CN103074356A - Vector for knocking out streptomycete gene as well as constructing method and application of same - Google Patents
Vector for knocking out streptomycete gene as well as constructing method and application of same Download PDFInfo
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- CN103074356A CN103074356A CN2013100232818A CN201310023281A CN103074356A CN 103074356 A CN103074356 A CN 103074356A CN 2013100232818 A CN2013100232818 A CN 2013100232818A CN 201310023281 A CN201310023281 A CN 201310023281A CN 103074356 A CN103074356 A CN 103074356A
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a vector for knocking out a streptomycete gene as well as a constructing method and an application of the vector. A recombinant vector is a recombinant DNA (Deoxyribose Nucleic Acid) obtained by inserting a locus ClaI of pKC1139 into a glucanase gene. In actual application, a streptomycete target gene knockout vector can be obtained after a DNA fragment for knocking out a streptomycete target gene is inserted in a multiple clone site of the recombinant vector; after the streptomycete target gene knockout vector is introduced into streptomycete with the target gene to be knocked out, transformants with thiostrepton resistances are screened; cellulose degradation and screening are carried out on the transformants with the thiostrepton resistances; and specifically, a homologous double-exchange transformant can be obtained through inserting the transformants with the thiostrepton resistances into a cellulose screening flat plate and then carrying out Congo red staining on the transformants with the thiostrepton resistances so as to directly observe degradation situation of the cellulose. Therefore, the phenomenon of incomplete knockout of the gene due to false positive antibiotics resistance can be avoided. Meanwhile, PCR verifications of a large amount of transformants can be avoided. The vector for knocking out the streptomycete gene, disclosed by the invention, can be used for screening any gene knockout double-exchange transformant of strains with no glucanase yield.
Description
Technical field
The present invention relates to be used to the carrier that knocks out streptomyces gene and construction process and application.
Background technology
Streptomycete (streptomyces) is a kind of aerobic Gram-positive soil bacteria with branched mycelium structure, belongs to Prokaryota actinomycetales Streptomycetaceae, is one of microbe groups main in the soil.Its G%+C% content is that the class that the known G%+C% content of occurring in nature is the highest up to now is biological up to 70%-80%.Although streptomycete belongs to prokaryotic organism, but very complicated mechanism of cell differentiation is arranged but, it is the good material of research gene expression and regulation mechanism on time, space and program, secondly streptomycete has abundant secondary metabolism diversity, the microbiotic of occurring in nature nearly 70% is produced by streptomycete and nearly edge actinomycetes thereof, streptomycete also produces other useful secondary metabolites such as antitumor drug, immunosuppressor, pest-resistant dose and the outer lytic enzyme of some other born of the same parents etc. in addition, is widely used in industry, agricultural and medical and health and life science field.Therefore, disclosing the research of streptomycete heredity, growth and metabolic regulation extremely payes attention to, but not clear for the research of the living metabolic regulation mechanism of streptomycete at present, along with finishing of the order-checking of in recent years streptomyces coelicolor, Avid kyowamycin and streptomyces griseus, more streptomyces gene group examining order is carried out, therefore the function of new gene and known is carried out genetic analysis and compel to cut at enzyme, and the most important method of research gene is carried out gene knockout exactly.Gene knockout is exactly by homology of chromosome restructuring, replaces or interrupts certain gene, then observes its phenotype and changes.But the method for this gene knockout directly depends on the foundation of efficient gene engineered vector in the streptomycete of a certain genus or kind and genetic conversion system thereof, obtains the research that gene knockout mutant strain carries out gene function in order to people can be screened more easily.Present genetic conversion system about streptomyces, the genetic system that comprises gene knockout is set up basically, basically all be that the plasmid that will carry goal gene upstream and downstream homology arm and resistant gene engages the method that shifts between belonging between intestinal bacteria and the streptomycete, utilize on the tra gene that comprises among the intestinal bacteria ET12567 helper plasmid pUZ8002 and the shuttle vectors and carry the conjugal transfer of inducing from RP4 conjugal transfer initiation site oriT.Wherein the screening of double exchange transformant all is according to antibiotic resistance and susceptibility basically, utilizes the technology of panel photocopy to engage that the methods such as pcr amplification, southern hybridization screen.But because for replica screening the time, there are significantly false positive in antibiotics resistance and susceptibility; And directly can consume a large amount of human and material resources by the pcr amplification screening, because streptomycete is as gram positive bacterium, its cell walls is thicker, and bacterium colony PCR's is stable very poor, so utilize the method bacterium of antibiotics resistance and pcr amplification can not improve the screening efficiency of double exchange transformant.
Summary of the invention
A technical problem to be solved by this invention provides with the carrier and the construction process thereof that are used for knock out streptomyces gene of glucanase gene as the selection markers gene.
Carrier be used to knocking out streptomyces gene provided by the present invention, name is called pKCG
+1139, be that glucanase gene is inserted the recombinant DNA that the Cla I site of pKC1139 obtains.
Wherein, the physical map of pKC1139 as shown in Figure 1, the pKC1139 plasmid belongs to streptomyces gene group damage type plasmid, can be used for the gene disruption test, realization is to the checking of gene function, and can improve antibiotic output or the active (Shen Fengying of product spore in some streptomycete by blocking some negative regulatory sequences, Li Yaning, Liu Liqiang, Wu Weigang, Liu Daqun. foundation and the optimization of biological and ecological methods to prevent plant disease, pests, and erosion streptomycete Men-myco-93-63 genetic conversion system. Chinese agronomy circular 2009,25 (13): 197-201).
The concrete codified of above-mentioned glucanase gene following a) or b) protein:
A) protein of aminoacid sequence shown in SEQ ID No.1;
B) with replacement and/or disappearance and/or the interpolation of one or several amino-acid residue among the SEQ ID No.1 and have the degraded cellulose activity by a) derivative protein.
Wherein, SEQ ID No.1 is comprised of 499 amino-acid residues.
The encoding sequence of described glucanase gene specifically can be shown in the 208-1707 position of SEQ ID No.2.
