CN117965377A - Method for improving threonine fermentation yield of escherichia coli by combining fed-batch culture medium with oxygen regulation - Google Patents
Method for improving threonine fermentation yield of escherichia coli by combining fed-batch culture medium with oxygen regulation Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 80
- 230000004151 fermentation Effects 0.000 title claims abstract description 80
- 239000004473 Threonine Substances 0.000 title claims abstract description 69
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 title claims abstract description 36
- 239000001301 oxygen Substances 0.000 title claims abstract description 36
- 229910052760 oxygen Inorganic materials 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 31
- 241000588724 Escherichia coli Species 0.000 title claims abstract description 25
- 239000001963 growth medium Substances 0.000 title claims abstract description 19
- 230000033228 biological regulation Effects 0.000 title claims abstract description 17
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 title abstract description 31
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 claims abstract description 106
- 229960002898 threonine Drugs 0.000 claims abstract description 68
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 26
- AEQDJSLRWYMAQI-UHFFFAOYSA-N 2,3,9,10-tetramethoxy-6,8,13,13a-tetrahydro-5H-isoquinolino[2,1-b]isoquinoline Chemical compound C1CN2CC(C(=C(OC)C=C3)OC)=C3CC2C2=C1C=C(OC)C(OC)=C2 AEQDJSLRWYMAQI-UHFFFAOYSA-N 0.000 claims abstract description 24
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- 239000000176 sodium gluconate Substances 0.000 claims abstract description 24
- 235000012207 sodium gluconate Nutrition 0.000 claims abstract description 24
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- 239000000843 powder Substances 0.000 claims abstract description 21
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims abstract description 18
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- 235000013619 trace mineral Nutrition 0.000 claims abstract description 10
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 9
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- 238000012258 culturing Methods 0.000 claims description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 6
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- 229910000365 copper sulfate Inorganic materials 0.000 claims description 3
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 3
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- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 3
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- RDQMORJTLICVPR-UHFFFAOYSA-N phosphoric acid;2-(trimethylazaniumyl)acetate Chemical compound OP(O)([O-])=O.C[N+](C)(C)CC(O)=O RDQMORJTLICVPR-UHFFFAOYSA-N 0.000 claims description 3
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Abstract
The application specifically relates to a method for improving fermentation yield of escherichia coli ESCHERICHIA COLI BIO-59193 threonine by using phosphobetaine and oxygen regulation. The culture medium consists of the following components: 1.7g/L of ammonium sulfate, 2.8g/L of sodium gluconate, 4.0g/L of magnesium sulfate heptahydrate, 7.0g/L of sodium dihydrogen phosphate, 2.0g/L of phosphoric betaine, 16.0g/L of corn steep liquor, 2.0mL/L of trace elements and the balance of water. The method comprises the following steps: inoculating the activated escherichia coli bacterial liquid into an optimized fermentation tank culture medium according to an inoculation amount of 5% for culture, controlling dissolved oxygen to be not lower than 15% through oxygen supply regulation in the fermentation process, and controlling the oxygen consumption rate (Oxygen Uptake Rate, OUR) of cells to be about 200mmol/L/h through feeding a mixed solution of sodium gluconate, phosphoric acid betaine and corn steep liquor dry powder in real time. On the basis of optimizing the configuration of a fermentation medium, the application provides a solution for optimizing fed batch by using a linear oxygen consumption rate control system and the action relation between feed control and OUR regulation, the yield of L-threonine is improved by 35%, the yield of L-threonine reaches 155g/L, and the application has important significance for the industrial production of L-threonine by escherichia coli.
Description
Technical Field
The invention relates to the field of fermentation, in particular to a method for improving fermentation yield of escherichia coli ESCHERICHIA COLI BIO-59193 threonine by means of phosphobetaine and oxygen regulation.
