CN101063105A - Engineering bacterium producing 5-glycyl ethylformic acid and construction and application method thereof - Google Patents
Engineering bacterium producing 5-glycyl ethylformic acid and construction and application method thereof Download PDFInfo
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- CN101063105A CN101063105A CN 200710068170 CN200710068170A CN101063105A CN 101063105 A CN101063105 A CN 101063105A CN 200710068170 CN200710068170 CN 200710068170 CN 200710068170 A CN200710068170 A CN 200710068170A CN 101063105 A CN101063105 A CN 101063105A
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Abstract
The invention discloses a method to prepare engineering bacteria of 5-glycyl propionic acid and methods for using them, which comprises the following steps: incorporating radial agrobacteriocin; setting the preserved number at CGMCC No. 1938; activating engineering bacteria with LB medium flat; getting monoclonal of the engineering bacteria; seeding to shake; culturing; getting first-grade seed; seeding the first-grade in shake; culturing; getting second-grade seed; seeding the second-grade seed in fermenter; culturing; cooling and evoking with isopropyl-beta-D-sulfo-galactose-glycoside; culturing continually; proceeding supplementing material culture. This engineering bacteria possesses higher ALA synthase expression, simple craft, low cost and good industrial prospect.
Description
Technical field
The present invention relates to produce the engineering bacteria of 5-amino-laevulic acid and make up and methods for using them.
Background technology
(5-aminolevulinic acid ALA) extensively is present in the organism 5-amino-laevulic acid, is synthetic protoheme, chlorophyll, vitamins B
12Common precursor Deng tetrapyrrole.(photodynamic agent PDT), has purposes widely in agrochemicals and medical field to ALA as a kind of photodynamics agent.At agriculture field, ALA has weeding, desinsection, increase stress resistance of plant and promotes multiple function such as plant-growth, and the noresidue of easily degrading, to people and animals' nontoxicity, become the nuisanceless green agrochemicals that have development prospect.At medical field, as photodynamics medicament of new generation its to have toxicity little, remarkable advantages such as selectivity height are used to treat multiple cancers such as skin carcinoma, bladder cancer, colorectal carcinoma and carcinoma of the pancreas, just progressively represent its huge applicability in photochemical treatment field.Based on the function of ALA and wide application prospect, its study on the synthesis has caused widely to be paid attention to.
The present more employing chemical synthesis of production of ALA, its complex steps, by product is many and productive rate is low; And the biological induced-mutation method is produced ALA, mainly is to optimize Rhodobacter sphaeroides mutant strain, and the production peak of the ALA that reports is 27mmol/L, but its culture cycle is long, and cost is higher.
Summary of the invention
An object of the present invention is to provide a kind of engineering bacteria and construction process thereof of the 5-of production amino-laevulic acid.
Another object of the present invention is that the engineering bacteria of using above-mentioned structure realizes that a kind of technology is simple relatively, output height, the method for environment amenable production 5-amino-laevulic acid.
The engineering bacteria of production 5-amino-laevulic acid of the present invention, it is the engineering bacteria that contains the 5-aminolevulinate synthetase gene of agrobacterium radiobacter, Chinese colon bacillus by name, the Latin formal name used at school is Escherichia coli Rosetta (DE3)-pET28a-A.R.hemA, in the common micro-organisms center preservation of China Committee for Culture Collection of Microorganisms of specified depositary institution of Patent Office of the People's Republic of China, deposit number is: CGMCCNo.1938.
Produce the construction process of the engineering bacteria of 5-amino-laevulic acid, its step is as follows:
1) be to obtain recon pET28a-A.R-hemA the engineering bacteria of CGMCC No.1332 from deposit number, the Chinese of said CGMCC No.1332 engineering bacteria is called colon bacillus, and the Latin formal name used at school is Escherichia coliBL21 (DE3)-pET28a-A.R-hemA;
2) recon pET28a-A.R-hemA is converted into the host bacterium Rosetta (DE3) (the commercialization bacterial strain is bought from German Novagen company), obtains engineering bacteria Rosetta (DE3)-pET28a-A.R.hemA, its deposit number is CGMCC No.1938.
