CN102517300B - DNA (deoxyribonucleic acid) molecule, recombinant plasmids and escherichia coli - Google Patents
DNA (deoxyribonucleic acid) molecule, recombinant plasmids and escherichia coli Download PDFInfo
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- CN102517300B CN102517300B CN201110460217.7A CN201110460217A CN102517300B CN 102517300 B CN102517300 B CN 102517300B CN 201110460217 A CN201110460217 A CN 201110460217A CN 102517300 B CN102517300 B CN 102517300B
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Abstract
The invention relates to the field of biotechnology, and discloses a DNA (deoxyribonucleic acid) molecule with a nucleotide sequence shown in SEQ ID NO: 1, a recombinant plasmid pKR-D and a recombinant plasmid pKTR-D. The recombinant plasmid pKR-D is prepared via inserting the DNA molecule between an Sma I cleavage site and an HindIII cleavage site of a plasmid pKK223-3, the recombinant plasmid pKTR-D is prepared via inserting a fragment between a BamHI cleavage site and an SphI cleavage site of a plasmid pKK223-3-ThrA'BC, and the fragment consists of a Tac promoter, the DNA molecule, an rrnB T1 Terminator and an rrnB Terminator. The recombinant plasmids are transferred into W3110 escherichia coli to obtain two strains with high L-threonine tolerance activity, and the strains can be applied to fermentation of L-threonine.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of DNA molecular and recombinant plasmid and intestinal bacteria.
Background technology
L-threonine is one of necessary 8 seed amino acids of human body, is widely used in medicine, food, feed etc., and L-threonine is mainly produced with the method for microorganism fermentation at present, and various bacteria can be used for L-threonine produces, as Corynebacterium glutamicum, intestinal bacteria etc.Because Escherichia coli fermentation production L-threonine has the advantage that breeding is rapid, leavening temperature is high, Physiology and biochemistry fundamental research is more deep, become gradually the conventional bacterial strain that L-threonine is produced.
Along with the continuous increase of the world to L-threonine demand, the research of L-threonine superior strain more and more comes into one's own.Wherein, a factor that affects the most critical of intestinal bacteria high yield is exactly the ability of its tolerance L-threonine, only has and removes the inhibition of L-threonine to pathways metabolism, could and then increase the output of L-threonine.Although the intestinal bacteria of wild-type have the gene of expressing L-threonine tolerance albumen, but the expressed protein content of this gene is lower, be only that intestinal bacteria normal growth is required, can not tolerate the L-threonine that is greater than 0.1M concentration, make the colibacillary acid producing ability of wild-type lower.
Therefore, utilize animal nutrition to transform wild-type e. coli, can there is higher L-threonine tolerance, the production tool of L-threonine is of great significance.
Summary of the invention
In view of this, the object of this invention is to provide a kind of DNA molecular and recombinant plasmid and intestinal bacteria, make described intestinal bacteria there is higher tolerance activity to L-threonine.
For achieving the above object, the invention provides following technical scheme:
A kind of DNA molecular, its nucleotide sequence is as shown in SEQ ID NO:1.
The present invention obtains the one section of protein sequence that comes from Yersinia-Yersinia enterocolitica subsp.enterocolitica 8081 bacterial strains from ncbi database, its aminoacid sequence is as shown in SEQ ID NO:4, NCBI albumen sequence number is Gi:123440599, this albumen concrete function not yet obtains open confirmation, there is and study it through applicant the activity that tolerates L-threonine, but express the gene source of this albumen in the larger Yersinia of same intestinal bacteria species variation, it can not this albumen of correction in intestinal bacteria.Therefore the present invention is according to the Preference of intestinal bacteria W3110 codon, optimizing codon, DNA molecular by nucleic acid synthesizer synthesizing ribonucleotide sequence as shown in SEQ ID NO:1, the exactness of this sequence of sequence verification, synthetic gene restriction enzyme site is SmaI/HindIII, and wherein optimization, synthetic work are completed by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
The present invention also provides a kind of recombinant plasmid pKR-D, between its SmaI and HindIII restriction enzyme site, inserted the DNA molecular of nucleotide sequence as shown in SEQ ID NO:1 obtains by plasmid pKK223-3, cut and verify and check order through PCR and enzyme, the DNA molecular that shows nucleotide sequence as shown in SEQ ID NO:1 successfully builds up in plasmid pKK223-3 between Sma I and HindIII restriction enzyme site, and its plasmid map is shown in Fig. 1.
