CN103320373A - Arthrobacter for overexpression of hypoxanthine phosphoribosyltransferase gene and construction method and application thereof - Google Patents
Arthrobacter for overexpression of hypoxanthine phosphoribosyltransferase gene and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses an arthrobacter for over-expressing hypoxanthine phosphoribosyl transferase gene, a construction method and application thereof, wherein a key enzyme hypoxanthine phosphoribosyl transferase gene (hgprt) in a purine salvage synthesis way is cloned, and escherichia coli-arthrobacter shuttle plasmid is utilized to construct recombinant expression plasmid. The recombinant plasmid is introduced into arthrobacter by an electrotransformation method, transformants are screened by kanamycin resistance, and genetic engineering bacteria are verified by plasmid PCR, so that the recombinant arthrobacter AR-HG is constructed. The recombinant arthrobacter AR-HG constructed by the invention has the advantages that the yield is increased by 16.6 percent compared with that of the original strain, the cAMP synthesis capacity of unit bacteria is increased by 27.0 percent, and the recombinant arthrobacter AR-HG can be directly used for industrial production of cAMP, the yield is increased, and the cost is reduced.
Description
Technical field
The invention belongs to genetically engineered and microbial fermentation technology field, be specifically related to Arthrobacter and construction process and application that a kind of mistake is expressed Hypoxanthine phospho-ribosyl transferase.
Background technology
Cyclic monophosphate (cyclic adenosine-3 ', 5 '-monophosphate, be called for short cAMP), it is a kind of small molecules with cell internal information transfer function, be called as the second messenger, extensively exist in vivo, the multiple physiological and biochemical procedures such as, protein synthesis synthetic to carbohydrate metabolism, metabolism of fat, nucleic acid and cytodifferentiation, canceration, reverse are being brought into play important regulating and controlling effect.Clinically, cAMP can be used for Cardiovarscular, hyperthyroidism, chronic renal insufficiency, nervous system disorders, liver and gall diseases and respiratory system disease etc.As animal feedstuff additive, cAMP had both had the effect of intending tethelin, can promote growth of animal, improved milk animal milk yield, livestock raised for meat rate of body weight gain and sheep wool production.As medicine intermediate, cAMP can derive a series of important derivatives, such as Meglumine Cyclic Adenylate and dibutyryl cyclic adenosine monophosphate, is used for the treatment of cardiovascular and cerebrovascular diseases and malignant tumour etc.CAMP has important using value and wide market outlook as a kind of typical fine chemicals, therefore is subject to extensive concern.
Chemical synthesis is that the main method of cAMP is produced in industrialization both at home and abroad at present, but has the major defects such as yield is low, environmental pollution is serious, production cost is high; And there are the problems such as enzyme stability is poor, substrate is expensive in enzyme process, and therefore, urgent need exploitation mild condition, cost cheapness, eco-friendly novel process substitute.
At present, the transformation for fermentative Production cAMP bacterial strain mainly is to screen substrate tolerance bacterial strain by the mode of traditional mutagenesis both at home and abroad, or imp dehydrogenase absence type bacterial strain, not yet has by Protocols in Molecular Biology transformation bacterial strain to improve cAMP output.Therefore make up the high yield strain excellent by genetic engineering technique, further improve cAMP output, have important economic worth and social effect.
Summary of the invention
The present invention puies forward technical problem to be solved and provides the Arthrobacter that expression Hypoxanthine phospho-ribosyl transferase (hgprt gene) crossed in a strain, expresses the output that the hgprt gene improves cAMP by crossing.
The technical problem that the present invention also will solve provides the construction process of above-mentioned Arthrobacter.
The technical problem that the present invention will solve at last provides the application of above-mentioned Arthrobacter.