Application deposit number of the present invention is the method that CGMCC No.1938 engineering bacteria is produced the 5-amino-laevulic acid, may further comprise the steps:
1) dips in from deposit number is the glycerine pipe of CGMCC No.1938 engineering bacteria Rosetta (DE3)-pET28a-A.R.hemA with inoculating needle and get bacterium liquid, after line on the LB culture medium flat plate that contains 30-40 μ g/ml kantlex and 30-40 μ g/ml paraxin, place 37 ℃ of baking oven incubated overnight;
2) mono-clonal on the flat board is inoculated in the 250ml that contains 30-50ml LB substratum and shakes in the bottle, rotating speed is 200-220rmp, cultivates 7-10h at 37 ℃ and obtains first order seed;
3) get the 2ml-4ml first order seed and be inoculated in the shaking of 500ml that contains the 100-150ml fermention medium and cultivate 3-4h in the bottle, obtain secondary seed;
4) the 100-150ml secondary seed is inoculated in the 15L fermentor tank that contains the 8-10L fermention medium carries out fermentation culture, make the initial bacterium liquid density OD on the jar
600Be 0.08-0.15, the fermentor tank rotating speed is 400-500rpm, and air flow quantity is 3-4.5L/min, and initial culture temperature is 37 ℃, reduces to 27-29 ℃ behind the 2h, induces with the 0.050.2mmol/L isopropyl-again;
5) flow feeding substratum behind the inducing culture 4-8h is at cell density OD
600When reaching 7-10, add the inhibitor of glucose as the 5-aminolevulinate dehydratase in batches;
6) the initial pH of fermentation culture adopts the dilute sulphuric acid of 10%-20% volume fraction to be controlled at 5.8-6.0; Inducing culture 4-8h is 6.1-6.3 by flow feeding substratum control pH.
The present invention produces in the 5-amino-laevulic acid, said initial fermention medium is respectively 1%-2% peptone, 0.5%-1% yeast powder, 0.3%-0.6% succsinic acid, 0.2%-0.4% glycine and 0.1%-0.5% glucose by mass volume ratio to be formed, and its pH value is 6.0-6.3.Contain 50-70g succsinic acid and 30-50g glycine in the said supplemented medium, volume is 800-1000ml.The inhibitor of said 5-aminolevulinate dehydratase is a 4-6g/L D-glucose.
Technology of the present invention is simple, controllability is high, cost is low, fermenting process is environmentally friendly, and the outer ALA output height of the born of the same parents of gained has wide industrial prospect.
Description of drawings
Fig. 1 is engineering bacteria Rosetta (DE3)-pET28a-A.R.hemA abduction delivering synoptic diagram.
Outer ALA concentration of Fig. 2 born of the same parents and cell density OD
600Relation curve with fermentation time.
Embodiment
Further specify the present invention below in conjunction with embodiment
Embodiment 1
The 5-amino-laevulic acid engineering bacteria that obtains, its structure may further comprise the steps:
1. the extraction of recombinant plasmid pET28a-A.R-hemA
1) be that the engineering bacteria of CGMCC No.1332 is inoculated in the liquid nutrient medium with deposit number, 37 ℃ of shaking culture 12hr can gather in the crops to the bacterium logarithmic phase;
2) get 3ml inoculum (dividing each 1.5ml 2 times) in the Eppendorf pipe, 13, the centrifugal 30s of 000rpm abandons supernatant liquor (complete as far as possible);
3) add solution I (the 50mmol/L glucose that 100ul ices precooling; 25mmol/L TrisCl (pH8.0); 10mmol/LEDTA (pH8.0) can prepare in batch, and steam sterilizing 15min is stored in 4 ℃), thermal agitation makes cell resuspended fully, leaves standstill 5～10min under the room temperature;
4) add new solution II (the 0.2mol/L NaOH for preparing of 200ul; 1%SDS), put upside down fast 4 times, mix with the mixing content ice bath 5min gently;
5) add solution III (the 5mol/L potassium acetate 60ml that 150ul ices precooling; Glacial acetic acid 11.5ml; Water 28.5ml; The solution final concentration potassium ion that is made into is 3mol/L, and acetate moiety is 5mol/L), gentle vibration is evenly dispersed in the bacterial lysate solution III 10 seconds, ice bath 5～10min;
6) 13, the centrifugal 5～10min of 000rpm moves into supernatant liquor in another centrifuge tube;
7) add equal-volume phenol: chloroform: primary isoamyl alcohol (25/24/1), the vibration mixing, 13, the centrifugal 5min of 000rpm, supernatant move into another clean centrifuge tube;
8) add 1/10 volume 3M NaAc earlier, mixing adds 2 times of volume dehydrated alcohols again, puts upside down mixing, leaves standstill 10 minutes deposit D NA in-20 ℃ of refrigerators;
9) 13, the centrifugal 5min of 000r/min;
10) carefully abandon supernatant, centrifuge tube is inverted on the filter paper remaining liq is dripped to the greatest extent;
11) with 1ml70% washing with alcohol DNA precipitation once, 13, the centrifugal 5min of 000r/min obtains the DNA precipitation; Remove supernatant, liquid is flow to end, in air, made precipitation dry 10 minutes;
12) with resolution of precipitate in 50ul TE damping fluid (containing RNase A 20ug/ml), 55 ℃ of following digestion 15min can obtain recombinant plasmid pET28a-A.R-hemA; Be stored in-20 ℃ of refrigerators standby.
