CN119242554A - Fermentation medium, fermentation method and application of ectoine-producing engineered strain - Google Patents
Fermentation medium, fermentation method and application of ectoine-producing engineered strain Download PDFInfo
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Abstract
本发明属于微生物发酵技术领域，公开了一种产依克多因工程菌株的发酵培养基、发酵方法及应用。本发明的产依克多因工程菌株的发酵培养基包括以下组分：葡萄糖5g/L‑35g/L，蛋白胨5g/L‑30g/L，酵母浸粉5g/L‑30g/L，天冬氨酸钠20mM‑250mM，硫酸铵2g/‑12g/L，磷酸二氢钾5g/L‑10g/L，磷酸氢二钾10g/L‑18g/L，硫酸镁0.5g/L‑2g/L，一水柠檬酸0.5g/L‑2g/L，复合维生素B0.05g/L‑0.5g/L。本发明的发酵方法采用同时流加碳源与氮源的双流加方式补料，发酵罐水平的依克多因产量显著提高，为依克多因工业化规模的生产提供技术支持。
The present invention belongs to the technical field of microbial fermentation, and discloses a fermentation medium, a fermentation method and an application of an engineered strain producing ectoine. The fermentation medium of the engineered strain producing ectoine of the present invention comprises the following components: 5g/L-35g/L of glucose, 5g/L-30g/L of peptone, 5g/L-30g/L of yeast extract powder, 20mM-250mM of sodium aspartate, 2g/-12g/L of ammonium sulfate, 5g/L-10g/L of potassium dihydrogen phosphate, 10g/L-18g/L of dipotassium hydrogen phosphate, 0.5g/L-2g/L of magnesium sulfate, 0.5g/L-2g/L of citric acid monohydrate, and 0.05g/L-0.5g/L of complex vitamin B. The fermentation method of the present invention adopts a dual-flow feeding method of simultaneously feeding a carbon source and a nitrogen source, and the ectoine yield at the fermentation tank level is significantly improved, providing technical support for the industrial-scale production of ectoine.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a fermentation medium for producing an engineering strain of exendin, a fermentation method and application.
Background
Ectoine (Ectoine) is a zwitterionic amino acid derivative that is an extreme enzymatic component. Ectoine is strongly hydrophilic, has strong water molecule complexing ability, and can tightly capture and orderly structure surrounding free water molecules to form a unique 'Ectoine-water complex'. The complex looks like a fine and smooth hydration shell with the functions of protection, nourishing and stabilization, and tightly surrounds cells, enzymes, proteins and other key biological molecules, so that the osmotic pressure balance of the cells is maintained. It can protect bioactive substances such as intracellular proteins, enzymes, nucleic acids, etc. under extreme environmental conditions such as high temperature roasting, dry invasion, radiation or severe cold freezing, etc., and resist invasion from external severe conditions. Therefore, the ectoin shows remarkable effect on resisting the damage of the environmental pressure to the skin, promotes the natural repair process of the skin damage, and builds a solid defense line for skin health. At present, the ectoin is recognized as an excellent biological function stability, and has obvious skin protection effects of repairing, anti-wrinkle, whitening, anti-inflammatory, ultraviolet-proof, antioxidant stress, long-acting moisturizing and the like, and is widely applied to the fields of skin care products, medicines and the like. The recommended dosage of the ectoin in the cosmetics is larger (about 2%), so that the market potential and the demand of the ectoin are highlighted, but the use cost of the ectoin in the cosmetics is higher, and the application expansion is limited.
The current technology field of the Ikeduo factor fermentation mainly uses two fermentation technologies, namely, a 'cell milking method' fermentation production technology of a wild strain 'high-salt induction synthesis-low-salt release' is utilized to produce Ikeduo factor, but the method has high equipment requirement, complex technology, high difficulty and low yield, for example, chinese patent document CN117070397A discloses a culture medium and a method for producing Ikeduo factor by utilizing the same, and the high-concentration NaCl is utilized to stimulate cells to produce Ikeduo factor, so that the fermentation period is long, the Ikeduo factor yield is low, and the practical application and feasibility in the industrial large-scale production of Ikeduo factor are limited. The other process is to construct engineering strain of the ectoin, optimize metabolic pathway, optimize fermentation medium formulation and fermentation condition to produce the ectoin. Carbon source, nitrogen source, inorganic salt, growth factor and the like are important components of a microbial fermentation culture system, the types of the microbial fermentation culture system are various, the components and the proportion directly influence the growth speed of a strain and the yield of metabolites, for example, chinese patent document CN115651943A discloses a fermentation preparation method of a micromolecular amino acid derivative ectoin, which only uses sodium glutamate as the carbon source, does not optimize fermentation conditions, and has long fermentation time and low yield.
