of increase (249bp) increases output
In the present embodiment, the schema that improves its terramycin output by the copy number of rgC gene in increase streptomyces rimosus is as Fig. 1.
, as parent, plasmid map is as Fig. 2 to adopt integrative vector pSET152 (streptomycete integrated plasmid, available from University of Strathclyde, Glasgow, UK).Phi C 31 integrase and corresponding attP sequence on the plasmid skeleton of pSET152, are included.The locus specificity recombination and integration that can directly be mediated by Φ C31 by the recombinant plasmid of its structure enters in genomic attB target sequence.
1, include the extraction of streptomyces rimosus genomic dna
First, streptomyces rimosus (Streptomyces rimosus SRI05) is seeded in trypticase soybean broth substratum (TSB), 30 ℃ of 220rpm cultivate 30h.Secondly, centrifugal collection mycelium, washs mycelium with sterilized water.Then adopt N,O-Diacetylmuramidase fracturing cell walls, remove the impurity such as protein with phenol-chloroform extracting.Finally obtain genomic dna with isopropanol precipitating.
The amplimer of design is as follows:
Upstream primer: 5 ' CGC
gGATCCaTGAAGTTCCGCCGAATGGA (underscore is BamHI site) (SEQ ID NO:3);
Downstream primer: 5 ' TGC
tCTAGAtCACGATGACAGCACCCACTG (underscore is XbaI site) (SEQ ID NO:4).
Take the genomic dna of above-mentioned acquisition as template, amplification obtains rgC gene order.
The ErmeP1 promoter sequence of design is as follows with primer:
Upstream primer: CCG
gAATTCgTACCAGCCCGACCCGAGC (underscore is EcoRI site) (SEQ ID NO:5);
Downstream primer: CGC
gGATCCtGTGGGGTCCTCCTGTGGAGT (underscore is BamHI site) (SEQ ID NO:6).
The DNA that adopts existing ermEp1 promotor is that template increases, and size is 301bp.
2, recombinant plasmid and strain construction
RgC gene fragment and ermEp1 promoter DNA that pcr amplification is obtained carry out purifying, remove after impurity, with using respectively BamHI/XbaI, EcoRI/BamHI restriction enzyme digests, pSET152 carrier is used to EcoRI and XbaI digestion with restriction enzyme equally, then reclaim postdigestive fragment for connecting, then will connect product and transform intestinal bacteria Top10 competent cell, converted product is coated with the LB flat board that contains Apramycin sulfate 50 μ g/mL, 37 ℃ leave standstill cultivation 12h, single bacterium colony of growing on picking flat board is to containing in the LB liquid nutrient medium of Apramycin sulfate 50 μ g/mL, 37 ℃ of 220rpm cultivate 12h, extract plasmid, adopt after digestion with restriction enzyme, loading 0.8% agarose gel electrophoresis, the correct plasmid of screening endonuclease bamhi size is recombinant vectors pSET152-ermEp1-rgC, as Fig. 3.
The recombinant plasmid transformed E.coli ET12567 (pUZ8002) that screening is obtained is (available from University ofStrathclyde, Glasgow, UK) competent cell, converted product is coated with the LB flat board that contains Apramycin sulfate 25 μ g/mL, kantlex 25 μ g/mL, paraxin 34 μ g/mL, 37 ℃ leave standstill cultivation 12h, single bacterium colony of growing on picking flat board is to the LB liquid nutrient medium that contains Apramycin sulfate 25 μ g/mL, kantlex 25 μ g/mL, paraxin 34 μ g/mL, 37 ℃ of 220rpm cultivate 12h, extract plasmid.Getting 10 μ L plasmids adds in 100 μ L streptomyces rimosus SRI05 competent cells, after mixing, add the electric revolving cup (diameter 1cm) of precooling, at voltage 2KV, electric capacity 25 μ F, streptomycete competent cell shocks by electricity under resistance 400Ohm condition, then add rapidly the liquid nutrient medium (CRM) of 1mL precooling, bacteria suspension is proceeded in test tube to 30 ℃, after 150rpm cultivation 3h, get 100 μ L bacterium liquid and be coated with the TSB flat board that contains Apramycin sulfate (Apramycin) 500 μ g/mL, leave standstill and cultivate 3 days in 30 ℃.The recon that screening contains Apramycin sulfate resistance.
The chromosomal DNA of the recombinant bacterium that extraction contains Apramycin sulfate resistance, adopts PCR checking.
