CN102747114B - Method for regulating recombinant escherichia coli metabolism by using transient anaerobic fermentation - Google Patents
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Abstract
The present invention discloses a method for regulating recombinant escherichia coli metabolism by using transient anaerobic fermentation. The yield of 5-aminolevulinic acid produced by the traditional method is not high. According to the method, transient anaerobic fermentation is performed at a certain stage of recombinant escherichia coli culture so as to change metabolic behavior of the recombinant escherichia coli, wherein the specific improvement measures comprise: carrying out anaerobic fermentation for 30-75 minutes at a certain stage of an engineering bacteria Rosetta (DE3)-pET28a-A.R.hema culture process so as to change physiological states of cells, appropriately reduce growth rate of cells, and promote accumulation of a product 5-aminolevulinic acid, wherein the preservation number of the engineering bacteria is CGMCC No.1939. According to the present invention, based on the invention 200710068169.0, the fermentation production technology for the 5-aminolevulinic acid is further improved, the resulting extracellular 5-aminolevulinic acid yield is high, and the method is suitable for industrial application and has broad prospects.
Description
Technical field
The invention belongs to microbial fermentation technology field, relate to the metabolism behavior that a kind of method of carrying out of short duration anaerobically fermenting by the certain phase in microbial culture regulates recombination bacillus coli, thereby improve optimizing fermentation and the regulation and control novel method of fermenting process object product output.
Background technology
5-ALA (5-amino1evulinic acid, ALA) is that the interior Pyrrolidine compounds of organism is (as hemachrome enzyme, porphyrin and vitamins B
12deng) common precursor, there is important physiologically active.ALA can be used as photosynthesis promoter, adverse-resistant agent, defoliant and weedicide etc. in agricultural, of many uses, environmentally friendly.At medical field, 5-ALA is having great using value as New Generation Optical kinetics medicine aspect the diagnosis of the disease such as brain tumor, skin carcinoma and treatment.
The production method of 5-ALA can be divided into chemical synthesis and microbe fermentation method.Chemosynthesis ALA has number of ways, the synthetic ALA taking urobenzoic acid, succsinic acid, tetrahydrofurfuryl amine and levulinic acid etc. as raw material respectively, but these method normal yields are low, by product is many, environmental pollution is serious.Utilize Rhodobacter sphaeroides mutant strain and recombination bacillus coli fermentative Production 5-ALA aspect production cost and quality product, all thering is significant competitive edge.Producing 5-ALA by structure genetic engineering bacterium is a kind of cheapness and effective means.In patent ZL 200710068169.0, disclose a kind of by genetic engineering bacterium Rosetta (DE3)-pET28a-A.R.
hemAcarry out the method for fermentative production ALA, wherein the output of ALA can reach 6.6g/L.
In the fermentation system of recombination bacillus coli, dissolved oxygen concentration is very complicated on the impact of cellular metabolism.2003; (the De Le ó n A such as Antonio De Le ó n; Hern á ndez V, Galindo E, Ram í rez OT; Enzyme Microb Technol; 2003,33:689-697) research show, when with recombination bacillus coli research overexpression penioillin acylase; the relation that growth is coupled type yield coefficient and dissolved oxygen concentration is saturation type curve, is issued to maximum value at the DOT of 1%-5% but not growth is coupled type yield coefficient.Accurately the oxygen dissolving value in controlled fermentation process can improve the output of object product in lower level.But the running cost of the oxygen dissolving value in the accurate controlled fermentation process of technical scale is higher.Intestinal bacteria are as facultative aerobic anaerobe, and in the time carrying out aerobic fermentation and anaerobically fermenting, its physiology and metabolism have very big-difference.(Vemuri GN, Eiteman MA, the Altman E such as Vemuri, J Ind Microbiol Biotechnol, 2002,28:325-332) research find, compared with aerobic fermentation completely, adopt aerobic/anaerobic two stage fermentations can increase substantially the output of succsinic acid.In two such stage fermentations, the conversion opportunity from aerobic fermentation to anaerobically fermenting, the impact that can not ignore colibacillary metabolism behavior generation, thus make succinic acid production occur significantly to change.Therefore, utilize dexterously the variation of physiology and metabolism between this aerobic fermentation and anaerobically fermenting, just likely on purpose realize the control of fermenting process metabolism stream.
