CN102747114A - 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 technical field of microbial fermentation, it is related to a kind of method of the of short duration anaerobic fermentation of certain phase progress by Bacteria Culture to adjust the metabolism behavior of recombination bacillus coli, so as to improve the optimizing fermentation and regulation and control new method of fermentation process purpose product yield.
Background technology
5-ALA (5-amino1evulinic acid, ALA) is tetrahydropyrrole compound in organism（Such as hemachrome enzyme, porphyrin and vitamin B12Deng）Common precursor, with important physiologically active.ALA agriculturally can be used as photosynthesis promoter, adverse-resistant agent, defoliant and herbicide etc., widely used, environmentally friendly.In medical domain, 5-ALA has great application value as New Generation Optical pharmacokinetic drug in terms of the diagnosis and treatment of the diseases such as brain tumor, cutaneum carcinoma.
The production method of 5-ALA can be divided into chemical synthesis and microbe fermentation method.Chemical synthesis ALA has number of ways, can be respectively with hippuric acid, butanedioic acid, tetrahydrofurfuryl amine and levulic acid etc. for Material synthesis ALA, but these method normal yields are low, accessory substance is more, environmental pollution is serious.Significant competitive advantage is respectively provided with terms of production cost and product quality using Rhodobacter sphaeroides mutant strain and recombination bacillus coli fermentation method production 5-ALA.It is a kind of cheap and effective method that 5-ALA is produced by building genetic engineering bacterium.One kind is disclosed in patent ZL 200710068169.0 by genetic engineering bacterium Rosetta (DE3)-pET28a-A.R.hemACarry out fermenting and producing ALA method, wherein ALA yield can reach 6.6g/L.
In the fermentation system of recombination bacillus coli, influence of the dissolved oxygen concentration to cell metabolism is extremely complex., Antonio De Le ó n etc. in 2003（De León A, Hernández V, Galindo E, Ramírez OT, Enzyme Microb Technol, 2003, 33: 689-697）Research show that when studying overexpression penioillin acylase with recombination bacillus coli, the relation that growth couples type yield coefficient and dissolved oxygen concentration is in saturation type curve, rather than growth couples type yield coefficient and is then issued to maximum in 1%-5% DOT.The oxygen dissolving value accurately controlled in fermentation process can improve the yield of purpose product in relatively low level.But, the running cost of the oxygen dissolving value in commercial scale precise control fermentation process is higher.Escherichia coli are as facultative aerobic anaerobic organism, and when carrying out aerobic fermentation and anaerobic fermentation, its physiology and metabolism have very big difference.Vemuri etc.（Vemuri GN, Eiteman MA, Altman E, J Ind Microbiol Biotechnol, 2002, 28: 325-332）Research find, compared with complete aerobic fermentation, using aerobic/anaerobic two benches fermentation can increase substantially the yield of butanedioic acid.In the fermentation of such two benches, the conversion opportunity from aerobic fermentation to anaerobic fermentation can produce the influence that can not ignore, so that succinic acid production is significantly changed to the metabolism behavior of Escherichia coli.Therefore, the change of physiology and metabolism between this aerobic fermentation and anaerobic fermentation is dexterously utilized, it is possible to purposefully realize the control of fermentation process metabolic fluxes.
The content of the invention
The purpose of the present invention is in view of the shortcomings of the prior art, a kind of method that recombination bacillus coli metabolism is adjusted with of short duration anaerobic fermentation to be proposed, further to improve the yield of 5-ALA.
The technical scheme that the inventive method is used comprises the following steps：
Step (1) is with transfer needle from engineering bacteria Rosetta (DE3)-pET28a-A.R. that deposit number is CGMCC No.1939hemAGlycerol tube in dip after bacterium solution, containing 30
50 μ g/ml kanamycins and 30
Rule on the LB culture medium flat plates of 50 μ g/ml chloramphenicol, and the LB culture medium flat plates after line are positioned over incubated overnight at 37 DEG C；
Described deposit number is CGMCC No.1939 engineering bacteria Rosetta (DE3)-pET28a-A.R.HemA'sDepositary institution's title：China Committee for Culture Collection of Microorganisms's common micro-organisms center；Depositary institution address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3；Preservation date：2007.01.25.