The present invention also provides a kind of recombinant plasmid pKTR-D, and it is prepared by following methods:
Take recombinant plasmid pKR-D as template, the downstream primer amplification as shown in SEQ ID NO:3 of upstream primer with nucleotide sequence as shown in SEQ ID NO:2 and nucleotide sequence, BamHI/SphI double digestion amplified production, obtain and stop molecular fragment by DNA molecular, rrnB T1 Terminator terminator, the rrnB Terminator of nucleotide sequence shown in Tac promotor, SEQ ID NO:1, then with through the plasmid pKK223-3-ThrA ' BC of BamHI/SphI double digestion be connected and get final product;
Wherein, described Tac promotor, rrnB T1 Terminator terminator are positioned at fragment two ends, the DNA molecular of nucleotide sequence shown in SEQ ID NO:1 is between Tac promotor, rrnBTerminator terminator, and rrnB Terminator terminator is between the DNA molecular and rrnB T1 Terminator terminator of nucleotide sequence shown in SEQ ID NO:1.
Cut and verify and check order through PCR and enzyme, show that described fragment successfully builds up in plasmid pKK223-3-ThrA ' BC between BamHI and SphI restriction enzyme site, its plasmid map is shown in Fig. 2.
Plasmid pKK223-3 and pKK223-3-ThrA ' BC are open in document " Kwang Ho Lee; Molec μ lar Systems Biology 3:149; 2007 ", and can be by commercially available acquisition, both are all strongly expressed plasmid, and the recombinant plasmid pKR-D take both as basic plasmid construction and pKTR-D contribute to the expression of DNA molecular of the present invention in intestinal bacteria.Wherein, pKR-D is with the Tac strong promoter of basic plasmid pKK223-3, can increase copy number and the expression amount of DNA molecular described in intestinal bacteria, and stop the expression of this gene by the original rrnB T1 Terminator terminator with strong termination structure of basic plasmid pKK223-3 and rrnB Terminator terminator, in case TAG terminator codon can not be expressed reading over of causing by terminator in described DNA molecular.
PKTR-D is take pKR-D as template, the above-mentioned described cloned dna molecule with Tac promotor, rrnB T1Terminator terminator and rrnB Terminator terminator, between BamHI in plasmid pKK223-3-ThrA ' BC and SphI restriction enzyme site, is had to above-mentioned advantage equally.In addition, pKTR-D is with rite-directed mutagenesis threonine operon-ThrA ' BC of basic plasmid pKK223-3-ThrA ' BC, the strong Tac promotor of this operon and introducing can strengthen the expression amount of described DNA molecular jointly, is conducive to the raising of improved colibacillary L-threonine tolerance.
The present invention is transformed into recombinant plasmid pKR-D in the intestinal bacteria W3110 competent cell of preparing through Calcium Chloride Method according to this area ordinary method, obtain a kind of escherichia coli that can tolerate L-threonine, called after MHZ-0211-1, cut and verify and check order through PCR and enzyme, show that described recombinant plasmid pKR-D successfully proceeds in intestinal bacteria W3110, and be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 29th, 2011, deposit number is CGMCC No:5665, Classification And Nomenclature is colon bacillus, Escherichia.coli.
The present invention is transformed into recombinant plasmid pKTR-D in the intestinal bacteria W3110 competent cell of preparing through Calcium Chloride Method according to this area ordinary method, obtain a kind of escherichia coli that can tolerate L-threonine, called after MHZ-0211-2, cut and verify and check order through PCR and enzyme, show that described recombinant plasmid pKR-D successfully proceeds in intestinal bacteria W3110, and be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 29th, 2011, deposit number is CGMCC No:5666, Classification And Nomenclature is colon bacillus, Escherichia.coli.
Described intestinal bacteria W3110 is purchased from US mode culture collection warehousing, and commodity article No. is
n μ mber:39936
tM.