Summary of the invention
The object of the invention is for the deficiencies in the prior art, propose the method for the of short duration anaerobically fermenting adjusting of a kind of use recombination bacillus coli metabolism, further to improve the output of 5-ALA.
The technical scheme that the inventive method adopts comprises the following steps:
Step (1). be engineering bacteria Rosetta (DE3)-pET28a-A.R. of CGMCC No.1939 from deposit number with inoculating needle
hemAglycerine pipe in dip after bacterium liquid, containing 30
50 μ g/ml kantlex and 30
on the LB culture medium flat plate of 50 μ g/ml paraxin, rule, and the LB culture medium flat plate after line is positioned over to incubated overnight at 37 DEG C;
Described deposit number is engineering bacteria Rosetta (DE3)-pET28a-A.R. of CGMCC No.1939
hemA'sdepositary institution's title: China Committee for Culture Collection of Microorganisms's common micro-organisms center; Depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Preservation date: 2007.01.25.
Step (2). the mono-clonal on LB culture medium flat plate is inoculated in containing 30
in the 250ml shaking flask of 50ml LB substratum, and to be placed on rotating speed be 200
220rpm, temperature is incubated overnight in the shaking table of 37 DEG C, obtains first order seed;
Step (3). get 1
5ml first order seed is inoculated in containing 50
in the shaking flask of the 500ml of 100ml LB substratum, and to be placed on rotating speed be 200
220rpm, temperature is to cultivate 3 in the shaking table of 37 DEG C
4h, obtains secondary seed;
Step (4). by 100
300ml secondary seed is inoculated in containing 7
in 15 L fermentor tanks of 9 L fermention mediums, carry out fermentation culture, the mixing speed of fermentor tank is 300
500rpm, air flow quantity is 5
10 L/min, initial culture temperature is 37 DEG C; 2
after 3h, culture temperature is down to 26
30 DEG C, and with 0.02
the induction of 0.2mmol/L isopropyl-β-D-thiogalactoside(IPTG);
Fermention medium in described step (4) is to be respectively 0.5 by mass volume ratio
2% peptone, 0.25
1% yeast powder, 0.3
1% succsinic acid, 0.2
0.4% glycine and 0.1
0.5% glucose composition, its pH value is 6.0
6.3; Fermention medium starts with 10 most
the dilute sulphuric acid of 20% volume fraction, is controlled at 5.8 by pH
6.0;
Step (5). when thalli growth to 5
after 6 hours, the mixing speed of fermentor tank is adjusted to 0, in fermented liquid, oxygen dissolving value becomes 0 thereupon, maintains 30
after the anaerobically fermenting of 75min, recover the mixing speed of fermentor tank;
Described fermented liquid, in secondary seed inoculation 4
after 8h, add supplemented medium by feedback flow, the pH of fermented liquid is adjusted to 6.1-6.3;
Described supplemented medium volume is 800
1100ml, and contain 70
90 g succsinic acids and 50
70 g glycine;
Step (6). as cell density OD
600reach 3
, add 5-ALA dehydratase inhibitor at 10 o'clock in batches or continuously;
The inhibitor of described 5-ALA dehydratase is 3
5g/L D-Glucose;
Deposit number in described step (1) is CGMCC No.1939 engineering bacteria, Classification And Nomenclature: Chinese colon bacillus by name, Latin name is
escherichia colirosetta (DE3)-pET28a-A.R.
hemA.