Background
L-threonine (Threonine) is one of 8 amino acids necessary for human and animal, and has the functions of improving immunity, promoting growth of human body and fowl and livestock, etc. Is widely used in the aspects of medicines, food reinforcing agents, feed additives and the like. At present, threonine is mainly prepared by microbial fermentation of escherichia coli (ESCHERICHIA COLI) and the like. The fermentation condition of the escherichia coli with high yield of L-threonine is optimized, so that the yield of L-threonine can be effectively improved. The fermentation method has the advantages of low cost, higher yield, less environmental pollution and the like, and promotes the large-scale production of L-threonine.
L-threonine is closely related to the life and health of the human body and its effect cannot be varied. The L-threonine has irreplaceable effects on the synthesis of organism proteins and the normal performance of life activities, whether the L-threonine is used for human bodies or other animals, plants and microorganisms, and has unique and wide application in the feed industry, the food industry, the medicine industry and the environmental protection industry as described above. The research and optimization of the direct fermentation method for producing L-threonine not only has positive effects on the development of the amino acid production industry in China, but also promotes the progress and the vigorous development of biotechnology research in China. The current research on the production of L-threonine has a large gap at home and abroad, and the current industrial bottleneck of L-threonine in China is mainly low production efficiency, unstable heredity and low efficiency of producing related enzyme systems in terms of production strains after decades of research; the fermentation and conversion process has the problems of difficult control, more byproducts, low conversion rate, high energy consumption, serious pollution and the like.
In order to improve the competitiveness of L-threonine production in China, besides the need of developing excellent production strains, the need of strengthening and developing innovative production technology, optimizing production technology and improving the control level of the L-threonine fermentation process is further needed, so that the method has important significance for optimizing the L-threonine fermentation process and researching metabolism.
Therefore, the invention optimizes the optimal configuration condition of the fermentation medium and further combines with oxygen regulation to improve the yield of the L-threonine.
Disclosure of Invention
The invention aims to provide a fermentation medium and a culture method for producing L-threonine. The fermentation medium can improve the yield of L-threonine in the process of producing L-threonine by fermentation.
A first object of the present invention is to provide a fermentation medium which can be used for producing L-threonine.
In order to achieve the above object, the present invention provides the following solutions:
The fermentation medium provided by the invention comprises the following components in percentage by mass: 0.5-3.0g/L of ammonium sulfate, 1.5-4.0g/L of sodium gluconate, 2.0-6.0g/L of magnesium sulfate heptahydrate, 5.0-10.0g/L of sodium dihydrogen phosphate, 1.0-5.0g/L of betaine phosphate, 8.0-25.0g/L of corn steep liquor dry powder, 2.0-5.0g/L of potassium dihydrogen phosphate, 1.5-2.5mL/L of trace elements and pH 6.0-7.0.
Preferably, the trace elements are as follows in g/L: copper sulfate 6.00, sodium iodide 0.10, manganese sulfate 4.00, biotin 0.50, sodium molybdate 0.60, boric acid 0.08, cobalt chloride 10.00 and ferrous sulfate 80.00.
Further, the optimal mass concentration values of the components of the synthetic medium are as follows: 1.7g/L of ammonium sulfate, 2.8g/L of sodium gluconate, 4.0g/L of magnesium sulfate heptahydrate, 7.0g/L of sodium dihydrogen phosphate, 2.0g/L of phosphoric betaine, 16.0g/L of corn steep liquor and 2.0mL/L of trace elements.
The invention also provides a method for producing L-threonine by escherichia coli fermentation, which comprises the steps of inoculating the activated escherichia coli bacterial liquid into a fermentation tank culture medium according to an inoculation amount of 5% for culture, controlling dissolved oxygen to be not lower than 25% by adjusting oxygen supply in the fermentation process, controlling oxygen uptake rate (Oxygen Uptake Rate mmol/L/h) by reasonably adding sodium gluconate, phosphoric acid betaine and corn steep liquor dry powder in the fermentation process, and controlling the fermentation period to be 42 hours.
Further, the optimum value of the oxygen supply regulation control is: in the fermentation process, dissolved oxygen is controlled to be not lower than 15% by oxygen supply regulation, and OUR is controlled to be 200mmol/L/h by feeding mixed solution of sodium gluconate, phosphoric acid betaine and corn steep liquor dry powder in real time.