Therefore, development of a new process for producing the ectoin by fermentation is needed to solve the problems of low yield, long production period and high production cost of the ectoin by microbial fermentation in the prior art.
Disclosure of Invention
The invention aims to overcome the defects of low yield, long production period and high production cost of microbial fermentation of the ectoin in the prior art, and provides a fermentation medium, a fermentation method and application of an ectoin-producing engineering strain.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides a fermentation medium for producing an engineering strain of ectoin, comprising the following components:
5g/L-35g/L of glucose, 5g/L-30g/L of peptone, 5g/L-30g/L of yeast extract powder, 20mM-250mM of sodium aspartate, 2g/-12g/L of ammonium sulfate, 5g/L-10g/L of monopotassium phosphate, 10g/L-18g/L of dipotassium phosphate, 0.5g/L-2g/L of magnesium sulfate, 0.5g/L-2g/L of citric acid monohydrate and 0.05g/L-0.5g/L of vitamin B complex.
The fermentation medium is designed based on the metabolic characteristics of the escherichia coli ectoine, and the average ectoine yield of the ectoine test group is 48.36g/L which is improved by 54.12 percent compared with a basic culture system (22.19 g/L) by adopting the fermentation medium of the ectoine-producing engineering strain as a small fermentation test, so that the ectoine yield is directly and obviously influenced.
As a preferred embodiment of the fermentation medium, the fermentation medium further comprises trace elements, wherein the trace elements comprise the following components:
HCI solution of 0.5g/L-2g/L of manganese sulfate, 5g/L-10g/L of ferrous sulfate, 0.5g/L-1.5g/L of cobalt chloride, 2g/L-5g/L of zinc chloride, 0.05g/L-0.5g/L of sodium molybdate, 0.5g/L-1.5g/L of calcium chloride and 2mol/L-8 mol/L.
As a preferred embodiment of the fermentation medium according to the invention, the pH of the fermentation medium is 6-7.8.
As a preferred embodiment of the fermentation medium according to the invention, the following components are included:
20g/L-25g/L glucose, 15g/L-21.5g/L peptone, 10g/L-11g/L yeast extract powder, 100mM-200mM sodium aspartate, 2 g/L ammonium sulfate-6 g/L, 5g/L-7g/L potassium dihydrogen phosphate, 10g/L-14g/L dipotassium hydrogen phosphate, 0.5g/L-1g/L magnesium sulfate, 0.5g/L-1.1g/L citric acid monohydrate and 0.05g/L-0.1g/L vitamin B complex.
In a second aspect, the invention provides a fermentation method of an engineering strain producing exendin, comprising the following steps:
(1) Activating and culturing the exendin-producing engineering strain to obtain an activated bacterial liquid;
(2) Transferring the activated bacteria liquid into a seed culture medium for culture to obtain seed liquid;
(3) Inoculating the seed liquid into the fermentation medium for fermentation.
As a preferred embodiment of the fermentation method of the present invention, in the step (3), the inoculation amount at the time of transfer is 1% -10%, and preferably, the inoculation amount is 6% -10%.
As a preferred embodiment of the fermentation method, in the step (3), the fermentation condition is that the temperature is 35-40 ℃, the initial rotating speed is 150-250 rpm, the initial air quantity is 50-150L/min, the pH value is controlled to be 6.5-7.5, and the related rotating speed is 200-800 rpm.
As a preferred embodiment of the fermentation method of the present invention, in the step (3), the fermentation time is 36h to 72h. Preferably, the fermentation time is 52h-68h.
As a preferred embodiment of the fermentation method of the present invention, in the step (3), the amount of dissolved oxygen at the time of the fermentation is not less than 20%.
As a further preferable embodiment of the fermentation method of the invention, glucose with concentration of 30% -40% is fed to control the residual sugar in the fermentation broth to be 1g/L-5g/L when the dissolved oxygen is lower than 70%, and peptone with concentration of 15% -25% is fed to be 0-160mL/h.
The fermentation method adopts a double-flow feeding mode of feeding carbon sources and nitrogen sources simultaneously, the yield of the ectoin at the level of the fermentation tank is obviously improved, especially when dissolved oxygen is lower than 70%, the whole process of feeding glucose with 40% concentration is started to control 2g/L of residual sugar, and the constant-speed feeding of 20% peptone is started to 160mL/h, fermentation is carried out for 52h, and the yield of the ectoin is 176.32g/L.
In a third aspect, the invention provides the fermentation medium, and the fermentation method is applied to the industrial scale production of the ectoin.