Beneficial effect of the present invention is as follows:
Technique of the present invention is workable, by of short duration anaerobically fermenting regulation and control engineering bacteria Rosetta (DE3)-pET28a-A.R.
hemAmetabolism, the outer 5-ALA output of born of the same parents of gained is high, is suitable for industrial applications, has a extensive future.
Brief description of the drawings
The outer ALA concentration of Fig. 1 born of the same parents, glycine, succsinic acid, glucose, cell density OD
600relation curve with fermentation time;
Fig. 2 dissolved oxygen (DO), the outer ALA concentration of born of the same parents, glycine, succsinic acid, glucose, acetic acid and cell density OD
600relation curve with fermentation time.
Fig. 3 dissolved oxygen (DO), the outer ALA concentration of born of the same parents, glycine, succsinic acid, glucose, cell density OD
600relation curve with fermentation time.
Embodiment
Below in conjunction with drawings and Examples, the invention will be further described.
Embodiment 1
The method that regulates recombination bacillus coli metabolism with of short duration anaerobically fermenting, comprises the following steps:
1) with inoculating needle from deposit number be engineering bacteria Rosetta (DE3)-pET28a-A.R. of CGMCC No.1939
hemAglycerine pipe in dip bacterium liquid, on the LB culture medium flat plate that contains 30 μ g/ml kantlex and 30 μ g/ml paraxin line after, incubated overnight at 37 DEG C;
2) mono-clonal on flat board is inoculated in the 250ml shaking flask containing 50ml LB substratum, rotating speed is 200rpm, and incubated overnight at 37 DEG C, obtains first order seed;
3) get 1ml first order seed and be inoculated in the shaking flask of multiple 500ml containing 100ml LB substratum and cultivate 3 h, obtain secondary seed;
4) 200ml secondary seed is inoculated in containing in 15 L fermentor tanks of 9 L fermention mediums and carries out fermentation culture, fermentor tank mixing speed is 400 rpm, air flow quantity is 7 L/min, initial culture temperature is 37 DEG C, after 2h, be down to 29 DEG C, then be that 0.05 mmol/L isopropyl-β-D-thiogalactoside(IPTG) is induced with final concentration; Wherein 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 and forms, and regulating pH value with sodium hydroxide is 6.1;
5) at cell density OD
600reach at 5 o'clock, within every 3 hours, add 4g/L D-Glucose as 5-ALA dehydratase inhibitor, totally 3 times in batches;
6) fermentation culture initially adopts the dilute sulphuric acid control pH of 20% volume fraction 5.8; After cultivation 6h, be 6.3 by flow feeding substratum control pH;
Described supplemented medium volume is 1000ml, and contains 70 g succsinic acids and 55 g glycine;
7) fermenting to 6h, carrying out 60 min anaerobically fermentings, is also after fermentation culture 5h, to stop stirring, and recovers to stir during to 6h.
Adopt the analytical procedure (with reference to Mauzerall D, Granick S. J. Bio. Chem., 1956,219:435-442) of Mauzerall and Granick to measure the concentration of ALA.As seen from Figure 1, by once of short duration anaerobically fermenting operation, the maximum concentration of the outer ALA of born of the same parents can reach 7.4 g/L.