In one embodiment of the invention, the nucleotide sequence of described glucanase gene specifically can be the 9-1707 position of SEQ ID No.2.
Wherein, SEQ ID No.2 is comprised of 1715 Nucleotide, and the 1-8 position is Cla I recognition site and protection base, and the 9-207 position is the erythromycin promotor; the encoding sequence of 208-1707 position glucanase gene, 1708-1715 are Cla I recognition site and protection base.
The method that makes up above-mentioned carrier be used to knocking out streptomyces gene also belongs to protection scope of the present invention.
The recombinant microorganism that contains above-mentioned carrier be used to knocking out streptomyces gene also belongs to protection scope of the present invention.
Described recombinant microorganism specifically can be bacterium, yeast, algae and fungi.Wherein, bacterium can be from Escherichia (Escherichia), streptomyces (Streptomyces) etc.
Above-mentioned carrier be used to knocking out streptomyces gene can be used for knocking out the gene of the streptomycete that does not produce dextranase.
Wherein, the described streptomycete that does not produce dextranase can be streptomyces lydicus, streptomyces chatanoogensis, Natal streptomycete, brown yellow spore streptomycete etc.
In one embodiment of the invention, described streptomyces lydicus (Streptomyces lydicus) is streptomyces lydicus (Streptomyces lydicus) A02CGMCC No.1654, and the described gene of the streptomycete that does not produce dextranase that knocks out is for knocking out the synthetic positive regulating gene of tennecetin.
Another technical problem to be solved by this invention provides streptomycete goal gene knockout carrier.
Streptomycete goal gene knockout carrier provided by the present invention is to insert the recombinant DNA that obtains for the dna fragmentation that knocks out the streptomycete goal gene in the multiple clone site of above-mentioned carrier be used to knocking out streptomyces gene.
Wherein, thus described dna fragmentation for knocking out the streptomycete goal gene substitutes described goal gene by homologous recombination knocks out the goal gene of streptomycete.
Described dna fragmentation be used to knocking out the streptomycete goal gene can be followed successively by the downstream homology arm of upstream homology arm, thiostrepton resistant gene (tsr) and the described goal gene of described goal gene to the downstream from the upstream.
Described streptomycete goal gene knockout carrier comprises thiostrepton resistant gene (tsr) and apramycin resistant gene (apm), does not therefore affect the screening of resistance transformant in intestinal bacteria and streptomycete in the vector construction process.
Another technical problem to be solved by this invention provides a kind of method for preparing the streptomycete mutant strain that goal gene is knocked.
The method of the streptomycete mutant strain that preparation goal gene provided by the present invention is knocked, comprising the steps: above-mentioned streptomycete goal gene knockout carrier imported waits to knock out in the streptomycete of described goal gene, obtain the transformant of thiostrepton resistance by the thiostrepton resistance screening, screen the not transformant of degraded cellulose from the transformant of described thiostrepton resistance, the transformant of the not degraded cellulose that obtains is the streptomycete mutant strain that candidate's goal gene is knocked;
In the aforesaid method, described degraded cellulose is presented as degradation of sodium carboxymethylcellulo, e.
In the aforesaid method, the described streptomycete of streptomycete for not producing dextranase of waiting to knock out described goal gene is such as streptomyces lydicus, streptomyces chatanoogensis, Natal streptomycete, brown yellow spore streptomycete etc.
In one embodiment of the invention, described streptomyces lydicus (Streptomyces lydicus) is streptomyces lydicus (Streptomyces lydicus) A02CGMCC No.1654, and described goal gene is the synthetic positive regulating gene slnM of tennecetin.The nucleotide sequence of described dna fragmentation be used to knocking out the streptomycete goal gene is SEQ ID No.5.
The encoding sequence of the synthetic positive regulating gene slnM of described tennecetin is SEQ ID No.4, the protein of encoding amino acid sequence shown in SEQ ID No.3.
Wherein, SEQ ID No.4 is comprised of 579 Nucleotide, and SEQ ID No.3 is comprised of 192 amino-acid residues.SEQ ID No.5 is comprised of 4144 Nucleotide, the 29-1221 position is the upstream homology arm of the synthetic positive regulating gene slnM of tennecetin, the 1265-2911 position is thiostrepton resistant gene (tsr), the downstream homology arm of the synthetic positive regulating gene slnM of 2956-4117 position tennecetin.
Carrier pKCG be used to knocking out streptomyces gene of the present invention
+1139 will place from the glucanase gene of bacillus amyloliquefaciens under the streptomycete erythromycin promotor commonly used, comprise the two negative selection markers apramycin resistant genes of gene knockout and glucanase gene.PKCG
+1139 can express glucanase gene in intestinal bacteria (such as Escherichia coli BL21, E.coli DH5 α, E.coli Top10 etc.), streptomycete (such as streptomyces lydicus, streptomyces chatanoogensis, Natal streptomycete, brown yellow spore streptomycete etc.), therefore can knock out negative selection markers as streptomyces gene and use.In actual applications, can be at pKCG
+1139 multiple clone site is inserted for obtaining streptomycete goal gene knockout carrier behind the dna fragmentation that knocks out the streptomycete goal gene, this streptomycete goal gene knockout carrier is imported the transformant of screening thiostrepton resistance behind the streptomycete that knocks out described goal gene, the transformant of thiostrepton resistance is carried out the cellulose degradation screening again, carry out again the cellulosic degraded situation of congo red staining direct viewing on specifically can the transformant incoming fiber element screening flat board with the thiostrepton resistance can obtain homology double exchange transformant.The phenomenon that does not knock out gene fully that can avoid like this antibiotics resistance false positive to cause.Can avoid simultaneously a large amount of transformant PCR checkings.Can be used in the screening of any gene knockout double exchange transformant that does not produce the dextranase bacterial strain.