Further, the preparation method comprises the following steps: ammonium sulfate, sodium gluconate, magnesium sulfate heptahydrate, dihydrogen phosphate, phosphoric acid betaine, corn steep liquor and microelements are added into water to be mixed, and the liquid amount of a 50L fermentation tank is 20L.
Further, the method comprises: the sterilization temperature after the uniform mixing is 121 ℃, and the heat preservation and sterilization time is 30min.
Further, the preparation method of the synthetic culture medium is characterized by comprising the following steps of: the sterilization condition of the feed supplement glucose is 115 ℃, and the sterilization time is 20min.
Further, the fermentation culture specifically comprises: fermenting and culturing in the culture medium under the conditions that the aeration rate is 30L/min, the rotating speed is 700r/min and the fermentation temperature is 37 ℃; the pH was controlled to 6.5.+ -. 0.1.
Further, the fermentation culture specifically comprises: dissolving sodium gluconate, phosphoric acid betaine and corn steep liquor dry powder with concentration of 4 times of fermentation medium into water, sterilizing at 121deg.C for 30min.
Further, the fermentation culture specifically comprises: and in the whole culture process, 25% ammonia water is fed in to control pH, and the rotation speed and ventilation are adjusted to control dissolved oxygen by more than 15%. When dissolved oxygen starts to rise, glucose with the mass concentration of 65% is fed in, the initial flow acceleration is 1g/L/h, and when dissolved oxygen continuously rises, the sugar supplementing amount is increased, and the concentration of residual sugar is controlled to be not more than 1.0g/L. In the whole fermentation process, a mass spectrometer is used for detecting tail gas, and Biostar software is used for collecting process parameters including temperature, pH, DO, rotating speed, CER, OUR, RQ and other cell physiological parameters on line.
The invention discloses the following technical effects:
The novel culture medium and the oxygen regulation control are used for culturing E.coli, and the yield of L-threonine in shake flask fermentation reaches 7.15g/L, so that the yield is improved by about 1.86 times. The fermentation process is optimized on a 50L tank, and the OUR maintained by the oxygen consumption in the middle and later stages of fermentation can be well maintained at the optimal level of 215mmol/L/h by optimizing the oxygen consumption rate and the feeding mode. The results show that the yield of L-threonine is improved by 35 percent compared with the original initial fermentation medium and the oxygen regulation process, and 155g/L is achieved.
According to the invention, optimal carbon source, nitrogen source, phosphorus source and other regulating factors are screened through a combined optimization experiment, and an optimal fed-batch solution is provided by utilizing the action relation of an online oxygen consumption rate control system, feed control and OUR regulation, so that the method has important significance for industrial production of L-threonine by escherichia coli.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a contour plot of the effect of phosphate betaine, sodium gluconate, and corn steep liquor dry powder interactions on E.coli product synthesis;
FIG. 2 is a graph showing the variation of physiological metabolic parameters during fermentation;
FIG. 3 is a graph of metabolic process variation for different feed modes;
FIG. 4 is a graph of metabolic process changes at different oxygen consumption rate levels.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
Example 1
1.1 Materials
1.1.1 Starting Strain
The experimental strain is Escherichia coli ESCHERICHIA COLI BIO-59193.
1.1.2 Seed culture and preservation methods:
Preparation of solid LB culture medium: 5g/L of sodium chloride, 5g/L of yeast powder, 10g/L of peptone and 15g/L of agar are respectively packaged in 500ml triangular bottles, each bottle is sealed by a sterile film, is practically eliminated for 30min at 121 ℃, is taken out and cooled to about 55 ℃, and is poured into a flat plate.
The lawn (or the sucked culture solution) is picked up and streaked on a culture dish of a solid LB culture medium, and is inverted for 12 hours in an incubator at 37 ℃. Scraping lawn, inoculating into shake tube containing 5mL of liquid LB medium, and culturing at 37deg.C for 6 hr. 0.9mL of culture solution is taken and added into a freezing tube, then 0.3mL of 80% glycerol is added for uniform mixing, and the mixture is rapidly placed into a refrigerator at the temperature of minus 80 ℃ for freezing.
