Fermentation medium for producing Ectoin engineering strain, fermentation method and application

Abstract

The invention belongs to the technical field of microbial fermentation, and discloses a fermentation medium for producing Ectoine engineering strains, a fermentation method and application. The fermentation medium for producing the Ectoin engineering strain 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 / L-12g / L of ammonium sulfate, 5g / L-10 g / L of monopotassium phosphate, 10g / L-18g / L of dipotassium phosphate, 0.5 g / L-2g / L of magnesium sulfate, 0.5 g / L-2g / L of citric acid monohydrate and 0.05 g / L-0. 5g / L of compound vitamin B. According to the fermentation method disclosed by the invention, a double-flow feeding mode of simultaneously feeding the carbon source and the nitrogen source is adopted for feeding, so that the yield of the Ectoin at a fermentation tank level is remarkably improved, and a technical support is provided for industrial-scale production of the Ectoin.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and in particular to a fermentation medium, a fermentation method and an application of an ectoine-producing engineering strain. Background Art

[0002] Ectoine is a zwitterionic amino acid derivative and an extreme enzyme component. Ectoine is highly hydrophilic and has a strong ability to complex water molecules. It can tightly capture and orderly structure the surrounding free water molecules to form a unique "Ectoine-water complex". This complex is like a delicate hydration shell with protective, nourishing and stabilizing functions, tightly surrounding cells, enzymes, proteins and other key biological molecules, thereby maintaining the osmotic pressure balance of cells. It protects biologically active substances such as intracellular proteins, enzymes, nucleic acids, etc. under extreme environmental conditions such as high temperature roasting, dry invasion, radiation or severe freezing, and resists the damage of harsh external conditions. Therefore, Ectoine shows significant effects in combating the damage to the skin caused by environmental pressure, promoting the natural repair process of skin damage, and building a solid line of defense for skin health. At present, Ectoine is recognized as an excellent biological functional stability, and it also has significant skin protection effects such as repair, anti-wrinkle, whitening, anti-inflammatory, UV protection, anti-oxidative stress, and long-term moisturizing. It has been widely used in skin care products, medicine and other fields. The recommended dosage of ectoine in cosmetics is relatively large (about 2%), which highlights its market potential and demand. However, this results in a high cost for the use of this ingredient in cosmetics, limiting its expanded application.

[0003] At present, there are two main fermentation processes in the field of ectoine fermentation technology: one is to use the "cell milking method" fermentation production process of "high salt induced synthesis-low salt release" of wild strains to produce ectoine; but this method has high requirements for equipment and is complex and difficult to process, and the yield is low. For example, Chinese invention patent document CN117070397A discloses a culture medium and a method for fermenting and producing ectoine using it, using high concentrations of NaCl to stimulate cells to produce ectoine, with a long fermentation cycle and low ectoine yield, which limits the practical application and feasibility of ectoine in industrial large-scale production. Another process is to construct ectoine engineering strains, optimize metabolic pathways, and optimize the fermentation medium formula and fermentation conditions to produce ectoine. Carbon source, nitrogen source, inorganic salt, growth factor, etc. are important components of microbial fermentation culture system. There are many types of microbial fermentation culture system. The components and ratios directly affect the growth rate of the strain and the yield of metabolites. For example, Chinese invention patent document CN115651943A discloses a fermentation preparation method of a small molecule amino acid derivative ectoine. This method only uses sodium glutamate as a carbon source, does not optimize the fermentation conditions, and has a long fermentation time and low yield.

[0004] Therefore, it is urgent to develop a new ectoine fermentation production process in order to overcome the problems of low ectoine yield, long production cycle and high production cost in the existing technology of microbial fermentation. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of low ectoine yield, long production cycle and high production cost in the prior art of microbial fermentation, and to provide a fermentation medium, fermentation method and application of an ectoine-producing engineered strain.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a fermentation medium for producing an ectoine-producing engineered strain, comprising the following components:

[0008] Glucose 5g / L-35g / L, peptone 5g / L-30g / L, yeast extract powder 5g / L-30g / L, sodium aspartate 20mM-250mM, ammonium sulfate 2g / -12g / L, potassium dihydrogen phosphate 5g / L-10g / L, dipotassium hydrogen phosphate 10g / L-18g / L, magnesium sulfate 0.5g / L-2g / L, citric acid monohydrate 0.5g / L-2g / L, vitamin B complex 0.05g / L-0.5g / L.