Be that the intestinal bacteria (being designated hereinafter simply as MHZ-0211-1 and MHZ-0211-2) of CGMCC No:5665 and CGMCC No:5666 carry out L-threonine resistance test with the two strain intestinal bacteria W3110 (being designated hereinafter simply as MHZ-0200-1 and MHZ-0200-2) that proceed to respectively pKK223-3 and pKK223-3-ThrA ' BC by deposit number, result shows, MHZ-0211-1 and MHZ-0211-2 two bacterial strains all can tolerate the L-threonine of 0.4M, and MHZ-0200-1 and MHZ-0200-2 two bacterial strains all do not tolerate within the scope of 0.1-0.5M, show the L-threonine of escherichia coli bacterium shell tolerance higher concentration of the present invention.
In addition, MHZ-0211-1, MHZ-0211-2, MHZ-0200-1 and MHZ-0200-2 are carried out respectively to L-threonine fermentation test under equivalent environment, result shows, MHZ-0211-1 and MHZ-0211-2 two bacterial strain L-threonine output are respectively 0.4g/L, 1.1g/L, and MHZ-0200-1 and MHZ-0200-2L-Threonine output are respectively 0,0.9g/L, escherichia coli L-threonine output of the present invention, all higher than control strain separately, shows that it can be applied in L-threonine fermentation.
From above technical scheme, the present invention is take the protein sequence of one section of Unknown Function as reference, optimize and synthesize one section of new DNA sequence dna, and be cloned in strongly expressed carrier pKK223-3 and pKK223-3-ThrA ' BC, be transformed into and in intestinal bacteria W3110, obtain two strains and there is high L-threonine and tolerate active escherichia coli, can be applied in the fermentation of L-threonine.
The explanation of biomaterial preservation information:
1, MHZ-0211-1 intestinal bacteria, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 29th, 2011, deposit number is CGMCC No:5665, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, Classification And Nomenclature is colon bacillus, Escherichia.coli.
2, MHZ-0211-2 intestinal bacteria, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 29th, 2011, deposit number is CGMCC No:5666, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, Classification And Nomenclature is colon bacillus, Escherichia.coli.
Accompanying drawing explanation:
Fig. 1 shows recombinant plasmid pKR-D plasmid map of the present invention;
Fig. 2 shows recombinant plasmid pKTR-D plasmid map of the present invention;
Fig. 3 shows the electrophoresis evaluation figure of recombinant plasmid pKR-D of the present invention;
Wherein, the rightmost side is Marker, under upper, is followed successively by 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp; Left side band is double digestion product band, is followed successively by from top to bottom 4296bp, 893bp;
Fig. 4 shows the electrophoresis evaluation figure of recombinant plasmid pKTR-D of the present invention;
Wherein, the rightmost side is Marker, under upper, is followed successively by 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp; Left side band is single endonuclease digestion product band, is followed successively by from top to bottom 5201bp, 4824bp.
Embodiment:
The invention discloses a kind of DNA molecular and recombinant plasmid and intestinal bacteria, those skilled in the art can use for reference content herein, suitably improve processing parameter and realize.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, they are all deemed to be included in the present invention.DNA molecular of the present invention, recombinant plasmid, intestinal bacteria are described by preferred embodiment, related personnel obviously can change methods and applications as herein described in content of the present invention, spirit and scope or suitably change and combination not departing from, and realizes and apply the technology of the present invention.
Below in conjunction with embodiment, further set forth the present invention.Wherein, embodiment part is as undeclared, and the biotechnological means such as all molecular clonings, conversion is all with reference to " molecular cloning test guide " (work such as J. Pehanorm Brooker, Science Press publishes, the third edition).
Embodiment 1: the optimization of DNA molecular of the present invention and synthetic
The present invention obtains the one section of protein sequence that comes from Yersinia-Yersinia enterocolitica subsp.enterocolitica 8081 from ncbi database, its aminoacid sequence is as shown in SEQ ID NO:4, and NCBI albumen sequence number is Gi:123440599.The present invention is according to the Preference of intestinal bacteria W3110 codon, optimizing codon, DNA molecular by nucleic acid synthesizer synthesizing ribonucleotide sequence as shown in SEQ ID NO:1, the exactness of this sequence of sequence verification, synthetic gene restriction enzyme site is SmaI/HindIII.Take SmaI/HindIII as insertion point, be cloned into high copy number plasmid carrier pUC57 above, increase take bacillus coli DH 5 alpha as F-strain.