Embodiment 2:
1) with inoculating needle from deposit number be engineering bacteria Rosetta (DE3)-pET28a-A.R. of CGMCC No.1939
hemAglycerine pipe in dip bacterium liquid, on the LB culture medium flat plate that contains 40 μ g/ml kantlex and 30 μ g/ml paraxin line after, incubated overnight at 37 DEG C;
2) mono-clonal on flat board is inoculated in the 250ml shaking flask containing 30ml LB substratum, rotating speed is 220rpm, and incubated overnight at 37 DEG C, obtains first order seed;
3) get 5ml first order seed and be inoculated in the shaking flask of multiple 500ml containing 75ml LB substratum and cultivate 3 .5h, obtain secondary seed;
4) 300ml secondary seed is inoculated in the 15 L fermentor tanks containing 7L fermention medium and carries out fermentation culture, fermentor tank mixing speed is 500 rpm, air flow quantity is 5L/min, initial culture temperature is 37 DEG C, after 3h, be down to 30 DEG C, then be that 0.2 mmol/L isopropyl-β-D-thiogalactoside(IPTG) is induced with final concentration; Wherein fermention medium is respectively 2% peptone, 0.25% yeast powder, 1% succsinic acid, 0.4% glycine and 0.5% glucose by mass volume ratio and forms, and regulating pH value with sodium hydroxide is 6. 3;
5) at cell density OD
600reach at 5 o'clock, within every 3 hours, add 3g/L D-Glucose as 5-ALA dehydratase inhibitor, totally 3 times in batches;
6) fermentation culture initially adopts the dilute sulphuric acid control pH of 20% volume fraction 5.8; After cultivation 6h, be 6.3 by flow feeding substratum control pH;
Described supplemented medium volume is 800ml, and contains 90 g succsinic acids and 70 g glycine;
7) fermenting to 6h, carrying out 30 min anaerobically fermentings, is also that 6h stops stirring, and recovers to stir during to 6.5h.
Adopt the analytical procedure (with reference to Mauzerall D, Granick S. J. Bio. Chem., 1956,219:435-442) of Mauzerall and Granick to measure the concentration of ALA.By once of short duration anaerobically fermenting operation, the maximum concentration of the outer ALA of born of the same parents can reach 7.2 g/L.
Embodiment 3
The method that regulates recombination bacillus coli metabolism with of short duration anaerobically fermenting, comprises the following steps:
1) with inoculating needle from deposit number be engineering bacteria Rosetta (DE3)-pET28a-A.R. of CGMCC No.1939
hemAglycerine pipe in dip bacterium liquid, on the LB culture medium flat plate that contains 40 μ g/ml kantlex and 50 μ g/ml paraxin line after, incubated overnight at 37 DEG C;
2) mono-clonal on flat board is inoculated in the 250ml shaking flask containing 50ml LB substratum, rotating speed is 200rpm, and incubated overnight at 37 DEG C, obtains first order seed;
3) get 1ml first order seed and be inoculated in the shaking flask of multiple 500ml containing 100ml LB substratum and cultivate 3 h, obtain secondary seed;
4) 200ml secondary seed is inoculated in containing in 15 L fermentor tanks of 9 L fermention mediums and carries out fermentation culture, fermentor tank mixing speed is 400 rpm, air flow quantity is 7 L/min, initial culture temperature is 37 DEG C, after 2h, be down to 27 DEG C, then be that 0.05 mmol/L isopropyl-β-D-thiogalactoside(IPTG) is induced with final concentration; Wherein 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 and forms, and regulating pH value with sodium hydroxide is 6.1;
5) at cell density OD
600reach at 5 o'clock, within every 3 hours, add 5g/L D-Glucose as 5-ALA dehydratase inhibitor, totally 3 times in batches;
6) fermentation culture initially adopts the dilute sulphuric acid control pH of 20% volume fraction 5.9; After cultivation 6h, be 6.2 by flow feeding substratum control pH;
Described supplemented medium volume is 1100ml, and contains 75 g succsinic acids and 70 g glycine;
7) fermenting to 5h, carrying out 45 min anaerobically fermentings, is also that 5h stops stirring, and recovers to stir during to 5.75h.
Adopt the analytical procedure (with reference to Mauzerall D, Granick S. J. Bio. Chem., 1956,219:435-442) of Mauzerall and Granick to measure the concentration of ALA.As seen from Figure 2, by once of short duration anaerobically fermenting operation, the maximum concentration of the outer ALA of born of the same parents can reach 8.6 g/L.