PCR checking primer is:
Primer pair 1:
Upstream: GTTCACCCACAGCTGGAGGCC (SEQ ID NO:7);
Downstream: GCTCGACTTCGCGCTGAAGGT (SEQ ID NO:8);
Primer pair 2:
Upstream: GCTATAATGACCCCGAAGCAG (SEQ ID NO:9);
Downstream: TCGTCATGCCCCGCAGT (SEQ ID NO:10);
Primer pair 3:
Upstream: GTGCAATACGAATGGCGAAAA (SEQ ID NO:11);
Downstream: TCAGCCAATCGACTGGCGA (SEQ ID NO:12).
Respectively by through primer pair 1 with 2 amplification PCR products be connected T carrier, check order, sequencing result conforms to expection, prove recombinant plasmid orientation be inserted in karyomit(e) bacterial attachment site attB.
The recombinant bacterial strain of above-mentioned acquisition and control strain (proceeding to the SRI05/pSET152 mutant strain of pSET152 empty carrier) are seeded to shaking flask, and fermentation, by high performance liquid phase-MS Performance in Oxytetracycline Fermentation Broth biological value.
3, conditions of flask fermentation
The recombinant bacterial strain of wild-type streptomyces rimosus strain SRI05 or aforementioned preparation is (7.5% wheat bran, 2.5% agar) on wheat bran slant medium, cultivates 7 days for 30 ℃, and growth spore, as the seed of liquid fermenting.
When liquid fermenting, get 1cm from slant medium
2size box is inoculated into 50mL ferment-seeded substratum (starch 3%, bean powder 0.3%, ammonium sulfate 0.4～0.5%, calcium carbonate 0.4%～0.5%, corn steep liquor 0.4%, sodium-chlor 0.5%, potassium primary phosphate 0.015%～0.017%) at 32 ℃, 220rpm cultivates 3 days.Then the seed culture fluid of 6mL is transferred to 50mL fermention medium (starch 15%, bean cake powder 2%, calcium carbonate 0.4%, ammonium sulfate 1.4%, sodium-chlor 0.4%, corn steep liquor 0.5%, potassium primary phosphate 0.01～0.02%, cobalt chloride 10ug/ml, foam killer 0.01%, amylase 1-2 ‰), 30 ℃ of 220rpm cultivate 4-5 days.
4, the mensuration of biological value
Pre-treatment: to pH1.5-1.7, get centrifugal 10 minutes of the fermented liquid 12000rpm of 1mL acidifying with 9mol/L hcl acidifying fermented liquid, get supernatant liquor, with after the membrane filtration of 0.22m, get 20 μ l loading analyses.The analysis condition of high performance liquid chromatography is to adopt 5 μ m (4.6 × 200nm) C18 posts; Moving phase is 60% water, 10% methyl alcohol l, and 20% acetonitrile and 10% phosphoric acid (2mM) mixed solution, flow velocity is 0.8 ml/min, detection wavelength is 350nm.
Analyze and measure tiring as shown in Figure 4 of terramycin in fermented liquid, under described shaking flask condition, knocked in the recombinant bacterium SRI05/pSET152-ermEp1-rgC of rgC gene terramycin yield increased bacterial strain height approximately 45%.
In aforesaid method, following (249bp) (the SEQ ID NO:1) of rgC fragment sequence for the series connection of knocking in:
ATGAAGTTCCGCCGAATGGACGGGCGCCCCGACTACTTCCTCCGAGTCACCGTGGCCGACCACACCGCGTACGAAGCCTTCCTGACCAGACAAGCTCAGCTGCCTGCCCGCCGTCCTGCGCCTCGAATCCCATATGACCATGAAGGAGATCAAGGCCGACCGGTGAGGCGCCGACGCCCAGCCGCCGACGGCCTGACCTGGGCTGTCGGCCGGCTGTCGGTCGTTTGTCAGTGGGTGCTGTCATCGTGA
Embodiment 2, increase the copy number of rgC regulatory factor on streptomyces rimosus karyomit(e) and an ABC transporter gene otrC, the ability that improves industrial producing strain and produce terramycin simultaneously
In the present embodiment, utilizing integrative vector pSE T152 is parent, connect rgC and otrC gene, co expression simultaneously.
1, extract streptomyces rimosus (Streptomyces rimosusSRI05) genomic dna
Extracting method as described in example 1 above.