Carrier pKCG be used to knocking out streptomyces gene of the present invention
+1139 have overcome the deficiency that present gene knockout carrier false positive occurs and need to expend a large amount of human and material resources, are a kind of gene knockout carriers with selection markers directly perceived, can carry out streptomycete chromosome gene fast, easily and knock out.This selection markers gene directly perceived is glucanase gene, can obtain by the method direct viewing of congo red staining gene knockout double exchange transformant, remedied because the false-positive shortcoming of antibiotics resistance, for the screening of transgenation strain provides more practical instrument.
With pKCG
+M
-1139 carriers transform demethylation E.coli ET12567 (pUZ8002), method by the amphiphilic joint, transform streptomyces lydicus, by the thiostrepton resistance screening, 500 strains of picking positive transformant, and went down to posterity for 5 generations in the thiostrepton resistant panel, carry out Xylo-Mucine plate screening, congo red staining.The result shows 496 strain transformants and shows dextranase activity, there are 4 strains not have dextranase activity, whether the bacterial strain that does not have dextranase activity is carried out pcr amplification checking goal gene knocks out, the result shows that 4 strains do not produce the bacterial strain of dextranase and all do not have amplification to obtain goal gene, and the fragment of the 1.0kb that obtains expecting, and the bacterial strain PCR result who the produces dextranase goal gene fragment that all increased, therefore utilize one step of this carrier can obtain intuitively the double exchange mutant strain, saved a large amount of manpower and materials.In order further to verify the reliability of the method, remaining 496 strain transformant shaking flask subcultures are carried out the dextranase biopsy after 5 generations survey, filter out the bacterial strain that 3 strains do not have dextranase activity, the stepping performing PCR of going forward side by side checking, the result shows, all dextranase negative strains all do not increase and obtain goal gene, therefore illustrate that the method is accurate, easy, can be applied to the screening operation of double exchange mutant strain.
Description of drawings
Fig. 1 is the physical map of pKC1139.
Fig. 2 is pKCG
+1139 physical map.
Fig. 3 is pKCG
+ M
-1139 physical map.
Fig. 4 is the streptomycete mutant strain that is knocked of slnM and the PCR electrophoretogram of wild strain.
Wherein, swimming lane M is dna molecular amount standard, and A02 is streptomyces lydicus (Streptomyces lydicus) A02CGMCC No.1654, A02-slnM
-Streptomyces lydicus (Streptomyces lydicus) the A02CGMCC No.1654 mutant strain that is knocked for slnM.
Fig. 5 is the bacterium colony photo of congo red staining on the Mierocrystalline cellulose culture medium flat plate.
Arrow shows streptomyces lydicus (Streptomyces lydicus) the A02CGMCC No.1654 mutant strain (A02-slnM that slnM is knocked
-).
Fig. 6 A is that the HPLC of Streptomyces lydicus A02 CGMCC No.1654 active result separates spectrogram for the first time.
Fig. 6 B is that the HPLC of Streptomyces lydicus A02 CGMCC No.1654 active result separates spectrogram for the third time.
Fig. 7 is the UV scanning collection of illustrative plates of tennecetin sample.
Fig. 8 is the infrared absorption spectrum of tennecetin sample.
Fig. 9 A is the high resolution mass spectrum figure (negative ion) of tennecetin sample.
Fig. 9 B is the high resolution mass spectrum figure (positive ion) of tennecetin sample.
Figure 10 A is the carbon-13 nmr spectra (500MHz) of tennecetin sample.
Figure 10 B is the proton nmr spectra (500MHz) of tennecetin sample.
Figure 11 is the bacterium colony photo of congo red staining on the Mierocrystalline cellulose culture medium flat plate of Comparative Examples.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Streptomyces expression vector pIB139(Christopher J.Wilkinson among the following embodiment, et al.Increasing the Efficiency of Heterologous Promoters in Actinomycetes.J Mol Microbiol Bioteh (2002) 4 (4): 417 – 426), pKC1139(Shen Feng English, Li Yaning, Liu Liqiang, Wu Weigang, Liu Daqun. foundation and the optimization of biological and ecological methods to prevent plant disease, pests, and erosion streptomycete Men-myco-93-63 genetic conversion system. Chinese agronomy circular 2009,25 (13): the 197-201 public can obtain from the Beijing City Agriculture and Forestry Institute, to repeat the application's experiment.
Demethylation E.coliET12567 (pUZ8002) (Sun-Uk Choi among the following embodiment, Chang-Kwon Lee, Yong-Il Hwang, Hiroshi Kinoshita, and Takuya Nihira (2004) Cloning and Functional Analysis by Gene Disruption of a Gene Encoding a γ-Butyrolactone Autoregulator Receptor from Kitasatospora setae.JOURNAL OF BACTERIOLOGY, 186:3423 – 3430.) public can obtain from the Beijing City Agriculture and Forestry Institute, to repeat the application's experiment.
Embodiment 1, structure are used for knocking out the knockout carrier of the streptomyces gene that does not produce dextranase
1, the clone of bacillus amyloliquefaciens glucanase gene
A strain that obtains take this laboratory screening has remarkable inhibiting bacillus amyloliquefaciens YW26 genomic dna as template to the various plants pathogenic fungi, according to bacillus amyloliquefaciens endo glucanase gene (DQ782954.1) primers among the GenBank: P1(5'-ATGAAACGGTCAATTTCTAT-3 ') and P2(5'-CTAATTGGGTTCTGATCC-3'), carry out pcr amplification.Reaction is at 95 ℃ of denaturation 5min; 30 circulations (94 ℃ of 40s, 55 ℃ of 30s, 72 ℃ of 1min); 72 ℃ are extended 10min.The fragment of the 1500bp size that amplification is obtained is connected with plasmid pMD18-T carrier, obtains pMD18-glu, transforms bacillus coli DH 5 alpha, the positive transformant that obtains is carried out PCR and enzyme is cut checking by ammonia benzyl resistance screening.Utilize the universal primer order-checking, GenBank database BLAST analyzes the inscribe β-1 that shows this sequence and a strain Bacillus subtilis strain C-36,4-glucanase gene sequence identity reaches 100%, thereby determines that this gene is glucanase coding gene as well as.The nucleotide sequence of the glucanase coding gene as well as PCR product that P1 and P2 obtain among the pMD18-glu is the 208-1707 position of SEQ ID No.2, the dextranase of coding SEQ ID No.1.