1.1.3 Seed shake flask cultivation process:
Preparing LB culture solution: 5g/L of sodium chloride, 5g/L of yeast powder and 10g/L of peptone are subpackaged in 500mL baffle shake flasks, 50mL of liquid is filled in each flask, a gauze is sealed, the mixture is subjected to actual digestion for 20min at 121 ℃, and the mixture is cooled to room temperature for standby.
The microelement mixture is prepared according to g/L: copper sulfate 6.00, sodium iodide 0.10, manganese sulfate 4.00, biotin 0.50, sodium molybdate 0.60, boric acid 0.08, cobalt chloride 10.00 and ferrous sulfate 80.00.
1.2 Method
1.2.1 Biomass OD 600 and dry weight determination
Dilution of stock fermentation broth to appropriate fold was performed on a spectrophotometer at 600nm to determine OD 600,OD600 as the dilution of the absorbance value measured.
1.2.2 Determination of the product L-threonine
Analysis conditions: column temperature 36 ℃, flow v=1 mL/min, absorption wavelength: 360nm, sample injection amount: 10 mu L.
The derivatization method comprises the following steps:
Placing the sample in boiling water bath for 10min to remove protein, centrifuging to obtain supernatant, adding 200 μl of derivatization buffer solution, 300 μl of derivatization agent, and 40 μl of sample to be tested, shaking for 1min, centrifuging to obtain supernatant. Cooling to room temperature after light-shielding water bath at 65 ℃ for 60min, and centrifuging to obtain supernatant. 660 mu L of constant volume buffer solution and constant volume to 1.2mL. The sample to be measured was subjected to membrane filtration treatment with a 0.2 μm organic needle filter.
1.2.3 Determination of residual sugar concentration
And (3) measuring by using an SBA-40E biosensing analyzer. After diluting the supernatant of the fermentation broth after centrifugation by a proper multiple, fully shaking and uniformly mixing, sucking 25 mu L of the diluted liquid by a microsyringe for measurement, directly reading, and obtaining the residual sugar concentration (g/L) =obtained index/100 times of dilution.
1.2.4 Seed plate activation
Under the aseptic condition, inoculating the strain in the glycerol seed-retaining tube to a resistant LB solid medium by a Z-shaped streaking method, and culturing for 16h at 37 ℃.
1.2.5 Seed shaking flask culture
Scraping fresh bacterial colonies, putting the bacterial colonies into a 500mL round bottom triangular flask, filling 100mL of liquid, sealing by 8 layers of gauze, placing the bacterial colonies on a circulating shaking table, 220r/min, and carrying out shake culture at 35 ℃ for 12h.
1.2.6 Shaking flask fermentation
The 500mL round bottom flask was filled with 50mL of liquid, and the fermentation flask was additionally charged with 0.5g/L of calcium carbonate for short-term pH stabilization, 2 drops/flask of defoamer, prior to sterilization. The inoculation amount is 8%,8 layers of gauze are sealed, and the mixture is placed on a circulating shaking table, 220r/min and cultured for 36h at 35 ℃.
1.2.7 50L fermentation tank culture
The pH electrode was calibrated and the dissolved oxygen was calibrated by 0% with saturated sodium sulfite solution. The liquid amount of the 50L fermentation tank is 20L, sterilization is carried out for 30min at 121 ℃, the temperature is reduced to 37 ℃ before inoculation, stirring is carried out for 700r/min, the ventilation is 30L/min, and the dissolved oxygen is 100%. The inoculation amount is 5% (V/V), 25% ammonia water is fed in the whole process to control pH to 6.5, defoamer is fed in, and the rotating speed is adjusted to control dissolved oxygen to be more than 20% in the fermentation process. When dissolved oxygen is reduced to the minimum, glucose with the mass concentration of 65% is fed back when the rising is started, and the initial flow acceleration is 1g/L/h. When dissolved oxygen continuously rises, the sugar supplementing amount is increased, and the concentration of residual sugar is controlled to be about 0.01 g/L. In the whole fermentation process, a mass spectrometer is used for detecting tail gas, and Biostar software is used for collecting process parameters including temperature, pH, DO, rotating speed, CER, OUR, RQ and other cell physiological parameters on line.