Compared with the prior art, the invention has the beneficial effects that:
According to the invention, the optimal fermentation medium of the ectoin is screened and optimized through a carbon source, a nitrogen source and a growth factor, the fermentation condition is optimized, and a response surface method is utilized to optimize a fermentation system by combining a Plackett-Burman experiment and a steepest climbing experiment on the basis of a single factor experiment. The double-flow fermentation feeding technology is adopted on the fermentation tank level to amplify the fermentation process, so that the yield of the ectoin is improved. According to the invention, in the fermentation process of the engineering strain of the ectoin, the induction agents such as high-price IPTG and the like are not required to be added to induce the strain to produce the ectoin, toxic substances are avoided from being introduced, unnecessary nutrient addition is avoided through model analysis, a fermentation system is optimized, the production cost is greatly reduced, and a double-flow-addition fermentation feeding process is adopted to provide technical support for the industrial scale production of the ectoin. The invention provides technical support for the industrial scale production of the ectoin.
Drawings
FIG. 1 is a standard curve of exendin;
FIG. 2 is a graph of response curves and contours of peptone and glucose interactions on the effect of ekinoine production;
FIG. 3 is a graph of response curves and contours of glucose and yeast dip interactions on the effect of Ikeduo production;
FIG. 4 is a plot of response curves and contours of initial pH and glucose interactions on the effect of Ikeduoine production;
FIG. 5 is a graph of response curves and contours of yeast extract and peptone interactions on the effect of ekinoine production;
FIG. 6 is a plot of response curves and contours of initial pH and peptone interactions on the effect of ekinoine production;
FIG. 7 response curves and contour plots of initial pH and yeast dip interactions on the effect of Ikeduoine production.
Detailed Description
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.
The term "comprising" is intended to include the stated element, integer or step, but does not exclude any other element, integer or step. In this document, the term "comprising" when used in the sense of "including" unless otherwise specified, also covers the case of consisting of the recited elements, integers or steps. For example, when referring to a medium "comprising" a component or components, it is also intended to encompass a medium consisting of the particular component or components.
In the case of the engineering strain producing the ectoin, the starting strain of the engineering strain is Escherichia coli capable of producing the ectoin or a downstream derivative thereof, the engineering strain is a genetically engineered strain obtained by mutating a gene related to the ectoin metabolic pathway, and the mutating includes a manner of at least partially inactivating a gene function by gene deletion, gene insertion or gene substitution. The fermentation medium and the fermentation process are designed based on the metabolic characteristics of the escherichia coli ectoin, so that the ectoin yield of the ectoin engineering strain can be remarkably improved, wherein the ectoin engineering strain comprises an escherichia coli ESCHERICHIA COLI ECT-W020 (GDMCCNo:65012) strain, an escherichia coli ESCHERICHIA COLI ET strain, an escherichia coli ESCHERICHIA COLI ET strain (refer to Metabolic Engineering of Escherichia coli for Ectoine Production With a Fermentation Strategy of Supplementing the Amino Donor.Frontiers in Bioengineering and Biotechnology.2022)、 escherichia coli ESCHERICHIA COLI MWZ003 strain (refer to Metabolic engineering of Escherichia coli for efficient ectoine production.Systems Microbiology and Biomanufacturing.2021) and the like).
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples. It will be appreciated by persons skilled in the art that the specific embodiments described herein are for purposes of illustration only and are not intended to be limiting.
The test methods used in the examples are conventional methods unless otherwise specified, and the materials, reagents, etc. used, unless otherwise specified, are commercially available. The invention is exemplified by the engineering strain E.coli ESCHERICHIA COLI ET11 producing Exclusive.
In the following examples, the method for testing the concentration of ectoin in the fermentation broth was:
(1) Pretreatment of sample of fermentation liquor containing ectoine culture medium
The fermentation broth was added with three volumes of absolute ethanol. Shaking for 30min, centrifuging at high speed for 10min at 6000r/min, removing precipitate, collecting supernatant, blowing nitrogen to remove solvent, and adding anhydrous ethanol for ultrasonic dissolution for 15min. Centrifuging at high speed for 10min at 6000r/min, removing precipitate, collecting supernatant, blowing nitrogen, adding water, and performing vortex dissolution by HPLC.
(2) Conditions for detecting ectoine by HPLC
ZORBAX StableBond aq columns (4.6X250 mm,5 μm);
100% of 0.02M potassium dihydrogen phosphate;
the flow rate is 0.5mL/min;
the loading amount is 10 mu L;
Column temperature 25 ℃;
The detection wavelength is 210nm;
the elution mode is isocratic elution.
(3) Excreta standard curve
500Mg of the ectoin standard substance is dissolved in an aqueous solution and the volume is fixed to 5mL, 100mg/mL ectoin mother solution is prepared, and the solution is stored at 4 ℃ in a dark place.