The primer of design otrC gene:
Upstream primer: 5 ' CGC
gAGGA GGGGGCaTGACGCGAAAGACGATATCC (double underline is NdeI site) (SEQ ID NO:13);
Downstream primer: 5 ' TGC
tCTAGAtCAGGTCTTCTTGCGGAACTT3 ' (underscore is XbaI site) (SEQ ID NO:14);
The otrC gene fragment that amplification obtains is with RBS sequence (GGAGGA).
The primer of design rgC gene:
Upstream primer: 5 '-CGC
gGATCCaTGAAGTTCCGCCGAATGGA (underscore is BamHI site) (SEQ ID NO:15);
Downstream primer: 5 ' CGC
cATATGtCACGATGACAGCACCCACTG (underscore is Nde I site) (SEQ ID NO:16);
Take the genomic dna of aforementioned acquisition as template, amplification obtains rgC fragment.
2, the ermEp1 promoter sequence series connection of " 1 " described acquisition in rgC and otrC gene and embodiment 1 is cloned between the EcoRI and XbaI site of shuttle plasmid pSE T152, obtain pSE T152-ermep1-rgC-otrC (pSETERO12) recombinant plasmid (Fig. 5), as transformed intestinal bacteria as described in 2 in embodiment 1, and verify recombinant plasmid.
As described in " 2 " in embodiment 1, will after recombinant plasmid pSETERO12 demethylation, transform streptomyces rimosus competent cell, utilize resistance screening to obtain mutant strain.
As described in " 2 " in embodiment 1, the integration of recombinant plasmid and the exactness of integration site in validation chain mould mutant strain, and verify that recombinant bacterium produces the ability of terramycin.
3, as described in " 3 " in embodiment 1, fermentation streptomycete mutant strain.
4,, as described in " 4 " in embodiment 1, detect the terramycin output of recombinant bacterium.
Analyze and measure tiring as shown in Figure 6 of terramycin in fermented liquid, under described shaking flask condition, knocked in the recombinant bacterium of rgC gene and otrC gene terramycin yield increased bacterial strain height approximately 55%.
In aforesaid method, the complete sequence following (SEQ ID NO:2) for the otrC gene knocked in:
ATGACGCGAAAGACGATATCCAACGGCGCGAGGAACGCCGTCGAAGTGCGGGGACTGGTCAAGCACTTCGGCGAGGTGAAGGCCGTGGACGGGGTGGATCTCGATGTGAGGGAAGGCACCGTGCTCGGTGTGCTCGGGCCGACCGGCGCGGCAACACAACGTGGTGCGCTGCCTGCCCACGTTGCTGGTCCGGACGCCGGCAGGCGACCGTGGCGGTTTCAAACGTGGTGCGCCAACCGGCGCGCGTTGCGCCGCACGATCGGCCGTCACCGGCCAGTACGCGTCGGTCGACGAGAAAGCTTCTCCGGCCGCGAGAACCTGTACATGATCGGCCGCCTGCTGGACCTCTCCCGCAAGGACGCCCGCGCGCGGGCCGACGAGCTGCTGGAGCGGTTCTCCCTCACCGAGGCCGCCGGCCGGGCCGCCGCCAAGTACTCCGGCGGTATGCGCCGCCGCCTCGACCTGGCCGCCTCCATGATCGGCAGGCCCGCGGTGCTGTATCTGGACGAGCCGACGACGGGCCTCGACCCCCGCACCCGCAACGAGGTGTGGGACGAGGTCCGCAGCATGGTGCGCGACGGCGCCACGGTCCTGCTCACCACCCAGTACATGGAAGAGGCCGAGCAGCTGGCCCACGAGCTGACGGTCATCGACCGCGGCCGGGTCATCGCCGACGGCAAGGTGGACGAGCTGAAGACCAAGGTCGGCGGCCGTACGCTCCAGATACGCCCGGCGCACGCCGCCGAGCTGGACCGGATGGTCGGCGCCATCGCGCAGGCCGGCCTGGACGGCATCGCGGGCGCCACCGCCGACCACGAGGACGGCGTGGTCAACGTCCCGATCGTCAGCGACGAGCAGCTGTCCGCCGTGGTCGGCATGCTCGGCGAGCGGGGCTTCACGATCTCCGGGCATCAACACCCATCTGCCCAGCTGGACGAGGTGTTCCTGGCCATCACCGGCCAGAAGACCTCGGAGGCCGCCGACGGCGGCCCGCAGGACGGACCGCAGGACCAGCAGGGCGTTCAGGACAAGCAGTACGAGGAGGTTCCGGCATGAGTGCCGCGACGGTGGCGGCGCCGGGCAAGCCGCAGCGGCCGGGTGCCGACGAGGGCCGGATCGGCCTGCGTGCCCATCTGCGTCACACGAGCGCCCTGGTGCGCCGCAACGCGATGCAGATCAAGAACGACCCCGAGTCGATGTTCGACGCCCTGTTGATGCCGATCGTCTTCACGCTGCTGTTCGTGTACGTCTTCGGCGGCGCGATGGGCGGCAGCCTCGGCGGCGACCGCTCCGCGTACGTGAACTACATCATCCCCGGCCTGCTGGCCATGGGCGGCCTGAACATCGCCATGTCGGTCGGCTCCGGCGTCAACGACGACTTCAACAAGGGGGTGATGGACCGCTTCCGCACCATGCCGATAGCCCGCTCCTCGGTGCTGACGGCGAAGATGCTCGTCGAGTCCGGGCGGATGCTGGTCTCCACCGTCATCCTGCTGGTGCTGGCGTTCCTGATCGGCTTCGACCTGAAGACCGGCCCGCTGGAGCTGCTGGCGGCCGTCGCCCTGGCGCTGCTCTTCGGCTCCTCGCTGACCTGGATATTCATGCTGCTGGGCCTGACCATGAAGACGCCGCAGGCGGTGCAGGGCGTCGGCCTTCATGGTATGATGCCGCTGCCCGTCGGCTCCTCCATCTTCGCCCCGCCGACCACGATGCCGGGCTGGATGGAGGCGTTCGCCAAGGTCAACCCGCTGTCGAACCTCGCGGACGCGGCACGGGCNNNGATGAACGGACAGGGCGCCGTCGCCCAGCCGGTGCTGATCACCCTGGCCTGGACCGTCGGCATCACCCTGGTGTTCGCGCCCCTGGCCGTGGCCAAGTTCCGCAAGAAGACCTGA