Implementation example 1 Single factor Key factor optimization Experimental design
The Plackett-Burman experiment was used to determine the important factor components in the medium during L-threonine synthesis, and the matrix Design was performed by Design Expert software, and the factors and levels of the Design are shown in Table 1. With the influence of the analysis software on the response values of the various medium components, the statistical results are shown in table 2, and the statistics show that the regression equation established for the biomass score is significant (x) because p-value=0.0105 <0.05 and model F-value=432.6. The P-value of 0.0046<0.01 is an extremely significant factor for threonine synthesis. Research indicates that nitrogen sources are important for improving threonine productivity and are indispensable elements in the anabolism of proteins, nucleotides and threonine. The P value of sodium gluconate, corn steep liquor dry powder and ammonium sulfate is less than 0.05, and the influence on the threonine synthesis of cells is a significant factor. Analysis of the Plackett-Burma test results gave an approximate reference range for the several factors examined, and the experimental results determined that the concentrations of the several media components were: 1.7g/L of ammonium sulfate, 2mL/L of trace elements, 4.0g/L of disodium hydrogen phosphate, 7.5g/L of sodium dihydrogen phosphate, 3.5g/L of betaine phosphate, 16g/L of corn steep liquor and 2.0mL/L of trace elements.
TABLE 1 factors and levels of Plackett-Burman design
TABLE 2Plackett-Burman design threonine fermentation test results
Example 2Central Composite Design test design and results analysis
Further, two factors that determine significant L-threonine production were examined using a center combination test: and (5) carrying out refinement investigation on the optimal addition amount of the phosphoric acid betaine, the sodium gluconate and the corn steep liquor dry powder. And taking threonine yield as an index, and examining the influence of three factors on dependent variables. Each set is provided with three sets of parallelism. The experimental design and results for each factor and level are shown in table 3.
The analysis results of these 3 factors are shown in table 4, in order to predict the optimal point, analysis of variance and quadratic polynomial regression fitting are performed on the test data using Design Expert 8.06 software, the model decision coefficient R 2 = 0.9878 with threonine yield as a response value, the correction decision coefficient R 2 Adj =0.9769, p <0.0001, and the model is highly significant. The p-values of the univariate quadratic terms are all less than 0.0001, indicating that three factors have a very significant effect on threonine product yield.
Table 3Central Composite Design designs and results
Performing multiple quadratic regression fitting on the experimental result by using Design Expert software to obtain a regression equation ：Threonine＝7.01-0.17*A-0.35*B+0.61*C-0.15*A*B-0.085*A*C-0.39*B*C-1.23*A2-0.79*B2-2.30*C2
Wherein the predicted threonine production, A is the phosphobetaine concentration, B is the sodium gluconate concentration, C is the corn steep liquor dry powder concentration, the coefficients preceding A, B, C are the linear correlation coefficients, the coefficients preceding A 2、B2、C2 are the square coefficients, and the coefficients preceding AB, AC, BC are the interaction coefficients. And generating an analysis chart according to a regression equation, and examining the shape of the response surface. The contour map of the response surface of each factor is shown in fig. 1, and the contour centers of the three response surfaces are all in a set range, which indicates that the optimal design condition exists in the designed factor level. The three-dimensional response curved surface is arc-shaped, and the interaction between the adding amount of the phosphoric acid betaine, the sodium gluconate and the corn steep liquor dry powder is as follows: and respectively fixing the phosphoric acid betaine, the sodium gluconate and the corn steep liquor dry powder, wherein the threonine yield tends to rise and then fall along with the increase of the concentration of the other two factors, and the curved surface top point is the highest threonine product yield point.