2. the recombinant plasmid pET28a-A.R-hemA that obtains is converted into intestinal bacteria Rosetta (DE3) competence and can obtains engineering bacteria Rosetta (DE3)-pET28a-A.R.hemA, it induces overexpression ALA synthetic enzyme as shown in Figure 1 with IPTG.
Method with engineering bacteria is produced the 5-amino-laevulic acid may further comprise the steps:
1. from being the glycerine pipe of CGMCC No.1938 engineering bacteria Rosetta (DE3)-pET28a-A.R.hemA, deposit number dips in the bacterium liquid that takes a morsel with inoculating needle, on the LB culture medium flat plate that contains 30 μ g/ml kantlex and 34 μ g/ml paraxin, rule incubated overnight in 37 ℃ of baking ovens;
2. the picking mono-clonal is inoculated in the 250ml that contains 50ml LB substratum and shakes in the bottle, and rotating speed is 200rmp, cultivates 8h at 37 ℃, obtains first order seed;
3. get the 2ml first order seed and be inoculated in shaking in the bottle of the 500ml that contains the 100ml fermention medium,, cultivate 3h under the 200rpm condition, obtain secondary seed at 37 ℃.Wherein initial fermention medium is respectively 1% peptone, 0.5% yeast powder, 0.3% succsinic acid, 0.2% glycine and 0.2% glucose by mass volume ratio to be formed, and regulating the pH value with sodium hydroxide is 5.9;
4. the 100ml secondary seed is inoculated in the 15L fermentor tank that contains the above-mentioned fermention medium of 9L, makes the initial bacterium liquid density OD on the jar
600Be approximately 0.1, the fermentor tank rotating speed is 400rmp, and air flow quantity is 3L/min, and initial culture temperature is 37 ℃, reduces to 28 ℃ behind the 2h, and (IPTG) induces with the 0.05mmol/L isopropyl-;
5. inducing culture 4h stream adds and contains 63g succsinic acid and 36g glycine, and volume is the supplemented medium of 850ml, with the measuring density thalli growth situation of thalline, as cell density OD
600Reach at 7 o'clock, beginning is added 4g/L D-glucose as 5-aminolevulinate dehydratase inhibitor every 3h, adds glucose altogether 3 times.
6. the initial pH of fermentation culture adopts the dilute sulphuric acid of 10%-20% volume fraction to be controlled at 5.9; Inducing culture 4h is 6.2 by flow feeding substratum control pH.The analytical procedure of employing Mauzerall and Granick (with reference to Mauzerall D, Granick S.J.Bio.Chem., 1956, the 219:435-442) concentration of mensuration ALA.Can reach 6.5g/L during the concentration 22h of the outer ALA of born of the same parents as seen from Figure 2.
Claims (6)
1. produce the engineering bacteria of 5-amino-laevulic acid, it is characterized in that this bacterium is the engineering bacteria that contains the 5-aminolevulinate synthetase gene of agrobacterium radiobacter, Chinese colon bacillus by name, the Latin formal name used at school is Escherichia coli Rosetta (DE3)-pET28a-A.R.hemA, in the common micro-organisms center preservation of China Committee for Culture Collection of Microorganisms of specified depositary institution of Patent Office of the People's Republic of China, deposit number is: CGMCCNo.1938.
2. the construction process of the engineering bacteria of production 5-amino-laevulic acid according to claim 1, its step is as follows:
1) be to obtain recon pET28a-A.R-hemA the engineering bacteria of CGMCC No.1332 from deposit number, the Chinese of said CGMCC No.1332 engineering bacteria is called colon bacillus, and the Latin formal name used at school is Escherichia coliBL21 (DE3)-pET28a-A.R-hemA;
2) recon pET28a-A.R-hemA is converted among the host bacterium Rosetta (DE3), obtains engineering bacteria Rosetta (DE3)-pET28a-A.R.hemA, its deposit number is CGMCC No.1938.
3. with the method for the described engineering bacteria production of claim 1 5-amino-laevulic acid, it is characterized in that may further comprise the steps:
1) dips in from deposit number is the glycerine pipe of CGMCC No.1938 engineering bacteria Rosetta (DE3)-pET28a-A.R.hemA with inoculating needle and get bacterium liquid, after line on the LB culture medium flat plate that contains 30-40 μ g/ml kantlex and 30-40 μ g/ml paraxin, place 37 ℃ of baking oven incubated overnight;
2) mono-clonal on the flat board is inoculated in the 250ml that contains 30-50ml LB substratum and shakes in the bottle, rotating speed is 200-220rmp, cultivates 7-10h at 37 ℃ and obtains first order seed;
3) get the 2ml-4ml first order seed and be inoculated in the shaking of 500ml that contains the 100-150ml fermention medium and cultivate 3-4h in the bottle, obtain secondary seed;
4) the 100-150ml secondary seed is inoculated in the 15L fermentor tank that contains the 8-10L fermention medium carries out fermentation culture, make the initial bacterium liquid density OD on the jar
600Be 0.08-0.15, the fermentor tank rotating speed is 400-500rpm, and air flow quantity is 3-4.5L/min, and initial culture temperature is 37 ℃, reduces to 27-29 ℃ behind the 2h, induces with the 0.05-0.2mmol/L isopropyl-again;
5) flow feeding substratum behind the inducing culture 4-8h is at cell density OD
600When reaching 7-10, add the inhibitor of glucose as the 5-aminolevulinate dehydratase in batches;
6) the initial pH of fermentation culture adopts the dilute sulphuric acid of 10%-20% volume fraction to be controlled at 5.8-6.0; Inducing culture 4-8h is 6.1-6.3 by flow feeding substratum control pH.
