CN1597974A - Method for producing L-threonine by Escherichia coli - Google Patents
Method for producing L-threonine by Escherichia coli Download PDFInfo
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Abstract
The invention discloses a method for producing L-threonine by Escherichia coli, adopting bacterial strain of Escherichia coli K12, and screening and obtaining isoleucine-deleted Escherichia coli strain, E.coli dIle by isogenous recombination and deletion of threonine deaminase gene; constructing three structural genes in the threonine manipulator in Escherichia coli strain VNIIgenetika M-1 (U.S.Pat.No.4321325) in high-expression prokaryotic plasmid, then transferring into E.coli dIle, and by streptomycin sulfate resistance screening, obtaining threonine-producing gene engineering bacterium E.coli THR6. The engineering bacterium can plentifully accumulate synthesized threonine, and increases the productivity.
Description
Technical field
The invention belongs to the microbiological genetic engineering field, not be transformed in the intestinal bacteria by the Threonine synthesis related gene of Threonine feedback inhibition, utilize the expression of the temperature sensitive promotor regulatory gene of intestinal bacteria, the L-Threonine can be synthesized in a large number intestinal bacteria.
Background technology
Threonine nineteen thirty-five separates from fibrinogen first and obtains, and so its similar threose is with its called after Threonine.Threonine is colourless to little yellow crystalline powder, and extremely weak specificity smell is arranged.Be soluble in very much formic acid, soluble in water, be insoluble in organic solvents such as ethanol and ether.The molecular weight of Threonine is 119.18, and 4 kinds of isomer are arranged, and wherein D-Threonine animal can not absorb (BakerDH etc., 1998); Naturally occurring is the L-Threonine.
Test shows, it is necessary that Threonine is that animal is kept growth.Threonine lacks, and can cause symptoms such as animal feed intake reduction, growth retardation, efficiency of feed utilization decline, immunity function inhibition.In most of vegetable protein feeds (especially cereals feed), Threonine is the second or the 3rd limiting amino acid (Eckert and Allce, 1974; Fuller, 1979; Allce and Hince, 1971).So in the vegetalitas low-protein diet, it is remarkable to add the Threonine effect, particularly replenished the daily ration of methionine(Met), Methionin, add Threonine more simultaneously and can obtain best effect.Additional Threonine can improve the effect of live pig food consumption, day weight gain, efficiency of feed utilization, reduction cost per gain weight.Be in the live pig low-protein daily ration of protein main source with rapeseed meal or soybean meal, Methionin is first limiting amino acid, and Threonine is second limiting amino acid.
Threonine is mainly used in fodder additives and medicine.Mainly by fermentative production, part is separated from protein hydrolystate as fodder additives L-Threonine.Produce bacterial strain by the L-Threonine that the nature induction mutation of bacterium is formed and brevibacterium flavum is arranged, Corynebacterium glutamicum and serratia marcescens.They are raw material with glucose all, and acid production rate is respectively 18.0g/L, 14.0g/L, 14.0g/L (agricultural biochemistry,, 37,653 in 1973).
In intestinal bacteria, Threonine is synthetic, and aspartic acid changes aspartylphosphate under E.C. 2.7.2.4. AKI catalysis from aspartic acid, through the last homoserine of producing of reduction.AKI is a multifunctional enzyme, and the metabolism from the aspartic acid to the homoserine is by AKI catalysis.AKI is by the thrA genes encoding.Homoserine is phosphorylation under homoserine kinase catalysis, and the phosphorylation homoserine is by the synthetic Threonine of threonine synthetase.Homoserine kinase is by the thrB genes encoding, and threonine synthetase is by the thrC genes encoding.ThrA, thrB and thrC belong to threonine operon, and enzyme amount of their codings and enzyme are lived and all be subjected to the feedback inhibition of Threonine and Isoleucine.