The ectoine mother solution with the concentration of 100mg/mL is diluted by water to prepare a series of standard working solutions of 1mg/mL,3mg/mL,5mg/mL,10mg/mL,30mg/mL and 50mg/mL respectively, and the solutions are filtered by a 0.22 mu m filter membrane and detected by HPLC. The standard curve is plotted with the concentration of the ectoin standard working solution on the abscissa and the HPLC peak area of ectoin on the ordinate (see fig. 1).
In the detection method, the concentration of the exendin is in a linear relation in the concentration range of 5-100 mug/mL, the obtained standard curve equation is y=46.696 x-1.3824, and the correlation coefficient R 2 =0.9999, which indicates that the concentration of the exendin in a sample can be quantified by using a standard curve.
Example 1 fermentation Medium for producing engineering strains of Eimeredi E.coli
1. Preparation of culture Medium
The chloramphenicol mother liquor is prepared by dissolving 0.34g chloramphenicol in 10mL absolute ethanol, filtering with sterile organic filter membrane, packaging, and preserving at-20deg.C.
LB plate, yeast extract 5g/L, tryptone 10g/L, sodium chloride 10g/L, agar 16g/L. Sterilizing at pH 7.0+ -0.1,121 deg.C for 20min;
LB broth, 5g/L yeast extract, 10g/L tryptone and 10g/L sodium chloride. Sterilizing at pH 7.0+ -0.1,121 deg.C for 20min;
the seed culture medium comprises 20g/L glucose, 5g/L yeast extract, 10g/L tryptone and 10g/L sodium chloride. Sterilizing at pH 7.2+ -0.1,121 deg.C for 20min.
Basic fermentation medium comprises 20g/L glucose, 10g/L yeast extract powder, 7g/L potassium dihydrogen phosphate, 14g/L dipotassium hydrogen phosphate, 1g/L magnesium sulfate, 1.1g/L citric acid monohydrate, 2mL/L microelements (1.5 g/L manganese sulfate, 7g/L ferrous sulfate, 0.8g/L cobalt chloride, 4g/L zinc chloride, 0.1g/L sodium molybdate, 1.1g/L calcium chloride, dissolved in 5mol/L HCI solution), 0.1g/L vitamin B complex, and sterilized at 121 ℃ for 20min;
Chloramphenicol was added to a final concentration of 34 μg/mL prior to use of the above medium.
2. Component optimization experiments of Excreta Medium
Strain activation, namely inoculating the stored engineering strain E.coli producing the ectoin on an LB plate for activation, culturing for 24 hours at 37 ℃, inoculating single bacteria into a 5mL LB test tube, and culturing for 12 hours at 200rpm at 37 ℃.
Seed liquid culture, namely transferring 1% of activated bacterial liquid into a seed culture medium to be cultured for 16 hours at 37 ℃ and 200rpm, wherein the liquid volume of the bacterial liquid is 50mL/250 mL.
The seed solution is inoculated into a 500mL shaking flask containing 200mL of basic fermentation medium according to 10% of inoculation amount, the initial pH value is regulated to 7.2 by NaOH, and the mixture is placed in a shaking incubator to set the fermentation temperature to 37 ℃ and 200rpm, and the shaking incubator is used for 43h of fermentation. On the basis of the basal fermentation medium, the influence of the following factors on the yields of escitalopram was examined, respectively.
The carbon source type screening is carried out by taking 10g/L yeast extract powder as nitrogen source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, citric acid monohydrate, microelements and vitamin B complex as basic fermentation culture medium, respectively preparing 5 culture mediums with 20g/L soluble corn starch, maltodextrin, glucose, sucrose and glycerin as carbon sources, and regulating pH to 7.2.
The results of the influence of different carbon sources are shown in Table 1, and glucose is most favorable for synthesizing the ectoin by the engineering strain, and the yield is 16.52g/L. Thus, glucose was selected as the carbon source.
TABLE 1 Effect of different carbon sources on Excreta production
The organic nitrogen source type screening comprises the steps of taking 20g/L glucose as a carbon source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, citric acid monohydrate, microelements and vitamin B complex as basic fermentation culture mediums, respectively preparing 5 culture mediums which take 10g/L yeast extract powder, corn steep liquor dry powder, peptone, beef extract and tryptone as organic nitrogen sources, and regulating pH to 7.2.
The results of the influence of the organic nitrogen source are shown in Table 2, and peptone is most favorable for synthesizing the ectoin by the engineering strain, and the yield is 20.85g/L. The yeast is soaked for powder times, and the yield is 18.26g/L.
TABLE 2 Effect of different Nitrogen sources on Excreta yield
The single nitrogen source and the combined nitrogen source are selected by taking 20g/L glucose as a carbon source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, citric acid monohydrate, microelements and vitamin B complex as basic fermentation culture mediums, respectively preparing 3 culture mediums which take 10g/L yeast extract powder, 10g/L peptone, 10g/L yeast extract powder and 10g/L peptone as organic nitrogen sources, and regulating the pH value to 7.2.