2, the structure of streptomyces expression vector
The promotor of utilizing the erythromycin promotor (ermE*) of carrying on the streptomyces expression vector pIB139 to express as glucanase gene makes up and obtains streptomycete dextranase expression vector pIB139-glu.Concrete grammar is as follows:
According to the analysis to pIB139 carrier restriction enzyme site, suitable restriction enzyme site NdeI and EcoRI are added to F15'-in the glucanase gene primer
GGAATTCCATATGATGAAACGGTCAATCTCTAT-3'(NdeI) and R15'-
CGGAATTCCTAATTTGGTTCTGTTC-3'(EcoRI), obtain the dextranase with restriction enzyme site take pMD18-glu as template amplification, by the NdeI/EcoRI double digestion and cut the streptomyces expression vector pIB139 that obtains carrying erythromycin promotor (ermE*) through same enzyme and be connected, obtain pIB139-glu, and conversion bacillus coli DH 5 alpha, with apramycin (apramycin) thus resistance screening obtains to take the transformant of glucanase gene, obtain the approximately fragment of 1.5kb by pcr amplification, enzyme is cut checking and is obtained the pIB139 plasmid of 5.9kb and the about glucanase gene fragment of 1.5kb, thereby determines that the fragment exact connect ion is in the pIB139 carrier.
3, the clone obtains the glucanase gene under the erythromycin promoters driven, and structure carries the streptomyces gene knockout carrier of dextranase selection markers
With erythromycin promoter sequence upstream primer p3(
CCATCGATCTAGTATGCATGCGAGTG) and promotor p4(arranged under the glucanase gene
CCATCGATCTAATTTGGTTCTGTTC) (the line part is restriction enzyme ClaI site and protection base) is primer; take the streptomyces expression vector pIB139-glu that carries erythromycin promotor (ermE*) as template; amplification obtains the fragment ermE-glu that nucleotide sequence is erythromycin promotor and the glucanase gene sequence of SEQ ID No.2; this fragment is inserted the ClaI site of pKC1139 after the ClaI enzyme is cut, obtained the recombinant expression vector pKCG that erythromycin promotor and glucanase gene insert pKC1139
+1139(Fig. 2).PKCG
+1139 are the knockout carrier that is used for knocking out the streptomyces gene that does not produce dextranase of the present invention's protection.
Embodiment 2, structure knock out the knockout carrier of the synthetic positive regulating gene slnM of tennecetin in the streptomycete that does not produce dextranase
The encoding sequence of the synthetic positive regulating gene slnM of tennecetin is SEQ ID No.4, the protein of encoding amino acid sequence shown in SEQ ID No.3.
At first making up nucleotide sequence is the dna fragmentation that knocks out streptomycete slnM of SEQ ID No.5; SEQ ID No.5 is comprised of 4144 Nucleotide; the 1-8 position is EcoRI recognition site and protection base; the 29-1221 position is the upstream homology arm of the synthetic positive regulating gene slnM of tennecetin; the 1265-2911 position is thiostrepton resistant gene (tsr); the downstream homology arm of the synthetic positive regulating gene slnM of 2956-4117 position tennecetin, 4136-4144 position are HindIII recognition site and protection base.
The dna fragmentation that knocks out streptomycete slnM that with nucleotide sequence is SEQ ID No.5 inserts pKCG after Hind III and EcoR I enzyme are cut
+1139 Hind III and EcoR I site obtain slnM knockout carrier pKCG
+M
-1139(Fig. 3).
One, with pKCG
+M
-1139 carriers transform demethylation E.coliET12567 (pUZ8002)
By the heat shock method with pKCG
+M
-1139 carriers import E.coliET12567 (pUZ8002) and obtain changing over to pKCG through 15 μ g/ml thiostrepton resistance screenings
+M
-The recombinant bacterium E.coliET12567 (pUZ8002) of 1139 carriers/pKCG
+M
-1139.
Two, slnM knocks out the preparation of strain
Reference (Bierman M et al.1992) carries out amphiphilic and engages pKCG
+M
-1139 import streptomyces lydicus, and concrete grammar is as follows:
With recombinant bacterium E.coliET12567 (pUZ8002)/pKCG
+M
-1139 are inoculated into the LB liquid nutrient medium that contains paraxin, kantlex and apramycin resistance, and 37 ℃, 200rpm, overnight incubation, receive in the fresh LB substratum by 1% inoculum size and be cultured to OD morning next day
600=0.4-0.6 collects bacterium liquid, and 4 ℃, 4000rpm, centrifugal 2min abandons supernatant, with the ice-cold pure resuspended precipitation of LB nutrient solution of 20mL, 4 ℃, 4000rpm, centrifugal 2min abandons supernatant, and purpose is the flush away microbiotic.With the ice-cold pure resuspended precipitation of LB nutrient solution of 2mL.(2) grown in PDA inclined-plane streptomyces lydicus (Streptomyces lydicus) the A02CGMCC No.1654(CN100467588C in 2 weeks) spore elutes with aseptic double-distilled water, and with sample injector piping and druming evenly, prepares spore suspension.(3) 250 μ L streptomyces lydicus (Streptomyces lydicus) A02CGMCC No.1654 spore suspensions add 500 μ L2 * YT nutrient solution, gently mixing.50 ℃ of thermal shock 10min, the activation spore.500 μ L recombinant bacterium E.coliET12567 (pUZ8002)/pKCG
+M
-Streptomyces lydicus (Streptomyces lydicus) the A02CGMCC No.1654 spore suspension that 1139 bacterium liquid and 500 μ L activate, gently mixing.4000rpm, the centrifugal 3min of room temperature removes supernatant, and the mixing precipitation is coated MS substratum+10mM/L MgCl
2, 28 ℃ of inversions are cultured to morning next day (18h), are coated with afterwards antibiotic solution and (0.5mg nalidixic acid and 60 μ g apramycins are dissolved in 1mLddH