Monoclonal on LB culture medium flat plates is inoculated in containing 30 by step (2)In the 250ml shaking flasks of 50ml LB culture mediums, and it is 200 to be placed on rotating speed
220rpm, temperature is incubated overnight in 37 DEG C of shaking table, obtains first order seed；
Step (3) takes 1
5ml first order seeds are inoculated in containing 50
In the 500ml of 100ml LB culture mediums shaking flask, and it is 200 to be placed on rotating speed
220rpm, temperature is cultivates 3 in 37 DEG C of shaking table4h, obtains secondary seed；
Step (4) is by 100
300ml secondary seeds are inoculated in containing 7
Fermented and cultured is carried out in 15 L fermentation tanks of 9 L fermentation mediums, the speed of agitator of fermentation tank is 300500rpm, air mass flow is 5
10 L/min, starting cultivation temperature is 37 DEG C；2
Cultivation temperature is down to 26 after 3h
30 DEG C, and use 0.02
0.2mmol/L isopropyl-β-D-thiogalactosides are induced；
It by mass volume ratio is respectively 0.5 that fermentation medium in described step (4), which is,
2% peptone, 0.25
1% dusty yeast, 0.3
1% butanedioic acid, 0.2
0.4% glycine and 0.1
0.5% glucose group is into its pH value is 6.0
6.3；Fermentation medium most starts to use 10
The dilute sulfuric acid of 20% volume fraction, by pH controls 5.8
6.0；
Step (5) is when thalli growth to 5
After 6 hours, the speed of agitator of fermentation tank is adjusted to oxygen dissolving value in 0, zymotic fluid and is changed into 0 therewith, 30 are maintained
Recover the speed of agitator of fermentation tank after 75min anaerobic fermentation；
Described zymotic fluid, 4 are inoculated with secondary seed
Supplemented medium is added by feedback flow after 8h, the pH of zymotic fluid is adjusted to 6.1-6.3；
Described supplemented medium volume is 800
1100ml, and contain 70
90 g butanedioic acids and 50
70 g glycine；
Step (6) works as cell density OD600Reach 3When 10,5-ALA dehydratase inhibitor is added in batches or continuously；
The inhibitor of described 5-ALA dehydratase is 3
5g/L D-Glucoses；
Deposit number in described step (1) is CGMCC No.1939 engineering bacterias, Classification And Nomenclature：The entitled ETEC of Chinese, Latin name isEscherichia coli Rosetta(DE3)-pET28a-A.R.hemA。
The present invention has the beneficial effect that：
Present invention process is workable, adjusts and control engineering bacteria Rosetta (DE3)-pET28a-A.R. by of short duration anaerobic fermentationhemAMetabolism, the extracellular 5-ALA yield of gained is high, is suitable for industrial applications, has a extensive future.
Brief description of the drawings
The extracellular ALA concentration of Fig. 1, glycine, butanedioic acid, glucose, cell density OD600With the relation curve of fermentation time；
Fig. 2 dissolved oxygens（DO）, extracellular ALA concentration, glycine, butanedioic acid, glucose, acetic acid and cell density OD600With the relation curve of fermentation time.
Fig. 3 dissolved oxygens（DO）, extracellular ALA concentration, glycine, butanedioic acid, glucose, cell density OD600With the relation curve of fermentation time.
Embodiment
Below in conjunction with drawings and Examples, the invention will be further described.