Further screening and comparing the effects of peptone, yeast extract powder and peptone and yeast extract powder, and finding that the effects of peptone and yeast extract powder are better than the effects of single nitrogen source.
TABLE 3 Effect of Single and Combined Nitrogen sources on Excreta yield
The inorganic nitrogen source type screening comprises the steps of taking 20g/L glucose as a carbon source, taking 10g/L peptone and 10g/L yeast extract powder as organic nitrogen sources, respectively preparing 5 culture mediums of 5g/L urea, ammonium sulfate, ammonium bicarbonate, ammonium citrate and sodium nitrite as inorganic nitrogen sources, and regulating pH to 7.2, wherein the basic fermentation culture mediums comprise potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, citric acid monohydrate, trace elements and vitamin B complex.
The results of the influence of the inorganic nitrogen source are shown in Table 4, and the ammonium sulfate is favorable for synthesizing the ectoine by the engineering strain, and the yield is 24.75g/L.
TABLE 4 influence of inorganic Nitrogen Source on the yields of Excreta
The substrate screening is carried out by taking 20g/L glucose as carbon source, taking 10g/L peptone, 10g/L yeast extract powder and 5g/L ammonium sulfate as composite nitrogen source, taking potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, citric acid monohydrate, microelements and vitamin B complex as basic fermentation culture medium, respectively preparing three culture mediums taking 50mM sodium aspartate, 50mM sodium fumarate and 50mM diaminobutyric acid as substrates, and regulating pH value to 7.2.
The effect of the substrate is shown in Table 5, and sodium aspartate is used as the substrate to facilitate the engineering strain to synthesize the ectoin, and the yield is 25.86g/L.
TABLE 5 effect of substrate on yields of exendin
3. Single factor optimization experiment of Ikeduo factor
The glucose adding amount screening comprises the steps of setting the glucose content to be 5g/L, 10g/L, 15g/L, 20g/L, 25g/L, 30g/L and 35g/L respectively, the nitrogen source to be 10g/L peptone, 10g/L yeast extract powder, 5g/L ammonium sulfate and 50mM sodium aspartate, and the pH value of the fermentation medium is 7.2 by using potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, citric acid monohydrate, trace elements and vitamin B complex as basic materials;
The effect of different glucose levels is shown in Table 6, with a maximum yield of 28.64g/L for the enzyme at a glucose concentration of 25 g/L.
TABLE 6 Effect of different glucose concentrations on Excreta production
Screening the addition amount of peptone, wherein the experiment sets the peptone content to be 5g/L,10g/L, 15g/L, 20g/L, 25g/L and 30g/L respectively, the glucose to be 25g/L,10g/L yeast extract powder, 5g/L ammonium sulfate and 50mM sodium aspartate, and the pH value of the potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, citric acid monohydrate, microelements and vitamin B complex to be 7.2 with a basic fermentation culture medium;
The results of the effect of the different peptone contents are shown in Table 7, and when the peptone concentration is 20g/L, the maximum of the ectoin yield is 31.83g/L.
TABLE 7 Effect of different peptone concentrations on Excreta production
The yeast extract powder adding amount screening comprises the steps of setting the yeast extract powder content to be 5g/L, 10g/L, 15g/L, 20g/L, 25g/L and 30g/L respectively, glucose to be 25g/L, peptone to be 20g/L,5g/L ammonium sulfate, 50mM sodium aspartate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, citric acid monohydrate, microelements and vitamin B complex to be the same as a basic fermentation culture medium, and the pH value to be 7.2;
The results of the effect of different yeast extract levels are shown in Table 8, with a maximum of 33.26g/L for the Ikeduo factor when the yeast extract concentration was 10 g/L.
TABLE 8 Effect of different Yeast extract concentrations on Excreta yield
The method comprises the steps of screening the addition amount of ammonium sulfate, namely, setting the ammonium sulfate to be 2g/L, 4g/L, 6g/L, 8g/L, 10g/L and 12g/L respectively, the glucose to be 25g/L, the peptone to be 20g/L, the yeast extract to be 10g/L and 50mM sodium aspartate according to experiments, and setting the pH value of the fermentation culture medium to be 7.2 with the basic fermentation culture medium including potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, citric acid monohydrate, trace elements and vitamin B complex;
the results of the effect of the different ammonium sulfate levels are shown in Table 9, with the maximum yields of ectoin being 34.98g/L at an ammonium sulfate concentration of 6 g/L.