Embodiment 4
1) with inoculating needle from deposit number be engineering bacteria Rosetta (DE3)-pET28a-A.R. of CGMCC No.1939
hemAglycerine pipe in dip bacterium liquid, on the LB culture medium flat plate that contains 30 μ g/ml kantlex and 50 μ g/ml paraxin line after, incubated overnight at 37 DEG C;
2) mono-clonal on flat board is inoculated in the 250ml shaking flask containing 50ml LB substratum, rotating speed is 200rpm, and incubated overnight at 37 DEG C, obtains first order seed;
3) get 1ml first order seed and be inoculated in the shaking flask of multiple 500ml containing 100ml LB substratum and cultivate 3 h, obtain secondary seed;
4) 200ml secondary seed is inoculated in containing in 15 L fermentor tanks of 9 L fermention mediums and carries out fermentation culture, fermentor tank mixing speed is 400 rpm, air flow quantity is 7 L/min, initial culture temperature is 37 DEG C, after 2h, be down to 28 DEG C, then be that 0.05 mmol/L isopropyl-β-D-thiogalactoside(IPTG) is induced with final concentration; Wherein 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 and forms, and regulating pH value with sodium hydroxide is 6.1;
5) at cell density OD
600reach at 4 o'clock, D-Glucose and precursor hybrid feedback stream are added in fermentation fermented liquid, the pH that controls lotion is 6.2;
6) fermentation culture initially adopts the dilute sulphuric acid control pH of 20% volume fraction 5.9; Cultivating after 6h the mixture control pH that adds D-Glucose and supplemented medium by stream is 6.2;
The content of described D-Glucose is 145g, and supplemented medium volume is 1100ml, and contains 80 g succsinic acids and 63 g glycine;
7) fermenting to 5.5h, carrying out 75 min anaerobically fermentings, is also that 5.5h stops stirring, and recovers to stir during to 6.45h.
Adopt the analytical procedure (with reference to Mauzerall D, Granick S. J. Bio. Chem., 1956,219:435-442) of Mauzerall and Granick to measure the concentration of ALA.By once of short duration anaerobically fermenting operation, and the method for utilizing D-Glucose and supplemented medium hybrid feedback stream to add, the maximum concentration of the outer ALA of born of the same parents can reach 8.2 g/L.
Embodiment 5
1) with inoculating needle from deposit number be engineering bacteria Rosetta (DE3)-pET28a-A.R. of CGMCC No.1939
hemAglycerine pipe in dip bacterium liquid, on the LB culture medium flat plate that contains 50 μ g/ml kantlex and 40 μ g/ml paraxin line after, incubated overnight at 37 DEG C;
2) mono-clonal on flat board is inoculated in the 250ml shaking flask containing 40ml LB substratum, rotating speed is 210rpm, and incubated overnight at 37 DEG C, obtains first order seed;
3) get 3ml first order seed and be inoculated in the shaking flask of multiple 500ml containing 50ml LB substratum and cultivate 4 h, obtain secondary seed;
4) 100ml secondary seed is inoculated in containing in 15 L fermentor tanks of 8 L fermention mediums and carries out fermentation culture, fermentor tank mixing speed is 300 rpm, air flow quantity is 10 L/min, initial culture temperature is 37 DEG C, after 2.5h, be down to 30 DEG C, then be that 0.02 mmol/L isopropyl-β-D-thiogalactoside(IPTG) is induced with final concentration; Wherein fermention medium is respectively 0.5% peptone, 1% yeast powder, 0.5% succsinic acid, 0.3% glycine and 0.1% glucose by mass volume ratio and forms, and regulating pH value with sodium hydroxide is 6.0;
5) at cell density OD
600reach at 6 o'clock, D-Glucose and precursor hybrid feedback stream are added in fermentation fermented liquid, the pH that controls lotion is 6.1;
6) fermentation culture initially adopts the dilute sulphuric acid control pH of 20% volume fraction 5.8; Cultivating after 5h the mixture control pH that adds D-Glucose and supplemented medium by stream is 6.1;
The content of described D-Glucose is 145g, and supplemented medium volume is 900ml, and contains 70 g succsinic acids and 50g glycine;
7) fermenting to 5h, carrying out 45 min anaerobically fermentings, is also that 5h stops stirring, and recovers to stir during to 5.75h.