Wherein optimization, synthetic work are completed by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
Embodiment 2: construction recombination plasmid pKR-D
Extract pUC57 plasmid in above-mentioned bacillus coli DH 5 alpha, SmaI/HindIII double digestion, 50 μ l systems, T b μ ffer+BSA, enzyme is cut product and is carried out 1% agarose gel electrophoresis, treats that the suitable time takes off gel, cuts glue and reclaims goal gene fragment, get 3 μ l and mix with the same pKK223-3 plasmid 1 μ l with SmaI/HindIII double digestion making, the linked system that adds efficient ligase enzyme 4 μ l to make 8 μ l.16 ℃ of connections of spending the night, transform the intestinal bacteria Trans T1 competent cell of having made next day, and final concentration 100 μ g/ml penbritin LB plate screenings are chosen single bacterium colony after cultivation 20h, and upgrading grain, with using HindIII and the checking of BamHI double digestion, is shown in Fig. 3.Result shows, described DNA molecular is built up to pKK223-3, forms new recombinant plasmid pKR-D, and plasmid map is shown in Fig. 1.
As shown in Figure 3, plasmid transformation escherichia coli can propose plasmid, enzyme slitting band correct position, big or small correct, and sequencing result is consistent with described DNA molecular sequence, illustrates that this plasmid successfully constructs.
Embodiment 3: construction recombination plasmid pKTR-D
Extract the plasmid pKR-D in intestinal bacteria in embodiment 2 and do template, with primer shown in SEQ ID NO:2 in sequence table and SEQ ID NO:3, amplifying target genes fragment, this fragment is with one section on pKTR-D Tac promoter sequence that comes from basic plasmid pKK223-3 and rrnB T1 Terminator, rrnB Terminator terminator sequence.BamHI/SphI enzyme is cut amplified production, gets 3 μ l and mixes with same pKK223-3-thrA ' the BC plasmid 1 μ l with BamHI/SphI double digestion making, the linked system that adds efficient ligase enzyme 4 μ l to make 8 μ l.16 ℃ of connections of spending the night, transform the intestinal bacteria Trans T1 competent cell of having made next day, and final concentration 100 μ g/ml penbritin LB plate screenings are chosen single bacterium colony after cultivation 20h, and upgrading grain HindIII enzyme is cut checking, sees Fig. 4.Result shows, described fragment is built up to pKK223-3-thrA ' BC, forms new recombinant plasmid pKTR-D, and plasmid map is shown in Fig. 2.
As shown in Figure 4, plasmid transformation escherichia coli can propose plasmid, enzyme slitting band correct position, big or small correct, and sequencing result is consistent with described DNA molecular sequence, illustrates that this plasmid successfully constructs.
Embodiment 4: transform recombinant plasmid pKR-D to intestinal bacteria W3110
Calcium Chloride Method is prepared intestinal bacteria W3110 competent cell, in intestinal bacteria Trans T1 competent cell from embodiment 2, extract plasmid pKR-D, transform W3110 bacterial strain, penbritin concentration 100 μ g/ml concentration plate screenings, obtain MHZ-0210-1 intestinal bacteria.
Cut and verify and check order through PCR and enzyme, show that described recombinant plasmid pKR-C successfully proceeds in intestinal bacteria W3110, and be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 29th, 2011, deposit number is CGMCC No:5665, Classification And Nomenclature is colon bacillus, Escherichia.coli.
Embodiment 5: transform recombinant plasmid pKTR-D to intestinal bacteria W3110
Calcium Chloride Method is prepared intestinal bacteria W3110 competent cell, in intestinal bacteria Trans T1 competent cell from embodiment 3, extract plasmid pKTR-D, transform W3110 bacterial strain, penbritin concentration 100 μ g/ml concentration plate screenings, obtain MHZ-0210-2 intestinal bacteria.