It is more clear that research is now regulated in the Threonine metabolism.In the Threonine metabolic process, the AKI of thrA coding plays a decisive role.The amount of the synthetic Threonine of general intestinal bacteria is limited, because Threonine suppresses the AKI enzymic activity, and regulates threonine operon by Threonine-tRNAs and transcribes.The Threonine analogue can be used for screening Threonine and produce bacterial classification, and in the resistant strain as the amino β hydroxypentanoic acid of α, the thrA gene is undergone mutation, and the AKI enzyme of mutator gene coding resists the Threonine feedback inhibition, thereby causes the accumulation of Threonine.Have in the intestinal bacteria of microbiotic borrelidin resistance, variation has taken place in the conformation of Threonine-tRNAs synthetic enzyme, greatly reduces the avidity with Threonine, and Threonine is accumulated in a large number.
Another principal element of a large amount of synthetic of restriction Threonine derives from Isoleucine.The synthetic of Isoleucine is raw material with the Threonine, if therefore its route of synthesis does not cut off, can only cause the accumulation of Isoleucine, and the content of Threonine is not high; On the other hand, Isoleucine has the intensive feedback inhibition to thrA gene transcription level, and the high-content Isoleucine is blocked the synthetic of AKI enzyme.
Summary of the invention
The objective of the invention is to seek a kind of genetic engineering bacterium of energy high yield Threonine.
Another object of the present invention discloses the production method of the genetic engineering bacterium of high yield Threonine.
The present invention is an original strain with e. coli k12 (available from the ATCC of American Type Culture Collecti).It is that phage P1 (available from the ATCC of American Type Culture Collecti) in the intestinal bacteria of carbon source infects e. coli k12 strain that utilization colonizes in sucrose, in the substratum that only contains sucrose, screen, picking is well-grown bacterial strain on sucrose medium, as the host bacterium of Threonine production.
The present invention flows to Isoleucine in order to block the Threonine product, by homologous recombination, and disappearance threonine deaminase gene.Utilize primer
Gctgactcgcaacccctgtccgtccggtgctccgg aaggtgccgaatatttaagagctcgcgcgtttcggtgatgacggtgaaaacc and gtcgtggcaatcgt
agcccagctctcattcagccgggtttcgaaatccggttcatggtcgccaagttcgaacgccgccagtcg
tggccttctgcttaatttgatgcctggcag
Amplification contains kalamycin resistance gene expresses unitary recombinant fragment.After will containing kalamycin resistance gene and expressing unitary recomposition unit pcr amplification, utilize electric shock instrument (Bio-rad Gene pulser) (U.S. BIO-RED company) electric shock transformed into escherichia coli, add the recomposition unit dna fragmentation that 2 μ g reclaim above in the 50 μ l intestinal bacteria.The conversion parameter is as follows: 25KV, 200 Ω, 25 μ F.The growth 1 hour on the rich medium of added with antibiotic not of electric shock back intestinal bacteria is coated in the LB substratum that contains 50 μ g/ml kantlex then.Cultivated 12 hours the bacterium colony that screening has resistance for 37 ℃.(contain Na in every liter of substratum at the M9 substratum that adds Isoleucine and do not add Isoleucine
2HPO
47H
2O12.8g; KH
2PO
43g; NaCl0.5g; NH
4Cl1g) the corresponding coli strain of differentiating the Isoleucine defective in.The bacterium line that will have kalamycin resistance, corresponding the drawing of same bacterium contained Isoleucine and do not contained on the M9 substratum of Isoleucine, 37 ℃ of growths are after 24-48 hour, the former bacterium colony that the latter do not grow of growing of picking, therefrom select a bacterium colony called after E.colidIle arbitrarily, be used for conversion and have threonine operon expression unit.
The present invention is building up to 3 structure genes in the threonine operon among the coli strain VNIIgenetika M-1 (U.S.Pat.No.4321325) in the protokaryon plasmid that efficiently expresses, and synthesize new TAC promotor according to cloning vector pGEX-6P (gene pool numbering U78874) (available from U.S. Promega company) design primer: design of primers is as follows.