TABLE 9 Effect of different ammonium sulfate concentrations on Excreta yield
Screening the addition amount of sodium aspartate, namely setting the sodium aspartate content to be 20mM, 50mM, 100mM, 150mM, 200mM and 250mM, the glucose to be 25g/L, the peptone content to be 10g/L, the yeast extract to be 10g/L and the ammonium sulfate to be 6g/L according to experiments, and setting the pH value of the fermentation medium to be 7.2 by using potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, citric acid monohydrate, trace elements and vitamin B complex;
The results of the effect of different sodium aspartate levels are shown in Table 10, with a maximum of 36.28g/L of ectoin yield at a sodium aspartate concentration of 100 mM.
TABLE 10 Effect of different sodium aspartate concentrations on Excreta production
Screening inoculum size, namely setting 1%, 2%, 4%, 6%, 8% and 10% of inoculum size, 25g/L of glucose, 20g/L of peptone, 10g/L of yeast extract powder, 6g/L of ammonium sulfate and 100mM of sodium aspartate, and setting pH value of 7.2 when potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, citric acid monohydrate, trace elements and vitamin B complex are used as basic fermentation culture medium;
the results of the effect of the different inoculum sizes are shown in Table 11, and when the inoculum size is 6%, the maximum production of the ectoin is 38.63g/L.
TABLE 11 Effect of different inoculum sizes on Excreta yield
The initial pH value screening is that the experimental setting pH values are 6.0, 6.6, 7.2, 7.8, 8.4 and 9.0, the glucose is 25g/L, the peptone content is 20g/L, the yeast extract powder is 10g/L, the ammonium sulfate is 6g/L, the sodium aspartate content is 100mM, the inoculation amount of the potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, citric acid monohydrate, microelements and vitamin B complex is 6 percent with the basic fermentation culture medium;
the results of the effect of different pH values are shown in Table 12, and when the pH value is 7.2, the maximum yield of the ectoin is 37.51g/L.
TABLE 12 Effect of different pH values on Excreta yield
The fermentation time is set to be 0h, 18h, 24h, 28h, 43h, 48h, 52h and 68h respectively, the glucose is 25g/L, the peptone content is 10g/L, the yeast extract is 10g/L, the ammonium sulfate is 6g/L, the sodium aspartate content is 100mM;
The results of the effect of fermentation time are shown in Table 13, and in view of the fact that the fermentation is performed for 12 hours more than for 68 hours, the yield of the ectoin is only increased by 1.07g, and the production cost and the economic benefit of the fermentation production are unbalanced. The optimal fermentation time of the engineering strain of the ectoine is 52 hours, and the yield of the ectoine is 45.76g/L.
TABLE 13 Effect of fermentation time on Ikeduo production
Finally, the optimized culture medium and fermentation process are obtained, namely, the seed solution of the escin engineering strain is inoculated into the optimized fermentation culture medium for cultivation for 52 hours with the inoculation amount of 6 percent.
The preferable fermentation medium formula comprises 25g/L glucose, 20g/L peptone, 10g/L yeast extract powder, 6g/L ammonium sulfate, 100mM sodium aspartate, 7g/L monopotassium phosphate, 14g/L dipotassium phosphate, 1g/L magnesium sulfate, 1.1g/L citric acid monohydrate, 0.1g/L vitamin B complex, 2mL trace elements (1.5 g/L manganese sulfate, 7g/L ferrous sulfate, 0.8g/L cobalt chloride, 4g/L zinc chloride, 0.1g/L sodium molybdate, 1.1g/L calcium chloride dissolved in 5mol/L HCI solution), 7.2 of initial pH value and 52h of culture.
4. Optimization experiment of response surface of Ikeduo factor
Based on the single factor test result, glucose (A), peptone (B), yeast extract powder (C) and initial pH value (D) which have great influence on the result are selected as investigation factors, the content (Y) of the exendin-producing engineering strain is taken as a response value, and a response surface test of Box-Behnken Design 4 factor 3 level is carried out by using Design-Expert 8.0.6 software, wherein the experimental factor level and experimental Design scheme are shown in tables 14 and 15:
table 14 response surface experimental factor level design
Table 15 response surface Experimental design scheme
And performing multiple quadratic regression fitting on the test results of the table 16 by adopting Design-Expert 12 software to obtain a multiple quadratic regression equation:
Y=
-1312.86+5.69A+8.636B+1.902C+329.72D-0.055AB-0.022AC+0.54AD-0.047BC-0.148BD+0.253CD-0.164A2-0.133B2-0.1C2-23.806D2.
table 16 response surface design test results
TABLE 17 response surface analysis of variance
Note that "+" indicates significant effect on the results (P < 0.05); "indicates significant effect on the results (P < 0.01).