All documents of mentioning in the present invention are all quoted as a reference in this application, are just quoted separately as a reference as each piece of document.In addition should be understood that 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.
Claims (10)
1. a method that increases the terramycin output of streptomyces rimosus, is characterized in that, described method comprises: the rgC gene that increases 1-8 copy in streptomyces rimosus genome.
2. the method for claim 1, is characterized in that, described method also comprises: the otrC that increases 1-8 copy in streptomyces rimosus genome.
3. the method for claim 1, is characterized in that, described method comprises:
(1) provide expression vector, in described expression vector, comprise the sequence of rgC gene;
(2) expression vector is transformed to streptomyces rimosus, in Select gene group, be integrated with the restructuring streptomyces rimosus of the rgC gene order of external source.
4. method as claimed in claim 3, is characterized in that, in (1), also comprises otrC gene in described expression vector; (2), in, in Select gene group, be integrated with the rgC gene order of external source, the restructuring streptomyces rimosus of otrC gene order.
5. method as claimed in claim 3, is characterized in that, in described expression vector, 5 ' end of rgC sequence, comprises ermEp1 promotor.
6. the method as described in as arbitrary in claim 3-5, is characterized in that, described expression vector is pSET152 carrier.
7. a streptomyces rimosus for restructuring, is characterized in that, is integrated with the rgC gene of 1-8 copy of external source in its genome.
8. the streptomyces rimosus of restructuring as claimed in claim 7, is characterized in that, also contains the otrC gene of 1-8 copy of the external source of 1-8 copy in the streptomyces rimosus of described restructuring.
9. the streptomyces rimosus of recombinating as claimed in claim 7 or 8, is characterized in that, it is to prepare by the arbitrary described method of claim 1-6.
10. a method of producing terramycin, is characterized in that, described method comprises: cultivate the streptomyces rimosus of the arbitrary described restructuring of claim 7-9, thereby produce terramycin.
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| ROBERTS MC ET AL: "Steptomyces rimosus putative transcription regulator gene, and putative ABC transporter ATP-binding component and putative ABC-transporter transmembrane component (otrC) gene, complete cds; and putative multi-domain regulatory protein gene, partial cds", 《GENBANK登录号：AY509111》, 2 May 2005 (2005-05-02) * |
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| CN105821053A (en) * | 2015-01-04 | 2016-08-03 | 中国科学院微生物研究所 | Method for constructing recombinant strain by utilization of terramycin positive regulator gene and raising yield of terramycin |
| CN105821053B (en) * | 2015-01-04 | 2019-09-20 | 中国科学院微生物研究所 | Method for constructing recombinant bacteria and improving oxytetracycline production by using oxytetracycline positive regulation gene |
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