Optimization analysis of the 3 factors of CCD (phosphobetaine, sodium gluconate, corn steep liquor dry powder) gave the following: 2.0g/L, sodium gluconate: 2.8g/L, corn steep liquor dry powder: 16.0g/L. The shaking flask experiment proves that the threonine yield is 7.15g/L in the culture medium under the optimal condition, and the model is reasonable. Provides good data support for further and deeply researching the physiological characteristics of the escherichia coli and efficiently producing threonine.
Table 4Central Composite Design test analysis of variance
EXAMPLE 3 batch fermentation Process for threonine production by E.coli
In a 15L seed tank, culturing Escherichia coli with seed culture medium, controlling dissolved oxygen not lower than 25%, culturing for 12 hr, and inoculating into a fermentation tank containing 20L fermentation culture medium according to 5% of inoculation amount when bacterial concentration OD 600 reaches above 10. And (3) performing fermentation verification experiments on the optimized culture medium under a fermentation system of a 50L tank, wherein physiological metabolic parameters in the fermentation process are shown in figure 2. Experimental results show that the optimal proportion obtained after the optimization of the culture medium is adopted for carrying out the batching and sterilization operation, the residual sugar of the fermentation liquid is controlled to be not higher than 1g/L by supplementing sugar in the fermentation process, the dissolved oxygen is controlled to be not lower than 25% by stirring rotation speed and ventilation quantity in the fermentation process, the result shows that the growth speed of thalli in the fermentation process is higher than 16h, the bacterial concentration OD 600 reaches more than 75, and the fermentation oxygen consumption rate OUR and the release rate of carbon dioxide reach more than 140 mmol/L/h. The fermentation time is 20 hours, and the yield of L-threonine reaches 92g/L. But after 16 hours, OUR shows a rapid decline trend, when the growth rate of the thalli is obviously reduced and fermentation is carried out for 32 hours, OUR is reduced to below 71mmol/L/h, and the content of L-threonine is not increased.
EXAMPLE 4 OUR-directed media fed-batch control Process
In order to better promote the maintenance of the OUR level after 16 hours, a regulation experiment of supplementing the mixed nutrient of the phosphoric acid betaine, the sodium gluconate and the corn steep liquor dry powder is implemented, the experimental result is shown in a figure 3, the added phosphoric acid betaine experimental group is added, the OUR in the fermentation process can be well maintained, and the increase of the bacterial concentration and the synthesis of threonine are promoted to a certain extent. On this basis, the synthesis rate of threonine was significantly increased in the experimental group to which sodium gluconate was added.
Meanwhile, under the condition of supplementing the dry powder of the phosphoric acid betaine, the sodium gluconate and the corn steep liquor, OUR can be well maintained at 175mmol/L/h, meanwhile, the concentration of thalli is obviously improved, the OD 600 value reaches more than 75, the synthesis rate of threonine is well maintained, and the highest threonine synthesis rate reaches 141g/L after 36 hours of fermentation.
Example 5 control of OUR levels at different oxygen consumption rates to enhance threonine production
Nutrition and oxygen supply are important factors affecting threonine production by escherichia coli fermentation, and the oxygen consumption rate level can be regulated by oxygen supply conditions without limitation of nutrient supply. OUR level after entering the stabilization period is controlled to be 215+ -13 mmol/L/h and 240+ -16 mmol/L/h respectively by adjusting the rotation speed and ventilation. As shown in fig. 4, the experimental results show that the time of the logarithmic growth phase of the cells was significantly prolonged and the cell concentration also showed a significant trend of increasing with the increase of the oxygen supply rate, but the fermentation unit of threonine was different from the trend of change in cell concentration. When OUR is controlled at 215+/-13 mmol/L/h, the cell concentration OD 600 can reach 80 at the highest, and the fermentation unit of threonine reaches 155g/L at the highest. However, as the oxygen consumption rate increased to 240.+ -.16 mmol/L/h, the threonine synthesis rate before 24 hours increased significantly although the cell concentration OD 600 reached 85 or more, but the highest threonine fermentation unit did not improve well.