Adopt the analytical procedure (with reference to Mauzerall D, Granick S. J. Bio. Chem., 1956,219:435-442) of Mauzerall and Granick to measure the concentration of ALA.By once of short duration anaerobically fermenting operation, and the method for utilizing D-Glucose and supplemented medium hybrid feedback stream to add, the maximum concentration of the outer ALA of born of the same parents can reach 7.6 g/L.
Embodiment 6
The method that regulates recombination bacillus coli metabolism with of short duration anaerobically fermenting, comprises the following steps:
1) with inoculating needle from deposit number be engineering bacteria Rosetta (DE3)-pET28a-A.R. of CGMCC No.1939
hemAglycerine pipe in dip bacterium liquid, on the LB culture medium flat plate that contains 50 μ g/ml kantlex and 40 μ g/ml paraxin line after, incubated overnight at 37 DEG C;
2) mono-clonal on flat board is inoculated in the 250ml shaking flask containing 50ml LB substratum, rotating speed is 200rpm, and incubated overnight at 37 DEG C, obtains first order seed;
3) get 1ml first order seed and be inoculated in the shaking flask of multiple 500ml containing 100ml LB substratum and cultivate 3 h, obtain secondary seed;
4) 200ml secondary seed is inoculated in containing in 15 L fermentor tanks of 9 L fermention mediums and carries out fermentation culture, fermentor tank mixing speed is 400 rpm, air flow quantity is 7 L/min, initial culture temperature is 37 DEG C, after 2h, be down to 28 DEG C, then be that 0.05 mmol/L isopropyl-β-D-thiogalactoside(IPTG) is induced with final concentration; Wherein 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 and forms, and regulating pH value with sodium hydroxide is 6.1;
5) at cell density OD
600reach at 4 o'clock, D-Glucose and precursor hybrid feedback stream are added in fermentation fermented liquid, the pH that controls lotion is 6.2;
6) fermentation culture initially adopts the dilute sulphuric acid control pH of 20% volume fraction 5.9; Cultivating after 6h the mixture control pH that adds D-Glucose and supplemented medium by stream is 6.2;
The content of described D-Glucose is 145g, and supplemented medium volume is 1100ml, and contains 80 g succsinic acids and 63 g glycine;
7) fermenting to 5h, carrying out 45 min anaerobically fermentings, is also that 5h stops stirring, and recovers to stir during to 5.75h.
Adopt the analytical procedure (with reference to Mauzerall D, Granick S. J. Bio. Chem., 1956,219:435-442) of Mauzerall and Granick to measure the concentration of ALA.As seen from Figure 3, by once of short duration anaerobically fermenting operation, and the method for utilizing D-Glucose and supplemented medium hybrid feedback stream to add, the maximum concentration of the outer ALA of born of the same parents can reach 9.4 g/L.