Embodiment 1
The method that recombination bacillus coli is metabolized is adjusted with of short duration anaerobic fermentation, is comprised the following steps：
1) with transfer needle from engineering bacteria Rosetta (DE3)-pET28a-A.R. that deposit number is CGMCC No.1939hemAGlycerol tube in dip bacterium solution, on the LB culture medium flat plates containing 30 μ g/ml kanamycins and 30 μ g/ml chloramphenicol rule after, the incubated overnight at 37 DEG C；
2) monoclonal on flat board is inoculated in the 250ml shaking flasks of the culture mediums of LB containing 50ml, rotating speed is 200rpm, the incubated overnight at 37 DEG C obtains first order seed；
3) take 1ml first order seeds to be inoculated in the 500ml of multiple culture mediums of LB containing 100ml shaking flask and cultivate 3 h, obtain secondary seed；
4) 200ml secondary seeds are inoculated in the 15 L fermentation tanks containing 9 L fermentation mediums and carry out fermented and cultured, fermentation tank speed of agitator is 400 rpm, air mass flow is 7 L/min, it is 37 DEG C to originate cultivation temperature, 29 DEG C are down to after 2h, then is induced with final concentration of 0.05 mmol/L isopropyl-β-D-thiogalactosides；It by mass volume ratio is respectively 1% peptone, 0.5% dusty yeast, 0.3% butanedioic acid, 0.2% glycine and 0.2% glucose group into being 6.1 with NaOH regulation pH value that wherein fermentation medium, which is,；
5) in cell density OD600During up to 5,4g/L D-Glucoses are added within every 3 hours in batches as 5-ALA dehydratase inhibitor, totally 3 times；
6) fermented and cultured initially controls pH 5.8 using the dilute sulfuric acid of 20% volume fraction；It is 6.3 to control pH by flow feeding culture medium after culture 6h；
Described supplemented medium volume is 1000ml, and contains 70 g butanedioic acids and 55 g glycine；
7) in fermentation to 6h, 60 min anaerobic fermentations are carried out, namely stop stirring after fermented and cultured 5h, to recovery stirring during 6h.
Using Mauzerall and Granick analysis method (with reference to Mauzerall D, Granick S. J. Bio. Chem., 1956,219:435-442) ALA concentration is determined.As seen from Figure 1, operated by once of short duration anaerobic fermentation, extracellular ALA maximum concentration can reach 7.4 g/L.
Embodiment 2：
1) with transfer needle from engineering bacteria Rosetta (DE3)-pET28a-A.R. that deposit number is CGMCC No.1939hemAGlycerol tube in dip bacterium solution, on the LB culture medium flat plates containing 40 μ g/ml kanamycins and 30 μ g/ml chloramphenicol rule after, the incubated overnight at 37 DEG C；
2) monoclonal on flat board is inoculated in the 250ml shaking flasks of the culture mediums of LB containing 30ml, rotating speed is 220rpm, the incubated overnight at 37 DEG C obtains first order seed；
3) take 5ml first order seeds to be inoculated in the 500ml of multiple culture mediums of LB containing 75ml shaking flask and cultivate 3 .5h, obtain secondary seed；
4) 300ml secondary seeds are inoculated in 15 L fermentation tanks of the fermentation medium containing 7L and carry out fermented and cultured, fermentation tank speed of agitator is 500 rpm, air mass flow is 5L/min, it is 37 DEG C to originate cultivation temperature, 30 DEG C are down to after 3h, then is induced with final concentration of 0.2 mmol/L isopropyl-β-D-thiogalactosides；It by mass volume ratio is respectively 2% peptone, 0.25% dusty yeast, 1% butanedioic acid, 0.4% glycine and 0.5% glucose group into being 6. 3 with NaOH regulation pH value that wherein fermentation medium, which is,；
5) in cell density OD600During up to 5,3g/L D-Glucoses are added within every 3 hours in batches as 5-ALA dehydratase inhibitor, totally 3 times；
6) fermented and cultured initially controls pH 5.8 using the dilute sulfuric acid of 20% volume fraction；It is 6.3 to control pH by flow feeding culture medium after culture 6h；
Described supplemented medium volume is 800ml, and contains 90 g butanedioic acids and 70 g glycine；
7) in fermentation to 6h, 30 min anaerobic fermentations are carried out, namely 6h stops stirring, to recovery stirring during 6.5h.
Using Mauzerall and Granick analysis method (with reference to Mauzerall D, Granick S. J. Bio. Chem., 1956,219:435-442) ALA concentration is determined.Operated by once of short duration anaerobic fermentation, extracellular ALA maximum concentration can reach 7.2 g/L.