The hgprt gene is the coding Hypoxanthine phospho-ribosyl transferase, and hypoxanthine phosphoribosyltransferase is the key enzyme of Purine salvage pathway.And Purine salvage pathway is the main path of producing cAMP, based on above theoretical analysis, crosses expression hgprt gene, can increase the metabolic flux of salvage route, is conducive to improve cAMP output, reduces cost.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
The Arthrobacter of expressing Hypoxanthine phospho-ribosyl transferase is crossed in one strain, it is the key enzyme Hypoxanthine phospho-ribosyl transferase hgprt that utilizes pcr amplification clone Purine salvage pathway, the said gene fragment is connected to over-express vector, then the method for utilizing electricity to transform transforms Arthrobacter, obtains the engineering bacteria of goal gene by the antibiotics resistance screening.
Wherein, described expression vector is the free plasmid carrier.Preferably, described expression vector is pARK.Constitutive promoter in the described expression vector is hndO.
Above-mentioned mistake is expressed the construction process of the Arthrobacter of Hypoxanthine phospho-ribosyl transferase, and the method comprises the steps:
(1) design PCR primer amplification Hypoxanthine phospho-ribosyl transferase hgprt;
(2) goal gene that the clone is obtained inserts the constitutive promoter hndO downstream multiple clone site of pARK, obtains pARK-HG and crosses expression plasmid;
(3) then pARK-HG is crossed expression plasmid and import in the escherichia coli DH5a, transform in order to electricity after the amplification;
(4) pARK-HG after extracting, concentrating is crossed the expression plasmid electricity and transform Arthrobacter, recovered 8 as a child for 30 ℃, coating kalamycin resistance flat board was cultivated 36 hours at 30 ℃ again, the screening transformant;
(5) transformant utilizes PCR to verify behind plasmid extraction, thereby obtains to express the Arthrobacter recombinant bacterium AR-HG of Hypoxanthine phospho-ribosyl transferase hgprt.
Above-mentioned mistake is expressed the application of Arthrobacter in preparation cAMP of Hypoxanthine phospho-ribosyl transferase.
Concrete preparation method comprises the steps:
(1) the single bacterium colony of Arthrobacter recombinant bacterium AR-HG is rule to the inclined-plane seed culture medium, cultivated 48-72 hour for 30 ℃;
(2) scrape a ring thalline and be inoculated in the 500mL shaking flask that the 50mL liquid seed culture medium is housed, 30 ℃, cultivated 24 hours the preparation seed liquor in the 200-250rpm shaking table;
(3) with the inoculum size of 10 (v/v) %, be inoculated in the fermention medium, under 25-35 ℃, pH7.0-7.2, ventilation 4-12L/min, mixing speed 250-350rpm condition, fermented 48-96 hour; Preferably under 30 ℃, pH7.0-7.2, ventilation 8L/min, mixing speed 350rpm condition, fermented 72 hours.
Wherein, described seed culture medium consists of: glucose 10g/L, peptone 10g/L, yeast extract paste 10g/L, extractum carnis 10g/L, pH7.2; Described inclined-plane seed culture medium is to add agar 2wt% in the seed culture medium.
Wherein, described fermention medium consists of: glucose 40g/L, K
2HPO
418g/L, KH
2PO
45g/L, MgSO
47H
2O0.1g/L, urea 10g/L, CoCl
20.005g/L, NaF0.4g/L, vitamin H 0.01g/L, xanthoglobulin 6g/L, pH7.0.
Beneficial effect: the construction process that the invention provides the genetic engineering bacterium that can improve cAMP output.The method comprises following process: the key enzyme Hypoxanthine phospho-ribosyl transferase hgprt of clone's Purine salvage pathway, utilize intestinal bacteria-Arthrobacter shuttle plasmid construction recombination plasmid (such as Fig. 1).Method by electricity transforms imports Arthrobacter with recombinant plasmid, by kalamycin resistance screening transformant, then by plasmid PCR genetic engineering bacterium is verified (such as Fig. 2), thereby has made up recombination nodule bacillus AR-HG.The recombination nodule bacillus AR-HG that the present invention makes up is than the output increased 16.6% of starting strain, and the thalline cAMP of unit synthesis capability improves 27.0%, can be directly used in the suitability for industrialized production of cAMP, improves the yield reducation cost.
Description of drawings
Fig. 1 crosses expression construction of recombinant vector flow process.