Cut and verify and check order through PCR and enzyme, show that described recombinant plasmid pKTR-D successfully proceeds in intestinal bacteria W3110, and be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 29th, 2011, deposit number is CGMCC No:5666, Classification And Nomenclature is colon bacillus, Escherichia.coli.
Embodiment 6:L-Threonine resistance test
With reference to the method for embodiment 4 or embodiment 5, plasmid pKK223-3 and plasmid pKK223-3-thrA ' BC are transformed into respectively in intestinal bacteria W3110, obtain MHZ-0200-1 and MHZ-0200-2 intestinal bacteria.
According to the M9 minimum medium of " molecular cloning test guide " preparation 1.5% agar, 115 ℃ of sterilizing 20min, be mixed with respectively containing 0, the substratum of the L-threonine of 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, to be cooled to 60 ℃ of left and right, add 100mg/ml penbritin to make final concentration 100 μ g/ml, the IPTG 0.1 μ l/ml of 1M, is down flat plate.Strain culturing is spent the night, and dilutes 10000 times and gets 100 μ l spread plates, and every 12h observes upgrowth situation, cultivates 72h colony growth situation and the results are shown in Table 1.
The each bacterial classification L-threonine of table 1 resistance test result
Note: ND represents not detect ,-represent not tolerate (not growing) ,+tolerance (growth) represented
As shown in Table 1, escherichia coli MHZ-0211-1 of the present invention and MHZ-0211-2 all can tolerate the L-threonine of 0.4M, but two kinds of bacterial strains of MHZ-0200-1 and MHZ-0200-2 cannot tolerate the L-threonine that exceedes 0.1M, the growth of bacterium colony also cannot definitely be detected in 0.
The test of embodiment 7:L-Threonine Fermentation
MHZ-0211-1, MHZ-0211-2, MHZ-0200-1 and MHZ-0200-2 are used to the test of fermenting of 200ml triangle shaking flask.Seed culture medium is as shown in table 2, and preparation process adopts revolution shaking table, rotating speed 220rpm, and 37 ℃ of culture temperature, liquid amount 25ml/200ml triangular flask adds the penbritin of final concentration 100 μ g/ml simultaneously.Fermention medium is as shown in table 3, and fermenting process adopts reciprocal shaking table, liquid amount 20ml/500ml triangular flask.In 37 ℃, 150rpm shaking culture, fermentation time 72h, inoculum size 10%.After fermentation ends by centrifugal fermented liquid removal thalline, dilution suitable multiple after in Fermentation Liquor by High Performance Liquid Chromatography L-threonine content, the results are shown in Table 4.
Table 2 seed culture medium
Table 3 fermention medium
The each bacterial classification L-threonine of table 4 output
| Bacterial strain (contained plasmid) | L-threonine output (g/L) |
| MHZ-0200-1(pKK223-3) | 0 |
| MHZ-0211-1(pKR-C) | 0.4 |
| MHZ-0200-2(pKK223-3-thrA’BC) | 0.9 |
| MHZ-0211-2(pKTR-C) | 1.1 |
As shown in Table 4, MHZ-0211-1 and MHZ-0211-2 two bacterial strain L-threonine output are respectively 0.4g/L, 1.1g/L, and MHZ-0200-1 and MHZ-0200-2L-Threonine output are respectively 0,0.9g/L, escherichia coli L-threonine output of the present invention, all higher than control strain separately, shows that it can be applied in L-threonine fermentation.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (6)
1. a DNA molecular, its nucleotide sequence is as shown in SEQ ID NO:1.
2. a recombinant plasmid pKR-D, is characterized in that, is inserted the DNA molecular of nucleotide sequence as shown in SEQ ID NO:1 obtain by plasmid pKK223-3 between its Sma I and Hind III restriction enzyme site.
3. intestinal bacteria, its deposit number is CGMCC No:5665, transforms intestinal bacteria W3110 obtain by recombinant plasmid pKR-D described in claim 2.
4. intestinal bacteria, its deposit number is CGMCC No:5666.
5. deposit number is the application in L-threonine fermentation of the intestinal bacteria of CGMCC No:5665.
6. deposit number is the application in L-threonine fermentation of the intestinal bacteria of CGMCC No:5666.
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