Embodiment 3
The method that recombination bacillus coli is metabolized is adjusted with of short duration anaerobic fermentation, is comprised the following steps：
1) with transfer needle from engineering bacteria Rosetta (DE3)-pET28a-A.R. that deposit number is CGMCC No.1939hemAGlycerol tube in dip bacterium solution, on the LB culture medium flat plates containing 40 μ g/ml kanamycins and 50 μ g/ml chloramphenicol rule after, the incubated overnight at 37 DEG C；
2) monoclonal on flat board is inoculated in the 250ml shaking flasks of the culture mediums of LB containing 50ml, rotating speed is 200rpm, the incubated overnight at 37 DEG C obtains first order seed；
3) take 1ml first order seeds to be inoculated in the 500ml of multiple culture mediums of LB containing 100ml shaking flask and cultivate 3 h, obtain secondary seed；
4) 200ml secondary seeds are inoculated in the 15 L fermentation tanks containing 9 L fermentation mediums and carry out fermented and cultured, fermentation tank speed of agitator is 400 rpm, air mass flow is 7 L/min, it is 37 DEG C to originate cultivation temperature, 27 DEG C are down to after 2h, then is induced with final concentration of 0.05 mmol/L isopropyl-β-D-thiogalactosides；It by mass volume ratio is respectively 1% peptone, 0.5% dusty yeast, 0.3% butanedioic acid, 0.2% glycine and 0.2% glucose group into being 6.1 with NaOH regulation pH value that wherein fermentation medium, which is,；
5) in cell density OD600During up to 5,5g/L D-Glucoses are added within every 3 hours in batches as 5-ALA dehydratase inhibitor, totally 3 times；
6) fermented and cultured initially controls pH 5.9 using the dilute sulfuric acid of 20% volume fraction；It is 6.2 to control pH by flow feeding culture medium after culture 6h；
Described supplemented medium volume is 1100ml, and contains 75 g butanedioic acids and 70 g glycine；
7) in fermentation to 5h, 45 min anaerobic fermentations are carried out, namely 5h stops stirring, to recovery stirring during 5.75h.
Using Mauzerall and Granick analysis method (with reference to Mauzerall D, Granick S. J. Bio. Chem., 1956,219:435-442) ALA concentration is determined.From Figure 2 it can be seen that being operated by once of short duration anaerobic fermentation, extracellular ALA maximum concentration can reach 8.6 g/L.
Embodiment 4
1) with transfer needle from engineering bacteria Rosetta (DE3)-pET28a-A.R. that deposit number is CGMCC No.1939hemAGlycerol tube in dip bacterium solution, on the LB culture medium flat plates containing 30 μ g/ml kanamycins and 50 μ g/ml chloramphenicol rule after, the incubated overnight at 37 DEG C；
2) monoclonal on flat board is inoculated in the 250ml shaking flasks of the culture mediums of LB containing 50ml, rotating speed is 200rpm, the incubated overnight at 37 DEG C obtains first order seed；
3) take 1ml first order seeds to be inoculated in the 500ml of multiple culture mediums of LB containing 100ml shaking flask and cultivate 3 h, obtain secondary seed；
4) 200ml secondary seeds are inoculated in the 15 L fermentation tanks containing 9 L fermentation mediums and carry out fermented and cultured, fermentation tank speed of agitator is 400 rpm, air mass flow is 7 L/min, it is 37 DEG C to originate cultivation temperature, 28 DEG C are down to after 2h, then is induced with final concentration of 0.05 mmol/L isopropyl-β-D-thiogalactosides；It by mass volume ratio is respectively 1% peptone, 0.5% dusty yeast, 0.3% butanedioic acid, 0.2% glycine and 0.2% glucose group into being 6.1 with NaOH regulation pH value that wherein fermentation medium, which is,；
5) in cell density OD600During up to 4, D-Glucose and precursor hybrid feedback stream are added in fermentation zymotic fluid, the pH for controlling lotion is 6.2；
6) fermented and cultured initially controls pH 5.9 using the dilute sulfuric acid of 20% volume fraction；It is 6.2 to control pH by the mixture of stream plus D-Glucose and supplemented medium after culture 6h；
The content of described D-Glucose is 145g, and supplemented medium volume is 1100ml, and contains 80 g butanedioic acids and 63 g glycine；
7) in fermentation to 5.5h, 75 min anaerobic fermentations are carried out, namely 5.5h stops stirring, to recovery stirring during 6.45h.
Using Mauzerall and Granick analysis method (with reference to Mauzerall D, Granick S. J. Bio. Chem., 1956,219:435-442) ALA concentration is determined.By once of short duration anaerobic fermentation operation, and the method added using D-Glucose and supplemented medium hybrid feedback stream, extracellular ALA maximum concentration can reach 8.2 g/L.