As can be seen from Table 17, the model is extremely remarkable (P < 0.0001), the P value of the mismatching term is 0.08, the mismatching term is not remarkable (P > 0.05), and the model has good fitting degree and high reliability. The determination coefficient R 2 of the model is 0.9670, which shows that the function relationship between the independent variable and the dependent variable of the model is very obvious, and almost no mismatching phenomenon shows that the regression equation and the curved surface have better fitting, and the method can be used for analyzing and predicting the Ikeduo-dependent yield. From the P values, the primary term A, B, D and the secondary term a 2、B2、C2、D2 had a very significant effect on the ectoin yield (P < 0.01), the interactive terms AB and BC had a significant effect on the ectoin yield (P < 0.05), and the other terms had no significant effect (P > 0.05).
The response curves and contour plots of the interactions between factors for the effect of the ekidon-production were plotted using Design-Expert 12 software, and the results are shown in fig. 2-7. From the graph, the interaction between glucose and yeast extract, peptone and initial pH, and yeast extract and initial pH was strong, the response surface was convex, the paraboloid was steeper, and the contour line was elliptical, which is consistent with the analysis of variance results of Table 16.
5. Response surface optimization verification
And optimally solving a regression equation by using Design-Expert 12 software to obtain the optimal fermentation condition of 25g/L of glucose, 21.37g/L of peptone, 10.83g/L of yeast extract powder and an initial pH value of 7.2. In order to facilitate practical operation, the optimal fermentation conditions are adjusted to 25g/L of glucose, 21.5g/L of peptone and 11g/L of yeast extract powder, and the initial pH value is 7.2. Under the condition, 4 verification tests are carried out, the average yield of the ectoin is 48.36g/L, the relative error is 1.73, and the model is proved to be reliable and effective as the predicted value is similar.
For comparison with the non-optimized fermentation medium, the non-optimized basal fermentation medium and the fermentation process were used as control groups (the seed solution of the ectoin engineering strain was inoculated into the preferred fermentation medium at an inoculum size of 10% for cultivation for 43h, the formulation of the basal fermentation medium was: glucose 20g/L, yeast extract 10g/L, potassium dihydrogen phosphate 7g/L, dipotassium hydrogen phosphate 14g/L, magnesium sulfate 1g/L, citric acid monohydrate 1.1g/L, trace elements 2mL/L (manganese sulfate 1.5g/L, ferrous sulfate 7g/L, cobalt chloride 0.8g/L, zinc chloride 4g/L, sodium molybdate 0.1g/L, calcium chloride 1.1g/L, dissolved in 5mol/L HCI solution), 0.1g/L of vitamin B complex, pH 7.2, etc.), using a response surface optimized culture medium and fermentation process as a test group (the method comprises inoculating the seed solution of the Ikeduo engineering strain in a preferred fermentation medium with an inoculum size of 6%, wherein the preferred fermentation medium comprises 25g/L of glucose, 21.5g/L of peptone, 11g/L of yeast powder, 6g/L of ammonium sulfate, 100mM sodium aspartate, 7g/L of potassium dihydrogen phosphate, 14g/L of magnesium dihydrogen phosphate, 1.2 g/L, pH 1.1g/L of magnesium chloride, 2 g/L of zinc chloride complex, pH 1.1g/L of 2, pH 1.5g/L of magnesium chloride, 2m 2 g/L of zinc chloride solution, pH 1g/L of 2, pH 1g/L of vitamin B complex, pH 1.1g/L of 2g of 2) to perform the test, and (5) comparing. The results show that the yields of the ectoin in the test group are 48.36g/L, which are respectively improved by 54.12% compared with the control group (22.19 g/L), thus showing that the optimizing effect is remarkable.
Example 2 fermentative production of Epideodoxin-producing engineering Strain in a 200L fermenter level
1. Experiment group 1
And (3) activating the thalli, namely streaking and activating the glycerol tube seeds of the ectoin engineering strains on an LB plate, culturing for 24 hours at 37 ℃, inoculating single bacterial colonies into 10mL of LB culture medium, and culturing for 12 hours at 37 ℃ and 200 rpm.
Seed culture, namely transferring 1% primary seed solution into 800mL LB culture medium, and culturing for 16h at 37 ℃ and 200 rpm. Transferring 10% secondary seed solution into 10L sterilized seed culture medium in 20L volume fermentation tank, culturing at 37deg.C and initial rotation speed of 200rpm under 100L/min for 6 hr.