2The liquid that obtains among the O).Picking list bacterium colony is to the MS flat board of the new above-mentioned antibiotic solution of adding after 2-3 days.Picking adds on the MS+15 μ g/ml thiostrepton dull and stereotyped (add in MS thiostrepton make its final concentration be 15 μ g/ml) at the bacterium colony on this MS flat board, screening obtains transformant 500 strains of thiostrepton resistance, 5 generations of continuous passage, transformant is inoculated on the Mierocrystalline cellulose culture medium flat plate 29 ℃ of cultivations 2 days, carry out congo red staining, show 496 strain transformants by congo red staining and produce obvious transparent circle, have the ability of degraded cellulose, be the degraded cellulose transformant; There are 4 strains not produce transparent circle, be degraded cellulose transformant not, with this four strain bacterial strain primer (forward primer atggcgagccttgataaaac of degraded cellulose not, reverse primer tcacttcacgaagtcgtcc) whether pcr amplification checking goal gene slnM is knocked, the result show 4 strains not the bacterial strain of degraded cellulose all do not have amplification to obtain goal gene, and the fragment of the 180bp that obtains expecting (Fig. 4), and wild type strain streptomyces lydicus (Streptomyces lydicus) A02CGMCC No.1654PCR amplification obtains the fragment of 579bp, show 4 plant mutant strain generation double exchanges, pKCG
+M
-Carrier after the 1139 carrier generation double exchanges from streptomyces lydicus (Streptomyces lydicus) thus disappear to obtain streptomyces lydicus (Streptomyces lydicus) the A02CGMCC No.1654 mutant strain (Fig. 5) that slnM is knocked the A02CGMCC No.1654, and the bacterial strain PCR result who the produces dextranase goal gene fragment (fragment of 579bp) that all increased illustrates that single cross has only occured changes.Therefore utilize one step of this carrier can obtain intuitively the double exchange mutant strain, saved a large amount of manpower and materials.In order further to verify the reliability of the method, remaining 496 strain transformant shaking flask subcultures are carried out the detection of cellulose degradation ability according to the method described above after 5 generations, filter out the not bacterial strain of degraded cellulose of 2 strains, and further use primer (forward primer atggcgagccttgataaaac, reverse primer tcacttcacgaagtcgtcc) whether pcr amplification checking goal gene slnM is knocked, the result shows, this 2 strain does not have the ability of degraded cellulose, all there is not amplification to obtain goal gene, and the fragment of the 180bp that obtains expecting, therefore illustrate that the method is accurate, easy, can be applied to the screening operation of double exchange mutant strain.
In addition, streptomyces lydicus (Streptomyces lydicus) A02CGMCC No.1654 is inoculated on the Mierocrystalline cellulose culture medium flat plate 29 ℃ of cultivations 2 days, carry out congo red staining, the result shows that wild-type streptomycete bacterial strain streptomyces lydicus (Streptomyces lydicus) A02CGMCC No.1654 does not produce transparent circle, degraded cellulose does not have dextranase activity.
MS substratum in the present embodiment:
Mierocrystalline cellulose substratum in the present embodiment: Xylo-Mucine 20g, Na
2HPO
42.5g, KH
2PO
42.5g, peptone 2.5g, yeast extract paste 0.5g, be settled to 1000mL with distilled water.
Three, streptomyces lydicus (Streptomyces lydicus) the A02CGMCC No.1654 mutant strain that is knocked of slnM and wild strain tennecetin synthesis capability are relatively
Among this embodiment, used substratum is as follows:
Gause I slant medium: K
2HPO
40.5g, NaCl0.5g, KNO
31.0g, FeSO
47H
2O0.01g, MgSO
47H
2O0.5g, Zulkovsky starch 20g, agar 20g, water 1000ml; PH7.2-7.4.
Seed culture medium: 15g soybean grain (adding distil water boils 0.5-1h, gets filtrate), 5g peptone, 2.5g ammonium sulfate, 20g glucose, 10g starch, 0.25g sal epsom, 0.2g potassium primary phosphate, 5g sodium-chlor, be made into the aqueous solution, behind the accent pH7-8, add 1g calcium carbonate, add water and be settled to 1000ml.
Fermention medium: 15g soybean grain (adding distil water boils 0.5-1h, gets filtrate), 5g peptone, 2.5g ammonium sulfate, 10g sucrose, 10g starch, 0.25g sal epsom, 0.2g potassium primary phosphate, 5g sodium-chlor, be made into the aqueous solution, behind the accent pH7-8, add 1g calcium carbonate, add water and be settled to 1000ml.
1, the preparation of streptomyces lydicus (Streptomyces lydicus) natamycin fermentation liquor
Streptomyces lydicus (Streptomyces lydicus) the A02CGMCC No.1654 mutant strain that the slnM that streptomyces lydicus (Streptomyces lydicus) A02CGMCC No.1654 and step 2 are obtained is knocked is inoculated into respectively on the Gause I slant medium, cultivated 7-10 days for 28 ℃, treat that it produces the spore of capacity, encircle in the 50ml seed culture medium that is inoculated in the 250ml triangular flask with aseptic its spore of platinum loop scraping 2-3, put on the temperature controllable shaking table, under 28 ℃ of conditions, 200rpm(rotation radius 13mm) constant-temperature shaking culture 24h-30h; Then under aseptic condition, its minute be connected to (every bottled liquid measure is 100ml) in 10 fermention mediums in the 500ml triangular flask, streptomyces lydicus (Streptomyces lydicus) A02CGMCC No.1654 is identical with the inoculum size of streptomyces lydicus (Streptomyces lydicus) the A02CGMCC No.1654 mutant strain that the slnM that step 2 obtains is knocked, OD after every bottle graft kind
600Value is 0.1; Postvaccinal shaking flask is under 31 ℃ of conditions, with 240rpm(rotation radius 13mm) speed oscillation cultivate 96h.