Embodiment 5
1) with transfer needle from engineering bacteria Rosetta (DE3)-pET28a-A.R. that deposit number is CGMCC No.1939hemAGlycerol tube in dip bacterium solution, on the LB culture medium flat plates containing 50 μ g/ml kanamycins and 40 μ g/ml chloramphenicol rule after, the incubated overnight at 37 DEG C；
2) monoclonal on flat board is inoculated in the 250ml shaking flasks of the culture mediums of LB containing 40ml, rotating speed is 210rpm, the incubated overnight at 37 DEG C obtains first order seed；
3) take 3ml first order seeds to be inoculated in the 500ml of multiple culture mediums of LB containing 50ml shaking flask and cultivate 4 h, obtain secondary seed；
4) 100ml secondary seeds are inoculated in the 15 L fermentation tanks containing 8 L fermentation mediums and carry out fermented and cultured, fermentation tank speed of agitator is 300 rpm, air mass flow is 10 L/min, it is 37 DEG C to originate cultivation temperature, 30 DEG C are down to after 2.5h, then is induced with final concentration of 0.02 mmol/L isopropyl-β-D-thiogalactosides；It by mass volume ratio is respectively 0.5% peptone, 1% dusty yeast, 0.5% butanedioic acid, 0.3% glycine and 0.1% glucose group into being 6.0 with NaOH regulation pH value that wherein fermentation medium, which is,；
5) in cell density OD600During up to 6, D-Glucose and precursor hybrid feedback stream are added in fermentation zymotic fluid, the pH for controlling lotion is 6.1；
6) fermented and cultured initially controls pH 5.8 using the dilute sulfuric acid of 20% volume fraction；It is 6.1 to control pH by the mixture of stream plus D-Glucose and supplemented medium after culture 5h；
The content of described D-Glucose is 145g, and supplemented medium volume is 900ml, and contains 70 g butanedioic acids and 50g glycine；
7) in fermentation to 5h, 45 min anaerobic fermentations are carried out, namely 5h stops stirring, to recovery stirring during 5.75h.
Using Mauzerall and Granick analysis method (with reference to Mauzerall D, Granick S. J. Bio. Chem., 1956,219:435-442) ALA concentration is determined.By once of short duration anaerobic fermentation operation, and the method added using D-Glucose and supplemented medium hybrid feedback stream, extracellular ALA maximum concentration can reach 7.6 g/L.
Embodiment 6
The method that recombination bacillus coli is metabolized is adjusted with of short duration anaerobic fermentation, is comprised the following steps：
1) with transfer needle from engineering bacteria Rosetta (DE3)-pET28a-A.R. that deposit number is CGMCC No.1939hemAGlycerol tube in dip bacterium solution, on the LB culture medium flat plates containing 50 μ g/ml kanamycins and 40 μ g/ml chloramphenicol rule after, the incubated overnight at 37 DEG C；
2) monoclonal on flat board is inoculated in the 250ml shaking flasks of the culture mediums of LB containing 50ml, rotating speed is 200rpm, the incubated overnight at 37 DEG C obtains first order seed；
3) take 1ml first order seeds to be inoculated in the 500ml of multiple culture mediums of LB containing 100ml shaking flask and cultivate 3 h, obtain secondary seed；
4) 200ml secondary seeds are inoculated in the 15 L fermentation tanks containing 9 L fermentation mediums and carry out fermented and cultured, fermentation tank speed of agitator is 400 rpm, air mass flow is 7 L/min, it is 37 DEG C to originate cultivation temperature, 28 DEG C are down to after 2h, then is induced with final concentration of 0.05 mmol/L isopropyl-β-D-thiogalactosides；It by mass volume ratio is respectively 1% peptone, 0.5% dusty yeast, 0.3% butanedioic acid, 0.2% glycine and 0.2% glucose group into being 6.1 with NaOH regulation pH value that wherein fermentation medium, which is,；
5) in cell density OD600During up to 4, D-Glucose and precursor hybrid feedback stream are added in fermentation zymotic fluid, the pH for controlling lotion is 6.2；
6) fermented and cultured initially controls pH 5.9 using the dilute sulfuric acid of 20% volume fraction；It is 6.2 to control pH by the mixture of stream plus D-Glucose and supplemented medium after culture 6h；
The content of described D-Glucose is 145g, and supplemented medium volume is 1100ml, and contains 80 g butanedioic acids and 63 g glycine；
7) in fermentation to 5h, 45 min anaerobic fermentations are carried out, namely 5h stops stirring, to recovery stirring during 5.75h.