Tac1:5 '-caactgcacttgttgacaattaatcatcggctcgtataatgtgtggatttgtgagc ggataac-3 ' and
tac2：5’-cctagtagcatgaattctgtttcctgtgtgaaattgttatccgctcacaaatccaca?c-3’。The pcr amplification condition is: 94 ℃, and 20 seconds; 60 ℃, 20 seconds; 72 ℃, 20 seconds, carry out 30 cyclic amplifications, increase back 72 ℃ and extended 5 minutes.Utilize PstI and ClaI (available from Japanese TAKARA company) enzyme to cut the back and insert pVic40 carrier (U.S.pat.No.4 synthetic TAC promotor, 321,325), make up intermediate carrier pTAC-thrA, after pVic40 carrier PstI and ClaI enzyme cut, reclaim enzyme and cut back DNA small segment, DNA small segment TagI part enzymolysis is connected with pTAC-thrA, insert intermediate carrier pTAC-thrA ClaI restriction enzyme site, make up the final expression vector pTAC-THR of Threonine.Utilize the electric shock conversion method expression vector pTAC-THR to be transformed among the bacterial strain E.colikdIle of Isoleucine deletion mutantion, screening resistance bacterium colony E.coli THR6 in the Vetstrep of 100mg/ml, the engineering strain E.coliTHR6 of Threonine is produced in acquisition.
The present invention utilizes Threonine production bacterial strain E.coli THR6 containing 0.2% glucose, growth is 48 hours in the 100mg/ml Vetstrep solid medium, 10ml 0.9% (weight percent) the sodium chloride solution wash-out and the bacterium that suspends add the 5000ml seed culture medium, and the bacterium amount in every liter is 10
8Culture medium prescription following (weight percent):
Syrup 1.0-4.0%
Ammonium sulfate 0.2-0.5%
Potassium primary phosphate 0.1-0.2%
7 Magnesium sulfate heptahydrate 0.02-0.05%
7 ferrous sulfate hydrate 0.001-0.002%
5 anhydrous manganese 0.001-0.002%
Yeast lysate 0.2-0.3%
Add water to 100ml
Substratum forwarded in the 5L fermentor tank cultivated 20 hours, culture condition is logical oxygen (0.5 liter/minute), and 37 ℃ of growth temperatures stir (1200 rev/mins), and the medium pH value is 6.9-7.2.
(ammoniacal liquor: syrup=2-3: 1) mixed solution is regulated pH value and feed supplement to utilize syrup ammoniacal liquor.Fermented 32 hours, the Threonine resultant quantity is 75g.
Beneficial effect of the present invention:
The present invention produces Threonine by genetically engineered and has the following advantages:
1. except having on the Threonine pathways metabolism a small amount of variation, other physiological characteristic is constant in the genetic engineering modified back of process for original strain.Therefore do not influence the fermentation of bacterial strain.
2. the threonine operon of this project bacterial strain employing is not subjected to the Threonine feedback inhibition, and therefore in the Threonine building-up process, bacterial growth and Threonine metabolism are unaffected.
3. engineering strain is the Isoleucine defective, and Isoleucine is synthetic to be precursor with the Threonine, and the Isoleucine defective can make the synthetic Threonine add up in a large number.
4. this project bacterium threonine operon is controlled by the protokaryon strong promoter.
5. engineering bacteria normal growth under the finite concentration microbiotic, plasmid wherein keep stable.
Description of drawings
Fig. 1 is that the Threonine synthetic vectors makes up.
Fig. 2 is the test of Threonine fermentation 5L jar.
Embodiment
The screening of embodiment 1 wide spectrum carbon source e. coli k12 strain
It is that phage P1 in the intestinal bacteria of carbon source infects e. coli k12 strain that utilization colonizes in sucrose, in the substratum that only contains sucrose, screen, picking is well-grown bacterial strain on sucrose medium, therefrom take out a coli strain and name and be E.coli-Sac, the host bacterium that produces as Threonine.
Threonine ammonialyase gene in the embodiment 2 homologous recombination disappearance intestinal bacteria E.coli-Sac Isoleucine synthetic system.
1. the structure of recomposition unit
Amplification kalamycin resistance gene NPTII from plasmid pBI121 (the biotech research center NCBI gene pool GenBank of national sanitary institute numbers AF485783), add BamHI and SacI site at its two ends, make up prokaryotic expression carrier pYPX251 (GenBank numbers AY178046) plasmid, utilize primer
Gctgactcgcaacccctgtccgtccggtgctccgg aaggtgccgaatatttaagagctcgcgcgtttcggtgatgacggtgaaaacc and gtcgtggcaatcgt
agcccagctctcattcagccgggtttcgaaatccggttcatggtcgccaagttcgaacgccgccagtcg
The tggccttctgcttaatttgatgcctggcag amplification contains kalamycin resistance gene expresses unitary recombinant fragment.The pcr amplification condition is: 94 ℃, and 30 seconds; 60 ℃, 1 minute; 72 ℃, 2 minutes, carry out 30 cyclic amplifications, increase back 72 ℃ and extended 10 minutes.