2, tennecetin volume analysis
The 1mL fermented liquid that step 1 is obtained adds 9mL methyl alcohol, fully after the vibration, carry out the 30min ultrasonic extraction, 5000rpm/min centrifugal 10-15min sedimentation mycelium and solid substance, 10 times of aseptic filtering with microporous membrane of using afterwards 0.45 μ m of supernatant liquor dilution, collect filtrate, the gained sample is used for HPLC and detects.Chromatographic column: the C18 post (5 μ m, 4.6mm * 200mm); Detect wavelength: 303nm; Flow velocity: 1.00mL/min; Sample size: 10 μ L; Detect column temperature: 30 ℃; Experiment moving phase is V (methyl alcohol): V (water)=65: 35.Adopt external standard method (calibration curve method) that tennecetin is carried out quantitatively take tennecetin (sigma-P9703) as standard substance.
Experimental result shows in the fermented liquid of streptomyces lydicus (Streptomyces lydicus) the A02CGMCC No.1654 mutant strain that slnM that step 2 obtains is knocked and can't detect tennecetin, and the tennecetin output of streptomyces lydicus (Streptomyces lydicus) A02CGMCC No.1654 is 2.546g/L.Positive regulating gene slnM successfully is knocked in the streptomyces lydicus that the slnM that description of step two obtains is knocked (Streptomyces lydicus) the A02CGMCC No.1654 mutant strain, so that the transgenation strain does not have the ability of synthetic tennecetin, this is consistent with the forfeiture that causes the tennecetin synthesis capability that knocks out of approach specificity positive regulating gene pinM gene (homologous gene of Streptomyces lydicus A02 slnM gene) in the Natal streptomycete of Ant ó n et al.2007 report, this experimental result shows that further dextranase is as the negative selection markers of gene knockout, fast, easy, save time, laborsaving, can be widely used in streptomycete and other gene knockout mutant strain seed selection work.
Wherein, in the Streptomyces lydicus A02 CGMCC No.1654 fermented liquid tennecetin slightly carry with authentication method as follows:
One) slightly the carrying of tennecetin in the fermented liquid
The fermented liquid of above-mentioned streptomyces lydicus is used respectively the dehydrated alcohol pre-treatment of 3 times of volumes, 4 ℃ leave standstill 2h, with precipitation thalline, solid particles, solubility sticking jelly, nucleic acid and heteroproteins and mesostate etc., supernatant liquor with 2 metafiltration paper with the Büchner funnel vacuum filtration, filtrate through Rotary Evaporators at 45 ℃ of lower concentrating under reduced pressure, concentrated solution is the tennecetin crude extract, and 4 ℃ save backup.
Two), the separation and purification of tennecetin crude extract
Carry out progressively separation and purification by macroporous resin column chromatography, silica gel column chromatography and high performance liquid chromatograph HPLC.
1, macroporous resin adsorption column chromatography
Select 40cm * 2.6cm glass chromatography column, X-5 macroporous resin (Tianjin Nankai university chemical plant).Macroporous resin is mixed well with appropriate amount of deionized water after by producer explanation pre-treatment, slowly adds and is equipped with in the chromatography column of 1/3 volumes of deionized water, emits distilled water from the column bottom with uniform speed slow simultaneously, makes that liquid level remains at above the resin layer in the post.Be filled to approximately 3/4 post height, natural subsidence 6～10h, making the dress column volume after the balance is 150ml.
Get tennecetin crude extract and resin and carry out dynamic adsorption by the volume ratio of 1 ﹕ 1.Elution process is: the deionized water wash-out remove portion pigment of 2 times of column volumes and a large amount of water-soluble impurity; The 30%(volumn concentration of 2 times of column volumes) the methanol-eluted fractions element that discolors; Use at last the 70%(volumn concentration of 2 times of column volumes) the ethanol elution activeconstituents.Be in charge of the collection elutriant, every pipe 15ml, the filter paper method is carried out determination of activity.
The result shows that active eluant concentrates on the 48th～56 pipe (i.e. elutriant between 4.8 times of column volume to 5.6 times column volumes).With active eluant, supply next step separation behind 45 ℃ of lower concentrating under reduced pressure.
2, silica gel adsorption column chromatography
Select 40cm * 2.6cm glass chromatography column, 100 orders～200 order silica gel, the mixed solution take volume ratio as ethanol, ammoniacal liquor and the water of 8 ﹕, 1 ﹕ 1 is as elutriant; Get approximately that 150g silica gel soaks 3h with deionized water, the fine particle that inclines, the Büchner funnel vacuum filtration is removed moisture; Soak 12h with 6mol/L HCl again, then be washed till neutrality with deionized water, vacuum is drained; Spend the night with soaked in absolute ethyl alcohol, vacuum is drained; Under 120 ° of C, activate 2h before use, be dried to constant weight; Then pack in the chromatography column elutriant of 1/3 column volume slowly adds the silica gel that mixes with elutriant, stops when approximately high to 3/4 post adding, and leaves standstill 6～10h, makes the slow sedimentation of silica gel.Then with the elutriant of 2～3 times of volumes flow velocity flushing cylinder with 1mL/min, make it balance.Column volume after the balance is 150ml.The active wash-out concentrated solution of the macroporous resin 10ml upper prop of getting step 1 carries out dynamic adsorption, carries out wash-out with the elutriant of 2～3 times of column volumes by the flow velocity of 0.5ml/min, is in charge of the collection elutriant with automatic Fraction Collector, every pipe 5ml.Take S. cervisiae ACCC20036 as indicator, utilize the filter paper agar diffusion method to detect every pipe elutriant active.The result shows: the active ingredient in its elutriant concentrates on the 7th～36 pipe (i.e. elutriant between 0.23 times of column volume to 1.2 times column volume).With active eluant, supply next step separation behind 45 ℃ of lower concentrating under reduced pressure.
3, preparation HPLC separation and purification
Adopt LC-9101 type circulation preparative HPLC, JAIGEL-ODS-AP type SP-120-15 preparative column.