The fermentation culture comprises transferring 6% seed solution to 200L volume fermentation tank, and filling 100L sterilized preferred fermentation medium (glucose 25g/L, peptone 21.5g/L, yeast extract 11g/L, ammonium sulfate 6g/L, sodium aspartate 100mM, potassium dihydrogen phosphate 7g/L, dipotassium hydrogen phosphate 14g/L, magnesium sulfate 1g/L, citric acid monohydrate 1.1g/L, vitamin B complex 0.1g/L, trace elements 2mL/L (manganese sulfate 1.5g/L, ferrous sulfate 7g/L, cobalt chloride 0.8g/L, zinc chloride 4g/L, sodium molybdate 0.1g/L, calcium chloride 1.1g/L, dissolved in 5mol/L HCI solution), and initial pH 7.2. The fermentation culture condition is that the temperature is controlled at 37 ℃, the initial rotating speed is 200rpm, and the initial air quantity is 100L/min. The pH value of the whole fermentation system is controlled at 7.2 by automatically feeding ammonia water, dissolved oxygen is maintained to be not lower than 20% by stirring and changing ventilation, the related rotating speed is 200rpm-800rpm, the fermentation is carried out for 52 hours, OD 600 is detected by sampling per hour, the wet weight of thalli is detected, the residual sugar amount is calculated, when the dissolved oxygen is lower than 70%, glucose with the concentration of 40% (mass percent) is fed, peptone is not fed, and the whole fermentation is controlled by adopting control feedback to adjust the feeding speed.
2. Experiment group 2
When the dissolved oxygen is lower than 70%, the whole process of feeding glucose with concentration of 40% is started to control the residual sugar to 5g/L, peptone is not fed, and the other steps refer to the experimental group 1.
3. Experiment group 3
When the dissolved oxygen is lower than 70%, the whole process of feeding glucose with the concentration of 40% is started to control the residual sugar to be 1g/L, peptone is not fed, and the other steps refer to the experimental group 1.
4. Experiment group 4
When the dissolved oxygen is lower than 70%, the whole process of feeding glucose with the concentration of 40% is started to control the residual sugar to be 2g/L, and the constant-speed feeding of 20% peptone to be 80mL/h is started, and the other steps refer to the experimental group 1.
5. Experiment group 5
When the dissolved oxygen is lower than 70%, the whole process of feeding glucose with the concentration of 40% is started to control the residual sugar to be 2g/L, and the constant-speed feeding of 20% peptone to be 160mL/h is started, and the other steps refer to the experimental group 1.
6. Experiment group 6
When the dissolved oxygen is lower than 70%, the whole process of feeding glucose with the concentration of 40% is started to control the residual sugar to be 2g/L, and the constant-speed feeding of 20% peptone to be 240mL/h is started, and the other steps refer to the experimental group 1.
The OD 600, the Ikeduopin yield, and the wet cell weight of the fermentation broths of each experimental group are shown in Table 18.
Table 18 comparison of OD 600, ectoin yield, and cell wet weight of each experimental group
The comparative analysis of the experimental group 1, the experimental group 2 and the experimental group 3 can obtain that the growth performance and the production performance of the engineering strain of the ectoin have a great relationship with the glucose content, when the glucose content is low, the lack of a carbon source leads to the reduction of the activity of the thallus, and conversely, the excessive residual sugar content leads to high osmotic pressure so as to influence the growth and the production performance of the thallus. In the experimental group 1, when the dissolved oxygen is lower than 70%, the whole process of feeding glucose with the concentration of 40% is started to control the residual sugar in the fermentation liquid to be 2g/L, the fermentation OD 600 value of the engineering strain reaches 119, the yield of the ectoin is 136.68g/L, and the fermentation performance is optimal.
Analysis of the comparison of experimental group 1, experimental group 4, experimental group 5 and experimental group 6 resulted in a dual stream addition of supplemental carbon source and nitrogen source with a positive effect of carbon to nitrogen ratio on the yields of ectoin. In the experimental group 6, too much nitrogen source can lead the thallus to grow too vigorously, the pH is too high, the accumulation of metabolic products is not facilitated, and the insufficient nitrogen source can lead the thallus to have little propagation quantity and influence the yield of the ectoin. In experiment group 4, too much carbon source tends to form a low pH, and if insufficient carbon source tends to cause cell senescence and autolysis. In the experimental group 5, when the dissolved oxygen is lower than 70%, the whole process of feeding glucose with the concentration of 40% is started to control the residual sugar to be 2g/L, and the constant-speed feeding of 20% peptone to be 160mL/h is started, the yield of the ectoin is 176.32g/L, and the fermentation performance is optimal. In addition, the yields of ectoin are improved by 32.89% in experimental group 5 compared with experimental group 1.
The invention does not need to use inducers such as IPTG to induce the strains to express the synthesis of the ectoine in the fermentation process, thereby avoiding the problems of high IPTG cost and toxicity which are not suitable for industrial fermentation production of the ectoine. The fermentation process of the invention greatly reduces the cost of each liter of culture medium, shortens the fermentation time effectively and provides technical support for realizing industrial production.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
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