2. the screening of Isoleucine defective bacterial strain
1) electricity of preparation intestinal bacteria E.coli-Sac bacterial strain swashs competence.Method is as follows: utilize 100ml LB substratum (1% peptone, 0.5% yeast powder, 1% sodium-chlor) culturing bacterium spend the night to bacterial concentration be OD600=0.5-0.8.5000 revolutions per seconds of cultured bacteriums of centrifugal collection utilize the distilled water of sterilization to wash centrifugal thalline twice, and each 25ml washes once with 10% glycerine of sterilization again, utilizes 1ml 10% glycerine suspension thalline at last.
2) will contain kalamycin resistance gene and express unitary recomposition unit pcr amplification after, utilize 1% agarose gel electrophoresis, downcut electrophoretic band, pack in the dialysis tubing of forming by semi-permeable membranes, the water that adds capacity is got rid of air in the dialysis tubing clean, 100V electrophoresis 30 minutes, whole aqueous solution in the recovery dialysis tubing are to new centrifuge tube, the sodium-acetate of the 3M of the dehydrated alcohol of 2 times of volumes of adding and 1/10 volume in centrifuge tube, placed 1 hour for-20 ℃, 12000 rev/mins centrifugal 30 minutes, the precipitation of acquisition is the recomposition unit dna fragmentation, the distilled water that adds the sterilization of 20 microlitres in the centrifuge tube precipitation that suspends.Dna fragmentation concentration reaches every microlitre 1 microgram.
3) utilize electric shock instrument (Bio-rad Gene pulser) (U.S. BIO-RED company) electric shock transformed into escherichia coli, add the recomposition unit dna fragmentation that 2 μ g reclaim above in the 50 μ l intestinal bacteria.The conversion parameter is as follows: 25KV, 200 Ω, 25 μ F.
4) electric shock back intestinal bacteria growth 1 hour on the rich medium of added with antibiotic not is coated in the LB substratum that contains 50 μ g/ml kantlex then.Cultivated 12 hours the bacterium colony that screening has resistance for 37 ℃.
5) (contain Na in every liter of substratum at the M9 substratum that adds Isoleucine and do not add Isoleucine
2HPO
47H
2O12.8g; KH
2PO
43g; NaCl 0.5g; NH
4Cl1g) the corresponding coli strain of differentiating the Isoleucine defective in.With 4) in have the bacterium line of kalamycin resistance, corresponding the drawing of same bacterium contained Isoleucine and do not contained on the M9 substratum of Isoleucine, 37 ℃ of growths are after 24-48 hour, the former bacterium colony that the latter do not grow of growing of picking, therefrom select a bacterium colony called after E.colidIle arbitrarily, be used for conversion and have threonine operon expression unit.
The genetically engineered that embodiment 3 Threonines are produced bacterial strain makes up
1) the TAC promotor design primer according to cloning vector pGEX-6P (gene pool numbering U78874) (available from U.S. Promega company) synthesizes new TAC promotor: design of primers is as follows.
Tac1:5 '-caactgcacttgttgacaattaatcatcggctcgtataatgtgtggatttgtgagc ggataac-3 ' and
tac2：5’-cctagtagcatgaattctgtttcctgtgtgaaattgttatccgctcacaaatccaca?c-3’。The pcr amplification condition is: 94 ℃, and 20 seconds; 60 ℃, 20 seconds; 72 ℃, 20 seconds, carry out 30 cyclic amplifications, increase back 72 ℃ and extended 5 minutes.