Fig. 2 AR-HG recombination nodule bacillus plasmid PCR checking (M:DL5000marker; 1: amplification pCGori replication origin; 2: amplification kan gene; 3: amplification hgprt gene).
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand, the described content of embodiment only is used for explanation the present invention, and should also can not limit the present invention described in detail in claims.
Embodiment 1: the structure of genetic engineering bacterium AR-HG.
Design primer PCR amplification hgprt.
The upstream and downstream primer is respectively:
Upstream primer: AA
CTGCAGThe gTTGGTGGATTCAAACGAC(underscore is the PstI restriction enzyme site);
Downstream primer: GC
GTCGACThe CTACTCGTAAACGTGCGG(underscore is the SalI restriction enzyme site).
PCR product and expression vector pARK are used respectively PstI and SalI double digestion, after the recovery, hgprt is connected the ratio of 3:1～5:1 connects 3 hours with the T4 ligase enzyme at 16 ℃ and make up recombinant expression vector pARK-HG(such as Fig. 1 with pARK).Then pARK-HG is crossed expression plasmid after extracting, concentrating, electricity transforms Arthrobacter, recovers 8 as a child for 30 ℃, and coating kalamycin resistance flat board was cultivated 36 hours at 30 ℃ again, the screening transformant.Transformant utilizes PCR to verify (such as Fig. 2) behind plasmid extraction, thereby obtains to express the Arthrobacter recombinant bacterium AR-HG of Hypoxanthine phospho-ribosyl transferase hgprt.
It is as follows that electricity transforms concrete steps:
The preparation of Arthrobacter competence:
(1) the dull and stereotyped activation of LB is chosen single bacterium colony to 5mL LB liquid nutrient medium, cultivates 24h for 30 ℃;
(2) switching 0.1% to 100mL LB liquid nutrient medium is cultivated approximately 18h for 30 ℃, to OD
600Near 0.6;
(3) adding penicillin G sodium salt to final concentration is 30 μ g/mL, continues to cultivate 1h;
(4) 4000 * g, 4 ℃ of centrifugal 10min collect thalline, with 30mL10% glycerine+0.5mol/L sorb alcohol wash;
(5) repeat to wash 3 times;
(6) glycerine+the 0.5mol/L sorbyl alcohol is resuspended with 1mL10%, is distributed into 10 pipes ,-80 ℃ of preservations.
The Arthrobacter electricity turns:
(1) concentrated with isopropanol precipitating behind the plasmid extraction, final concentration is to the 500ng/ μ L;
(2) get 2 μ L plasmids and add 100 μ L competent cells, mixing gently adds the 0.2mm electric shock cup of precooling;
(3) 2.5kV, 25 μ F, 400 Ω, electric shock;
(4) add immediately the 900 μ L LB substratum that contain the 0.5mol/L sorbyl alcohol, 30 ℃ of recovery 8h;
(5) that 150 μ g/mL of card-coating is dull and stereotyped after the dilution suitable multiple, and 36h can see obvious bacterium colony.
Embodiment 2: the fermentation of Arthrobacter recombinant bacterium AR-HG is used.
The single bacterium colony line of Arthrobacter recombinant bacterium AR-HG to the inclined-plane seed culture medium, was cultivated 48-72 hour for 30 ℃; Scrape a ring thalline and be inoculated in the 500mL shaking flask that the 50mL liquid seed culture medium is housed, 30 ℃, cultivated 24 hours the preparation seed liquor in the 200-250rpm shaking table; With the inoculum size of 10 (v/v) %, being inoculated into liquid amount is in the automatic controlled fermentation tank of 5L of 3L, 30 ℃ of cultivations in fermentation culture, and control pH7.0-7.2, ventilation 8L/min, mixing speed 350rpm fermented 72 hours.By the cAMP concentration in the HPLC detection fermented liquid.Recombination nodule bacillus AR-HG is 6.04g/L than the output increased 16.6% of starting strain; The thalline cAMP of unit synthesis capability improves 27.0%, is the every gram thalline of 0.606g cAMP/.