[0009] The fermentation medium of the present invention is designed based on the metabolic characteristics of Escherichia coli ectoine. A small-scale fermentation test using the fermentation medium of the ectoine-producing engineered strain of the present invention obtains an average ectoine yield of 48.36 g / L in the ectoine test group, which is 54.12% higher than that of the basic culture system (22.19 g / L), directly and significantly affecting the yield of ectoine.

[0010] As a preferred embodiment of the fermentation medium of the present invention, the fermentation medium further comprises trace elements; the trace elements comprise the following components:

[0011] Manganese sulfate 0.5g / L-2g / L, ferrous sulfate 5g / L-10g / L, cobalt chloride 0.5g / L-1.5g / L, zinc chloride 2g / L-5g / L, sodium molybdate 0.05g / L-0.5g / L, calcium chloride 0.5g / L-1.5g / L, 2mol / L-8mol / L HCI solution.

[0012] As a preferred embodiment of the fermentation medium of the present invention, the pH value of the fermentation medium is 6-7.8.

[0013] As a preferred embodiment of the fermentation medium of the present invention, it includes the following components:

[0014] Glucose 20g / L-25g / L, peptone 15g / L-21.5g / L, yeast extract powder 10g / L-11g / L, sodium aspartate 100mM-200mM, ammonium sulfate 2g / -6g / L, potassium dihydrogen phosphate 5g / L-7g / L, dipotassium hydrogen phosphate 10g / L-14g / L, magnesium sulfate 0.5g / L-1g / L, citric acid monohydrate 0.5g / L-1.1g / L, vitamin B complex 0.05g / L-0.1g / L.

[0015] In a second aspect, the present invention provides a fermentation method for producing an ectoine-producing engineered strain, comprising the following steps:

[0016] (1) activating and culturing the ectoine-producing engineered strain to obtain an activated bacterial solution;

[0017] (2) transferring the activated bacterial solution to a seed culture medium for culturing to obtain a seed solution;

[0018] (3) taking the seed liquid and inoculating it into the above-mentioned fermentation medium for fermentation.

[0019] As a preferred embodiment of the fermentation method of the present invention, in step (3): the inoculation amount during the transfer is 1%-10%; preferably, the inoculation amount is 6%-10%.

[0020] As a preferred embodiment of the fermentation method of the present invention, in step (3): the fermentation conditions are: temperature 35°C-40°C, starting speed 150rpm-250rpm, starting air volume 50L / min-150L / min; pH value is controlled at 6.5-7.5; associated speed 200rpm-800rpm.

[0021] As a preferred embodiment of the fermentation method of the present invention, in step (3): the fermentation time is 36h-72h. Preferably, the fermentation time is 52h-68h.

[0022] As a preferred embodiment of the fermentation method of the present invention, in step (3): the dissolved oxygen content during the fermentation is ≥ 20%.

[0023] As a further preferred embodiment of the fermentation method of the present invention, when the dissolved oxygen content is lower than 70%, 30%-40% concentration of glucose is added to control the residual sugar in the fermentation liquid to 1g / L-5g / L, and 15%-25% concentration of peptone is added at 0-160mL / h.

[0024] The fermentation method of the present invention adopts a dual-flow addition method of simultaneously adding a carbon source and a nitrogen source for feeding, and the ectoine production at the fermentation tank level is significantly improved, especially when the dissolved oxygen is lower than 70%, 40% concentration of glucose is started to be added to control the residual sugar at 2g / L throughout the process, and 20% peptone is started to be added at a constant rate of 160mL / h. After fermentation for 52h, the ectoine production is 176.32g / L.