Get the active wash-out concentrated solution behind the silica gel column chromatography of step 2, filter automatic sampler sample introduction, each sample size 6ml with 0.45 μ m millipore filter; Take methyl alcohol: water (volume ratio is as 7:3) separates as moving phase, utilizes the UV detector to detect at wavelength 305nm place and automatically forms separating spectrum; Utilize run tank to collect respectively each corresponding elutriant in curve peak in the collection of illustrative plates; Take S. cervisiae ACCC20036 as indicator, utilize the filter paper agar diffusion method to detect every pipe elutriant active.After carrying out separating the first time with the flow rate pump of 2ml/min, the flow rate pump with 3ml/min separates 2 times in succession again.
Experimental result shows, HPLC separates and detects altogether 30 peaks for the first time, and wherein retention time is that the strong absorption peak of 57.866min is Peak Activity (Fig. 6 A), and its relative peak area is 35.121%; To its separation detection to the 6 peak second time that carries out, wherein retention time is that the peak of 41.699min is Peak Activity, and its relative peak area is 97.020%; Separation detection is that the peak of 39.766min is Peak Activity (Fig. 6 B) to retention time for the third time, is 99.845% by its purity of calculated by peak area.
Carry out vacuum concentration with separating for the third time the Peak Activity sample that obtains, be white in color after the drying or cream-colored powder, this sample is the tennecetin sample.Utilize Shimadzu analysis mode HPLC to adopt following method that it is carried out purity checking: the sample that takes a morsel is dissolved in 70% methanol aqueous solution, carries out gradient elution take methyl alcohol (A) and water (B) as moving phase.Chromatographic condition is: C
18Reversed-phase column, 30 ℃ of column temperatures, the UV detector detects wavelength 305nm, and SIL-10ADVP automatic sampler sample introduction 10 μ l are with the flow velocity wash-out 60min of 1ml/min.The gradient elution step is as follows:
The result shows that elution curve is single peak, illustrates that it is one-component, and purity reaches the requirement of determination of chemical structure.
Four, the parsing of purification of samples chemical structure is identified
1, ultra-violet absorption spectrum (UV)
The tennecetin sample denier of above-mentioned steps three purifying is dissolved in the ultrapure water, use the UV of Hitachi-the VIS3010 ultraviolet-visible pectrophotometer carries out full wavelength scanner take ultrapure water as blank in 190nm～400nm wavelength region, automatically form ultraviolet absorpting spectrum.
By the UV scanning collection of illustrative plates as seen, the tennecetin sample shows typical tetraenes microbiotic spectral pattern, near the absorption peak that typical conjugation tetraene chromophoric group is namely all arranged wavelength 281nm, 291nm, 305nm and 319nm, its medium wavelength 305nm place absorption value is maximum, 281nm place absorption value minimum (Fig. 7) illustrates that this material belongs to the tetraene microbiotic in the polyenoid class.
2, infrared absorption spectrum (IR)
Adopt the KBr pressed disc method, carry out 400cm with German BRUKER company's T ENSOR27 Fourier infrared spectrograph
-1-4000cm
-1The zone interscan.
The infrared spectra of tennecetin sample as shown in Figure 8, ν wherein
Max3416.78cm
-1Charateristic avsorption band for-OH; ν
Max3288.23cm
-1Stretching vibration charateristic avsorption band for N-H; ν
Max2940.44 and 2980.27cm
-1Be-CH
3Charateristic avsorption band; ν
Max3017.23cm
-1Be-CH
2Charateristic avsorption band; ν
Max1715.38cm
-1Show the strong absorption peak of typical carbonyl; ν
Max1571.44cm
-1The strong absorption peak of performance-C=C-; ν
Max1634.40cm
-1The weak absorption peak of performance-C=C-.
3, high resolution mass spectrum
Adopt the German BRUKER ultrahigh resolution 9.4T of company mixed type level Four bar fourier tandom mass spectrometer (9.4T Q-FT-MS); Condition: capillary4000, Dry Gas:4.0l/s, the source temperature: 180 ° of C, scan range:300～2000, syringe pump:1.5ml/min, data analysis software is Bruker Daltonics DataAnalysis3.4.
The result shows, in the collection of illustrative plates that the tennecetin sample is analyzed, adopts the positive ion detection mode to detect adduct ion [M+Na]
+Be m/z688.2937(Fig. 9 B); Adopt the negative ion detection mode to detect quasi-molecular ion [M-H]
+Compound (Fig. 9 A) for m/z664.2975; Adopt the positive and negative ion detection mode that the tennecetin sample is detected analysis, determine that 664.2975 are molecular ion peak.
In sum, the molecular formula of the main active component of tennecetin sample is C
33H
47NO
13, molecular weight is 665; By formula: degree of unsaturation (n)=1+Nc+(Nn-Nh)/2(Nc: carbonatoms; Nn: nitrogen-atoms number; Nh: the degree of unsaturation of number of hydrogen atoms) calculating its molecular formula is 11, shows and contains a plurality of unsaturated link(age)s and ring etc. in its molecular structure.
4, nuclear magnetic resonance spectrum (NMR)
Take deuterium generation-dimethyl formamide (d-DMF) as solvent, take tetramethylsilane (TMS) as interior mark, measure under the room temperature.
Adopt Bruker AVANCE DRX-500 nuclear magnetic resonance spectrometer (German Bruker spectral instrument company), take deuterium generation-dimethyl formamide (d-DMF) as solvent, tetramethylsilane (TMS) is interior mark, carry out hydrogen compose (
1HNMR) and carbon spectrum (
13CNMR) mensuration; The former resonant frequency is 500.1325156MH
Z, sampling number 32768 times; Latter's resonant frequency 125.7577612MH
Z, sampling number is 65536 times.