Claims (8)
1. the Arthrobacter of expressing Hypoxanthine phospho-ribosyl transferase is crossed in a strain, it is characterized in that, it is the key enzyme Hypoxanthine phospho-ribosyl transferase hgprt that utilizes pcr amplification clone Purine salvage pathway, the said gene fragment is connected to over-express vector, then the method for utilizing electricity to transform transforms Arthrobacter, obtains the engineering bacteria of goal gene by the antibiotics resistance screening.
2. mistake according to claim 1 is expressed the Arthrobacter of Hypoxanthine phospho-ribosyl transferase, it is characterized in that, described expression vector is the free plasmid carrier.
3. mistake according to claim 2 is expressed the Arthrobacter of Hypoxanthine phospho-ribosyl transferase, it is characterized in that, described expression vector is pARK.
4. mistake claimed in claim 1 is expressed the construction process of the Arthrobacter of Hypoxanthine phospho-ribosyl transferase, it is characterized in that, the method comprises the steps:
(1) design PCR primer amplification Hypoxanthine phospho-ribosyl transferase hgprt;
(2) goal gene that the clone is obtained inserts the constitutive promoter hndO downstream multiple clone site of pARK, obtains pARK-HG and crosses expression plasmid;
(3) then pARK-HG is crossed expression plasmid and import in the escherichia coli DH5a, transform in order to electricity after the amplification;
(4) pARK-HG after extracting, concentrating is crossed the expression plasmid electricity and transform Arthrobacter, recovered 8 as a child for 30 ℃, coating kalamycin resistance flat board was cultivated 36 hours at 30 ℃ again, the screening transformant;
(5) transformant utilizes PCR to verify behind plasmid extraction, thereby obtains to express the Arthrobacter recombinant bacterium AR-HG of Hypoxanthine phospho-ribosyl transferase hgprt.
5. mistake claimed in claim 1 is expressed the application of Arthrobacter in preparation cAMP of Hypoxanthine phospho-ribosyl transferase.
6. application according to claim 5 is characterized in that, the preparation method comprises the steps:
(1) the single bacterium colony of Arthrobacter recombinant bacterium AR-HG is rule to the inclined-plane seed culture medium, cultivated 48-72 hour for 30 ℃;
(2) scrape a ring thalline and be inoculated in the 500mL shaking flask that the 50mL liquid seed culture medium is housed, 30 ℃, cultivated 24 hours the preparation seed liquor in the 200-250rpm shaking table;
(3) with the inoculum size of 10 (v/v) %, be inoculated in the fermention medium, under 25-35 ℃, pH7.0-7.2, ventilation 4-12L/min, mixing speed 250-350rpm condition, fermented 48-96 hour.
7. application according to claim 6 is characterized in that, described seed culture medium consists of: glucose 10g/L, peptone 10g/L, yeast extract paste 10g/L, extractum carnis 10g/L, pH7.2; Described inclined-plane seed culture medium is to add agar 2wt% in the seed culture medium.
8. application according to claim 6 is characterized in that, described fermention medium consists of: glucose 40g/L, K
2HPO
418g/L, KH
2PO
45g/L, MgSO
47H
2O0.1g/L, urea 10g/L, CoCl
20.005g/L, NaF0.4g/L, vitamin H 0.01g/L, xanthoglobulin 6g/L, pH7.0.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104745616A (en) * | 2015-04-17 | 2015-07-01 | 南京工业大学 | Inosinic acid dehydrogenase gene-deficient arthrobacterium and construction method and application thereof |
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| CN104745616B (en) * | 2015-04-17 | 2018-10-23 | 南京工业大学 | Inosinic acid dehydrogenase gene-deficient arthrobacterium and construction method and application thereof |
| CN104962574A (en) * | 2015-06-10 | 2015-10-07 | 南京工业大学 | Arthrobacter expression plasmid and application |
| CN104962574B (en) * | 2015-06-10 | 2017-11-24 | 南京工业大学 | Arthrobacter expression plasmid and application |
| US11959116B2 (en) | 2021-10-27 | 2024-04-16 | Asahi Kasei Pharma Corporation | Method for producing nicotinamide mononucleotide |
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| CN103320373B (en) | 2015-09-23 |
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