2) synthetic TAC promotor is utilized PstI and ClaI (available from Japanese TAKARA company) enzyme cut the back and inserted pVic40 carrier (U.S.pat.No.4,321,325), make up intermediate carrier pTAC-thrA, after pVic40 carrier PstI and ClaI enzyme cut, reclaim enzyme and cut back DNA small segment, with DNA small segment TagI part enzymolysis, every micrograms of DNA fragment adds the TagI of 0.1-0.5 unit, 37 ℃ enzymolysis 30-60 minute, the DNA small segment that enzymolysis is good is connected with pTAC-thrA, and the DNA small segment is inserted intermediate carrier pTAC-thrA ClaI restriction enzyme site, makes up the final expression vector pTAC-THR of Threonine.
3) utilize electric shock conversion method (method is with embodiment 2), expression vector pTAC-THR is transformed among the bacterial strain E.colikdIle of Isoleucine deletion mutantion, screening resistance bacterium colony E.coli THR6 in the Vetstrep of 100 mg/ml.
Embodiment 4 Threonines are produced strain growth and fermentation (weight percent)
Threonine is produced bacterial strain E.coli THR6 and is being contained 0.2% glucose, growth is 48 hours in the 100mg/ml Vetstrep solid medium, 10ml 0.9% (weight percent) the sodium chloride solution wash-out and the bacterium that suspends add the 5000ml seed culture medium, and the bacterium amount in every liter is 10
8The following 1000ml substratum of culture medium prescription nutritive ingredient is as follows:
Syrup 30ml
Ammonium sulfate 4g
Potassium primary phosphate 1g
7 Magnesium sulfate heptahydrate 0.3g
7 ferrous sulfate hydrate 0.01g
5 anhydrous manganese 0.01g
Yeast lysate 2g
Substratum forwarded in the 5L fermentor tank cultivated 20 hours, culture condition is logical oxygen (0.5 liter/minute), and 37 ℃ of growth temperatures stir (1200 rev/mins), and the medium pH value is 6.9-7.2.
(ammoniacal liquor: syrup=2.92: 1) mixed solution is regulated pH value and feed supplement to utilize syrup ammoniacal liquor.Fermented 32 hours, the Threonine resultant quantity is 75g.
Claims (8)
1, a kind of Threonine product of blocking flows to Isoleucine disappearance threonine deaminase gene threonine operon and is not subjected to the Threonine feedback inhibition, and utilizing sucrose by protokaryon strong promoter control threonine operon is the coli strain of the production L-Soviet Union chloric acid of carbon source.
2, the coli strain of production according to claim 1 L-Threonine, the performance deficiencies threonine deaminase gene that it is characterized in that this bacterial strain has blocking-up Threonine product and flows to Isoleucine.
3, the coli strain of production L-Threonine according to claim 1 is characterized in that three structure genes in the threonine operon are that E.C. 2.7.2.4. AKI gene, Kos ammonia kinase gene and Threonine synthetic gene are not expressed by the Threonine feedback inhibition.
4, the preparation method of the coli strain of production as claimed in claim 1 L-Threonine, comprise by:
A, filter out the well-grown coli strain of sucrose medium;
B, the amplification of utilization design primer PCR obtain to be used for homologous recombination disappearance threonine deaminase gene;
The dna fragmentation of c, above-mentioned disappearance Threonine deaminase gene is by the electric shock transformed into escherichia coli;
D, 3 structure genes in the threonine operon in the intestinal bacteria bacterium bar are building up in the protokaryon plasmid that efficiently expresses, the synthetic TAC promotor of design, pcr amplification will synthesize the TAC promotor and be built into intermediate carrier PTAC-ThrA and be connected with DNA small segment after enzyme is cut, insert PTAC-ThrA ClaI restriction enzyme site and be built into the final expression vector PTA-THR of Threonine;
E, more above-mentioned PTAC-THR expression vector utilization electric shock conversion method is transformed in the coli strain of Isoleucine deletion mutantion, resistance screening obtains the Threonine genetic engineering bacterium.
5,, it is characterized in that designing primer and be according to the preparation method of the coli strain of the described production of claim 4 L-Threonine:
Gctgactcgcaacccctgtccgtccggtgctccgg aaggtgccgaatatttaagagctcgcgcgtttcggtgatgacggtgaaaacc and
gtcgtggcaatcgt
agcccagctctcattcagccgggtttcgaaatccggttcatggtcgccaagttcgaacgccgccagtcg
tggccttctgcttaatttgatgcctggcag
The pcr amplification homologous recombination obtains to be used to lack the dna fragmentation of Threonine deaminase gene.