Using Mauzerall and Granick analysis method (with reference to Mauzerall D, Granick S. J. Bio. Chem., 1956,219:435-442) ALA concentration is determined.As seen from Figure 3, by once of short duration anaerobic fermentation operation, and the method added using D-Glucose and supplemented medium hybrid feedback stream, extracellular ALA maximum concentration can reach 9.4 g/L.
Claims (1)
1. a kind of adjust the method that recombination bacillus coli is metabolized with of short duration anaerobic fermentation, it is characterised in that following steps：
Step (1) is with transfer needle from engineering bacteria Rosetta (DE3)-pET28a-A.R. that deposit number is CGMCC No.1939hemAGlycerol tube in dip after bacterium solution, containing 3050 μ g/ml kanamycins and 30
Rule on the LB culture medium flat plates of 50 μ g/ml chloramphenicol, and the LB culture medium flat plates after line are positioned over incubated overnight at 37 DEG C；
Monoclonal on LB culture medium flat plates is inoculated in containing 30 by step (2)
In the 250ml shaking flasks of 50ml LB culture mediums, and it is 200 to be placed on rotating speed
220rpm, temperature is incubated overnight in 37 DEG C of shaking table, obtains first order seed；
Step (3) takes 1
5ml first order seeds are inoculated in containing 50
In the 500ml of 100ml LB culture mediums shaking flask, and it is 200 to be placed on rotating speed220rpm, temperature is cultivates 3 in 37 DEG C of shaking table4h, obtains secondary seed；
Step (4) is by 100
300ml secondary seeds are inoculated in containing 7
Fermented and cultured is carried out in 15 L fermentation tanks of 9 L fermentation mediums, the speed of agitator of fermentation tank is 300500rpm, air mass flow is 5
10 L/min, starting cultivation temperature is 37 DEG C；2
Cultivation temperature is down to 26 after 3h
30 DEG C, and use 0.02
0.2mmol/L isopropyl-β-D-thiogalactosides are induced；
It by mass volume ratio is respectively 0.5 that fermentation medium in described step (4), which is,2% peptone, 0.251% dusty yeast, 0.3
1% butanedioic acid, 0.2
0.4% glycine and 0.1
0.5% glucose group is into its pH value is 6.0
6.3；Fermentation medium most starts to use 10
The dilute sulfuric acid of 20% volume fraction, by pH controls 5.8
6.0；
Step (5) is when thalli growth to 5After 6 hours, the speed of agitator of fermentation tank is adjusted to oxygen dissolving value in 0, zymotic fluid and is changed into 0 therewith, 30 are maintained
Recover the speed of agitator of fermentation tank after 75min anaerobic fermentation；
Described zymotic fluid, 4 are inoculated with secondary seed
Supplemented medium is added by feedback flow after 8h, the pH of zymotic fluid is adjusted to 6.1
6.3；
Described supplemented medium volume is 800
1100ml, and contain 7090 g butanedioic acids and 5070 g glycine；
Step (6) works as cell density OD600Reach 3
When 10,5-ALA dehydratase inhibitor is added in batches or continuously；
The inhibitor of described 5-ALA dehydratase is 3
5g/L D-Glucoses；
Deposit number in described step (1) is CGMCC No.1939 engineering bacterias, Classification And Nomenclature：The entitled ETEC of Chinese, Latin name isEscherichia coli Rosetta(DE3)-pET28a-A.R.hemA。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104195190A (en) * | 2014-09-04 | 2014-12-10 | 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 |
| CN114854809A (en) * | 2022-05-31 | 2022-08-05 | 可孚医疗科技股份有限公司 | Method for fermenting recombinant protein by micro-oxygen induced escherichia coli |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104195190A (en) * | 2014-09-04 | 2014-12-10 | 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 |
| CN114854809A (en) * | 2022-05-31 | 2022-08-05 | 可孚医疗科技股份有限公司 | Method for fermenting recombinant protein by micro-oxygen induced escherichia coli |
| 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 |
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