Experimental result shows, the nucleus magnetic resonance of tennecetin sample
13Can find out in the C collection of illustrative plates (Figure 10 A) that the chemical shift (δ 165.217) of a carboxyl carbon atom is arranged in the molecule; The chemical shift of a carbonylic carbon atom (δ 178.603); The chemical shift of one group of conjugation tetraene carbon atom (δ 125.089～145.447); The chemical shift of one group of carbon atom that links to each other with hydroxyl (δ 66.102～70.326); Sugar ring upper five carbon atom resonance peaks (δ 71.194～97.894); Methine carbon atom resonance peak (δ 18.062, and δ 20.360).The nucleus magnetic resonance of 500 megahertzes
1Can find out in the H collection of illustrative plates (Figure 10 B), ((δ 5.686～6.625ppm) in chemical shift CH=CH-) for the proton hydrogen that links to each other with four two keys on the polyenoid ring; (δ 4.187～4.741ppm) for the chemical displacement value of the proton hydrogen in five hydroxyls; (δ 1.274～2.439ppm) in the chemical shift of the proton hydrogen in five methylene radical and two methyl.
The above-mentioned experimental data of analysis-by-synthesis, the main active component of tennecetin sample are tennecetin, and its chemical structural formula is:
Comparative Examples, the glucanase gene under the erythromycin promoters driven is inserted the ClaI of pKC1139 and the recombinant vectors that the KpnI site obtains is not expressed glucanase gene
With erythromycin promoter sequence upstream primer p3(
CCATCGATCTAGTATGCATGCGAGTG) under (the line part is restriction enzyme ClaI site and protection base) and the glucanase gene promotor p4(is arranged
GGGTACCGGATCAGAACCCAATTAG) (the line part is restriction enzyme KpnI site and protection base) is primer; take the streptomyces expression vector pIB139-glu that carries erythromycin promotor (ermE*) as template; amplification obtains the fragment ermE-glu of erythromycin promotor and glucanase gene sequence; this fragment is inserted between the ClaI and KpnI site of pKC1139 after ClaI and KpnI enzyme are cut, obtained the recombinant expression vector pKCG1139-2 of erythromycin promotor and glucanase gene insertion pKC1139.
By the pKCG of heat shock method with embodiment 1
+1139 and pKCG1139-2 transform respectively bacillus coli DH 5 alpha and through 100 μ g/ml apramycin resistance screenings, obtain carrying carrier pKCG
+1139 bacillus coli DH 5 alpha transformant (called after DH5 α-pKCG
+1139) and carry bacillus coli DH 5 alpha transformant (the called after DH5 α-pKCG1139-2) of pKCG1139-2.With DH5 α-pKCG
+1139 and DH5 α-pKCG1139-2 be inoculated in respectively on the Mierocrystalline cellulose culture medium flat plate of embodiment 3 and cultivated 2 days at 29 ℃, carry out congo red staining, the result shows that DH5 α-pKCG1139-2 does not produce transparent circle, degraded cellulose does not have dextranase activity; DH5 α-pKCG
+1139 produce transparent circle, have dextranase activity.The recombinant vectors pKCG that ClaI site single endonuclease digestion obtains is described
+1139 express glucanase gene, and the recombinant vectors pKCG1139-2 that ClaI and KpnI site obtain does not express glucanase gene (Figure 11), show that ClaI and KpnI double digestion may cut away the essential site on the pKC1139 carrier, cause that dextranase can not express.
Claims (10)
1. making up the method for recombinant vectors, is the Cla I site of glucanase gene being inserted pKC1139, and the recombinant DNA that obtains is described recombinant vectors.
2. method according to claim 1 is characterized in that: described glucanase gene encode following a) or b) protein:
A) protein of aminoacid sequence shown in SEQ ID No.1;
B) with replacement and/or disappearance and/or the interpolation of one or several amino-acid residue among the SEQ ID No.1 and have the degraded cellulose activity by a) derivative protein.
3. method according to claim 1 and 2 is characterized in that: the 208-1707 position of the encoding sequence of described glucanase gene such as SEQID No.2.
4. method according to claim 1 and 2, it is characterized in that: the nucleotide sequence of described glucanase gene is the 9-1707 position of SEQID No.2.
5. the recombinant vectors that is made up by arbitrary described method among the claim 1-4.
6. the recombinant microorganism that contains the described recombinant vectors of claim 5.
7. the application of recombinant vectors claimed in claim 5 in knocking out the streptomyces gene that does not produce dextranase.
8. application according to claim 7 is characterized in that: the described streptomycete that does not produce dextranase is streptomyces lydicus, streptomyces chatanoogensis, Natal streptomycete or brown yellow spore streptomycete.
9. streptomycete goal gene knockout carrier is to insert the recombinant DNA that obtains for the dna fragmentation that knocks out the streptomycete goal gene in the multiple clone site of recombinant vectors claimed in claim 5.
10. method for preparing the streptomycete mutant strain that goal gene is knocked, comprising the steps: streptomycete goal gene knockout carrier claimed in claim 9 imported waits to knock out in the streptomycete of described goal gene, obtain the transformant of thiostrepton resistance by the thiostrepton resistance screening, screen the not transformant of degraded cellulose from the transformant of described thiostrepton resistance, the transformant of the not degraded cellulose that obtains is the streptomycete mutant strain that candidate's goal gene is knocked; The described streptomycete of streptomycete for not producing dextranase of waiting to knock out described goal gene.
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| WO2017121902A1 (en) * | 2016-01-15 | 2017-07-20 | Cic Nanogune - Asociación Centro De Investigación Cooperativa En Nanociencias | Endocellulases and uses thereof |
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| WO2017121902A1 (en) * | 2016-01-15 | 2017-07-20 | Cic Nanogune - Asociación Centro De Investigación Cooperativa En Nanociencias | Endocellulases and uses thereof |
| US11242514B2 (en) | 2016-01-15 | 2022-02-08 | Asociación Centro De Investigación Cooperativa En Nanociencias “Cic Nanogune” | Endocellulases and uses thereof |
| CN106434731A (en) * | 2016-09-20 | 2017-02-22 | 云南大学 | Method for increasing yield of elaiophylin and derivatives by knocking out almRIII gene of streptomyces autolyticus wild strain and conducting fermenting |
| CN106434731B (en) * | 2016-09-20 | 2019-05-28 | 云南大学 | Knock out the almRIII gene of autolytic streptin wild-type strain, the method that fermentation improves elaiophylin and derivative yield |
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