6, according to the described preparation method who produces the coli strain of L-Threonine of claim 4, it is characterized in that making up Threonine synthetic plasmid method and be in the threonine operon three and be aspartic acid kinases AKI gene, homoserine kinase gene and threonine synthetase gene also are building up in the protokaryon plasmid that efficiently expresses, the synthetic TAC promotor of design, primer is:
Tac1:5 '-caactgcacttgttgacaattaatcatcggctcgtataatgtgtggatttgtgagc ggataac-3 ' and
tac2：5’-cctagtagcatgaattctgtttcctgtgtgaaattgttatccgctcacaaatccaca?c-3’
Utilize PstI and ClaI enzyme to cut the back synthetic TAC promotor and insert pvic40 vector construction intermediate carrier Ptac-ThrA and is connected with DNA small segment after enzyme is cut, insertion PTAC-ThrA ClaI restriction enzyme site is built into the final expression vector PTAC-THR of Threonine.
7, according to the described preparation method who produces the coli strain of L-Threonine of claim 4, it is characterized in that Threonine expression vector PTAC-THR utilizes the electric shock conversion method to be transformed in the coli strain of Isoleucine deletion mutantion, resistance screening obtains Threonine producer gene engineering bacteria E.coli THR6.
8, according to the described preparation method who produces the coli strain of L-Threonine of claim 4, it is characterized in that Threonine genetic engineering bacterium E.coli THR6 at substratum (weight percent) is:
Syrup 1.0-4.0%
Ammonium sulfate 0.2-0.5%
Potassium primary phosphate 0.1-0.2%
7 Magnesium sulfate heptahydrate 0.02-0.05%
7 ferrous sulfate hydrate 0.001-0.002%
5 anhydrous manganese 0.001-0.002%
Yeast lysate 0.2-0.3%
Add water 100ml
Middle fermentation culture, temperature is at 37 ℃, and PH is 6.9-7.2, extracts to obtain.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104845995A (en) * | 2014-02-14 | 2015-08-19 | 中国科学院微生物研究所 | Method for dynamic regulation and control of threonine efflux transport protein gene expression for production of L-threonine |
| CN106867951A (en) * | 2011-04-01 | 2017-06-20 | Cj 第制糖株式会社 | The method for preparing L amino acid with the bar bacterium of the fructokinase genetic transformation from Escherichia and using the bar bacterium |
| CN108473992A (en) * | 2015-10-23 | 2018-08-31 | Cj第制糖株式会社 | The method for producing the recombinant microorganism of L-threonine and producing L-threonine using it |
| CN111019878A (en) * | 2020-01-13 | 2020-04-17 | 江南大学 | Recombinant escherichia coli with improved L-threonine yield as well as construction method and application thereof |
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2003
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106867951A (en) * | 2011-04-01 | 2017-06-20 | Cj 第制糖株式会社 | The method for preparing L amino acid with the bar bacterium of the fructokinase genetic transformation from Escherichia and using the bar bacterium |
| CN104845995A (en) * | 2014-02-14 | 2015-08-19 | 中国科学院微生物研究所 | Method for dynamic regulation and control of threonine efflux transport protein gene expression for production of L-threonine |
| CN104845995B (en) * | 2014-02-14 | 2018-11-13 | 中国科学院微生物研究所 | A kind of method that dynamic regulation threonine arranges transporter gene Expression product L-threonine outside |
| CN108473992A (en) * | 2015-10-23 | 2018-08-31 | Cj第制糖株式会社 | The method for producing the recombinant microorganism of L-threonine and producing L-threonine using it |
| CN111019878A (en) * | 2020-01-13 | 2020-04-17 | 江南大学 | Recombinant escherichia coli with improved L-threonine yield as well as construction method and application thereof |
| CN111019878B (en) * | 2020-01-13 | 2021-04-16 | 江南大学 | Recombinant escherichia coli with improved L-threonine yield as well as construction method and application thereof |
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