Claims (1)
1. a method that regulates recombination bacillus coli metabolism with of short duration anaerobically fermenting, is characterized in that following steps:
Step (1). from being the glycerine pipe of engineering bacteria Rosetta (DE3)-pET28a-A.R.hemA of CGMCC No.1939, deposit number dips after bacterium liquid with inoculating needle, on the LB culture medium flat plate that contains 30～50 μ g/ml kantlex and 30～50 μ g/ml paraxin, rule, and the LB culture medium flat plate after line is positioned over to incubated overnight at 37 DEG C;
Step (2). the mono-clonal on LB culture medium flat plate is inoculated in containing in the 250ml shaking flask of 30～50ml LB substratum, and to be placed on rotating speed be 200～220rpm, temperature is incubated overnight in the shaking table of 37 DEG C, obtains first order seed;
Step (3). get 1～5ml first order seed and be inoculated in the shaking flask containing the 500ml of 50～100ml LB substratum, and to be placed on rotating speed be 200～220rpm, temperature is to cultivate 3～4h in the shaking table of 37 DEG C, obtains secondary seed;
Step (4). 100～300ml secondary seed is inoculated in containing in the 15L fermentor tank of 7～9L fermention medium and carries out fermentation culture, and the mixing speed of fermentor tank is 300～500rpm, and air flow quantity is 5～10L/min, and initial culture temperature is 37 DEG C; After 2～3h, culture temperature is down to 26～30 DEG C, and induces with 0.02～0.2mmol/L isopropyl-β-D-thiogalactoside(IPTG);
Fermention medium in described step (4) is respectively 0.5～2% peptone, 0.25～1% yeast powder, 0.3～1% succsinic acid, 0.2～0.4% glycine and 0.1～0.5% glucose by mass volume ratio and forms, and its pH value is 6.0～6.3; Fermention medium starts the dilute sulphuric acid by 10～20% volume fractions most, and pH is controlled to 5.8～6.0;
Step (5). when behind thalli growth to 5～6 hour, the mixing speed of fermentor tank is adjusted to 0, in fermented liquid, oxygen dissolving value becomes 0 thereupon, maintains the mixing speed that recovers fermentor tank after the anaerobically fermenting of 30～75min;
Described fermented liquid adds supplemented medium by feedback flow after secondary seed inoculation 4～8h, and the pH of fermented liquid is adjusted to 6.1～6.3;
Described supplemented medium volume is 800～1100ml, and contains 70～90g succsinic acid and 50～70g glycine;
Step (6). in the time that cell density OD600 reaches 3～10, add 5-ALA dehydratase inhibitor in batches;
The inhibitor of described 5-ALA dehydratase is 3～5g/L D-Glucose;
Deposit number in described step (1) is CGMCC No.1939 engineering bacteria, Classification And Nomenclature: Chinese colon bacillus by name, Latin name is Escherichia coli Rosetta (DE3)-pET28a-A.R.hemA.
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| CN104195190B (en) * | 2014-09-04 | 2017-04-26 | 山东大学 | Method for producing 5-aminolevulinic acid by carrying out anaerobic fermentation by utilizing recombinant escherichia coli |
| CN108410914A (en) * | 2018-05-16 | 2018-08-17 | 山东兰典生物科技股份有限公司 | A method of producing sodium succinate by raw material of glucose |
| CN114854809B (en) * | 2022-05-31 | 2023-10-31 | 可孚医疗科技股份有限公司 | Method for fermenting recombinant protein by micro-oxygen induced escherichia coli |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0718405A2 (en) * | 1994-12-20 | 1996-06-26 | Cosmo Research Institute | 5-Aminolevulinic acid producing microorganism, and process for producing it |
| CN101041839A (en) * | 2007-04-20 | 2007-09-26 | 浙江大学 | Method for producing 5-glycyl ethylformic acid by using engineering bacterium |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0718405A2 (en) * | 1994-12-20 | 1996-06-26 | Cosmo Research Institute | 5-Aminolevulinic acid producing microorganism, and process for producing it |
| CN101041839A (en) * | 2007-04-20 | 2007-09-26 | 浙江大学 | Method for producing 5-glycyl ethylformic acid by using engineering bacterium |
Non-Patent Citations (3)
| Title |
|---|
| Succinate production in dual-phase Escherichia coli fermentations depends on the time of transition from aerobic toanaerobic conditions;Vemuri GN et al;《Journal of Industrial Microbiology ＆ Biotechnology》;20021231;第28卷;325-332 * |
| Vemuri GN et al.Succinate production in dual-phase Escherichia coli fermentations depends on the time of transition from aerobic toanaerobic conditions.《Journal of Industrial Microbiology ＆ Biotechnology》.2002,第28卷325-332. |
| 秦钢.重组大肠杆菌生产ALA的培养优化研究.《中国优秀博硕士学位论文全文数据库（硕士）工程科技Ⅰ辑》.2006,B016－141. * |
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