The application specifically relates to a method for improving fermentation yield of escherichia coli ESCHERICHIA COLI BIO-59193 threonine by using phosphobetaine and oxygen regulation. The culture medium consists of the following components: 1.7g/L of ammonium sulfate, 2.8g/L of sodium gluconate, 4.0g/L of magnesium sulfate heptahydrate, 7.0g/L of sodium dihydrogen phosphate, 2.0g/L of phosphoric betaine, 16.0g/L of corn steep liquor and 2.0mL/L of trace elements. The balance being water. The method comprises the steps of inoculating the activated escherichia coli bacterial liquid into an optimized fermentation tank culture medium according to an inoculation amount of 5% for culture, controlling dissolved oxygen to be not lower than 15% through oxygen supply regulation in the fermentation process, and feeding a mixed solution of sodium gluconate, phosphoric acid betaine and corn steep liquor dry powder in real time, wherein OUR is about 200 mmol/L/h. On the basis of optimizing the configuration of a fermentation medium, the application provides a solution for optimizing fed batch by using a linear oxygen consumption rate control system and the action relation between feed control and OUR regulation, the yield of L-threonine is improved by 35%, the yield of L-threonine reaches 155g/L, and the application has important significance for the industrial production of L-threonine by escherichia coli.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (8)
1. A medium for producing L-threonine by fermentation, wherein the medium comprises the following components: 0.5-3.0g/L of ammonium sulfate, 1.5-4.0g/L of sodium gluconate, 2.0-6.0g/L of magnesium sulfate heptahydrate, 5.0-10.0g/L of sodium dihydrogen phosphate, 1.0-5.0g/L of betaine phosphate, 8.0-25.0g/L of corn steep liquor dry powder, 2.0-5.0g/L of potassium dihydrogen phosphate, 1.5-2.5mL/L of trace elements and pH 6.0-7.0.
2. The method of claim 1, wherein the medium employed comprises the following composition: 1.7g/L of ammonium sulfate, 2.8g/L of sodium gluconate, 4.0g/L of magnesium sulfate heptahydrate, 7.0g/L of sodium dihydrogen phosphate, 2.0g/L of phosphoric betaine, 16.0g/L of corn steep liquor dry powder and 2.0mL/L of trace elements. The trace elements are calculated according to g/L: copper sulfate 6.00, sodium iodide 0.10, manganese sulfate 4.00, biotin 0.50, sodium molybdate 0.60, boric acid 0.08, cobalt chloride 10.00 and ferrous sulfate 80.00.
3. A fermentation method for producing L-threonine by fermentation, comprising the step of inoculating an activated escherichia coli bacterial liquid to the medium according to any one of claims 1 to 2 in an inoculum size of 5% to perform fermentation to produce L-threonine.
4. A method according to claim 3, wherein the medium according to any one of claims 1-2 is fermented on a 50L fermenter. Fermenting and culturing under the conditions of ventilation of 30L/min, rotating speed of 700r/min and fermentation temperature of 37 ℃; the pH was controlled to 6.5.+ -. 0.1. The dissolved oxygen is controlled to be not lower than 15% by oxygen supply regulation in the fermentation process, and the oxygen consumption rate (Oxygen Uptake Rate, OUR) is controlled to be 90-300mmol/L/h in the process.
5. The method of claim 3, wherein the mixed solution of sodium gluconate, phosphoric acid betaine and corn steep liquor dry powder is fed in real time during the fermentation process, and the OUR is controlled at 200mmol/L/h.
6. A culture medium according to any one of claims 1-2, characterized in that an L-threonine-producing escherichia coli engineering strain (ESCHERICHIA COLI BIO-59193) is cultivated.
7. A culture medium according to any one of claims 1-2, characterized by its use in the fermentative production of L-threonine.
8. A fermentation process according to claim 3 to 5, wherein L-threonine is produced by fermentation.
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