4. the method for production 5-amino-laevulic acid according to claim 3, it is characterized in that said initial fermention medium is respectively 1%-2% peptone, 0.5%-1% yeast powder, 0.3%-0.6% succsinic acid, 0.2%-0.4% glycine and 0.1%-0.5% glucose by mass volume ratio and forms, its pH value is 6.0-6.3.
5. the method for production 5-amino-laevulic acid according to claim 3 is characterized in that containing in the said supplemented medium 50-70g succsinic acid and 30-50g glycine, and volume is 800-1000ml.
6. the method for production 5-amino-laevulic acid according to claim 3, the inhibitor that it is characterized in that said 5-aminolevulinate dehydratase is a 4-6g/L D-glucose.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103710374A (en) * | 2014-01-14 | 2014-04-09 | 中国科学院天津工业生物技术研究所 | Bacterial strain produced by 5-aminolevulinic acid as well as preparation method and application thereof |
| CN104561158A (en) * | 2015-01-13 | 2015-04-29 | 江南大学 | Method for enhancing engineered escherichia coli synthesized 5-aminolevulinic acid by adding Fe<2+> |
| CN106967627A (en) * | 2017-04-11 | 2017-07-21 | 济南大学 | A kind of fermenting and producing ALA recombinant bacterial strain and preparation method thereof |
| CN114369562A (en) * | 2022-03-21 | 2022-04-19 | 中国农业科学院北京畜牧兽医研究所 | Method for improving expression quantity of 5-aminolevulinic acid |
| US11526233B2 (en) | 2018-08-23 | 2022-12-13 | Analogix (China) Semiconductor, Inc | Method for controlling touch screen, apparatus and device, storage medium and processor |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1699584A (en) * | 2005-04-19 | 2005-11-23 | 浙江大学 | Process for producing 5-aminolevulinic acid from engineering bacterium |
| CN1322132C (en) * | 2005-04-19 | 2007-06-20 | 浙江大学 | Engineering bacteria for producing 5-amino acetyl propionic acid and its constructing method |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103710374A (en) * | 2014-01-14 | 2014-04-09 | 中国科学院天津工业生物技术研究所 | Bacterial strain produced by 5-aminolevulinic acid as well as preparation method and application thereof |
| CN103710374B (en) * | 2014-01-14 | 2018-02-06 | 中国科学院天津工业生物技术研究所 | A kind of 5 amino-laevulic acid producing bacterial strains and preparation method and application |
| CN104561158A (en) * | 2015-01-13 | 2015-04-29 | 江南大学 | Method for enhancing engineered escherichia coli synthesized 5-aminolevulinic acid by adding Fe<2+> |
| CN104561158B (en) * | 2015-01-13 | 2018-01-16 | 江南大学 | One kind addition Fe2+Improve the method that colibacillus engineering synthesizes 5 amino-laevulic acids |
| CN106967627A (en) * | 2017-04-11 | 2017-07-21 | 济南大学 | A kind of fermenting and producing ALA recombinant bacterial strain and preparation method thereof |
| US11526233B2 (en) | 2018-08-23 | 2022-12-13 | Analogix (China) Semiconductor, Inc | Method for controlling touch screen, apparatus and device, storage medium and processor |
| CN114369562A (en) * | 2022-03-21 | 2022-04-19 | 中国农业科学院北京畜牧兽医研究所 | Method for improving expression quantity of 5-aminolevulinic acid |
| CN114369562B (en) * | 2022-03-21 | 2022-05-31 | 中国农业科学院北京畜牧兽医研究所 | Method for improving expression quantity of 5-aminolevulinic acid |
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Application publication date: 20071031 Assignee: Suzhou Yian Biotech Co., Ltd. Assignor: Zhejiang University Contract record no.: 2013320010038 Denomination of invention: Engineering bacterium producing 5-glycyl ethylformic acid and construction and application method thereof Granted publication date: 20120307 License type: Exclusive License Record date: 20130319 |
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