[0025] In a third aspect, the present invention applies the fermentation medium and the fermentation method to the industrial-scale production of ectoine.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention optimizes the most suitable fermentation medium of ectoine by screening carbon sources, nitrogen sources and growth factors, optimizes the fermentation conditions, and optimizes the fermentation system by using the response surface method based on the single factor experiment in combination with the Plackett-Burman experiment and the steepest climbing experiment. A dual-flow fermentation feeding process is adopted at the fermentation tank level to amplify the fermentation process and increase the yield of ectoine. During the fermentation process of the ectoine engineering strain of the present invention, there is no need to add expensive inducers such as IPTG to induce the strain to produce ectoine, and the introduction of toxic substances is also avoided. Unnecessary nutrient addition is avoided through model analysis, the fermentation system is optimized, the production cost is greatly reduced, and the dual-flow fermentation feeding process is adopted to provide technical support for the industrial-scale production of ectoine. The present invention provides technical support for the industrial-scale production of ectoine. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the standard curve of ectoine;

[0029] Figure 2 The response surface and contour map of the interaction between peptone and glucose on the yield of ectoine;

[0030] Figure 3 The response surface and contour map of the interaction between glucose and yeast extract on the yield of ectoine;

[0031] Figure 4 The response surface and contour map of the interaction between initial pH value and glucose on the yield of ectoine;

[0032] Figure 5 The response surface and contour map of the interaction between yeast extract powder and peptone on the yield of ectoine;

[0033] Figure 6 The response surface and contour map of the interaction between initial pH value and peptone on the yield of ectoine;

[0034] Figure 7Response surface and contour plot of the effect of initial pH value and yeast extract powder interaction on ectoine yield. DETAILED DESCRIPTION

[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0036] The term "comprising" means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps. In the text, when the term "comprising" is used, unless otherwise indicated, the situation consisting of the stated elements, integers or steps is also covered. For example, when referring to a culture medium "comprising" a certain component or components, it is also intended to cover a culture medium consisting of the specific certain component or components.

[0037] When referring to an engineered strain that produces ectoine, the starting strain of the engineered strain is Escherichia coli that is capable of producing ectoine or its downstream derivatives; the engineered strain is a genetically engineered strain obtained by mutating the relevant genes in the ectoine metabolic pathway; the mutation includes at least partial inactivation of gene function caused by gene deletion, gene insertion or gene replacement. The fermentation medium and fermentation process of the present invention are designed based on the metabolic characteristics of Escherichia coli ectoine, which can significantly improve the ectoine production of ectoine engineering strains, including Escherichia coli ECT-W020 (GDMCC No: 65012) strain, Escherichia coli ET08 strain, Escherichia coli ET11 strain (refer to Metabolic Engineering of Escherichia coli for Ectoine Production With a Fermentation Strategy of Supplementing the AminoDonor. Frontiers in Bioengineering and Biotechnology. 2022), Escherichia coli MWZ003 strain (refer to Metabolic engineering of Escherichia coli for efficientectoine production. Systems Microbiology and Biomanufacturing. 2021), and the like.

[0038] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. It should be understood by those skilled in the art that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0039] Unless otherwise specified, the test methods used in the examples are all conventional methods; the materials and reagents used, unless otherwise specified, can all be obtained from commercial sources. The present invention is described by taking the ectoine-producing engineered strain Escherichia coli ET11 as an example.

[0040] In the following examples, the test method for the concentration of ectoine in the fermentation broth is:

[0041] (1) Pretreatment of fermentation broth samples containing ectoine

[0042] Add three times the volume of anhydrous ethanol to the fermentation broth. Shake the reaction for 30 minutes, centrifuge at high speed for 10 minutes at a speed of 6000 r / min, remove the precipitate, take the supernatant, blow nitrogen to remove the solvent, add anhydrous ethanol and ultrasonically dissolve for 15 minutes. Centrifuge at high speed for 10 minutes at a speed of 6000 r / min, remove the precipitate and take the supernatant, blow nitrogen, add water and vortex to dissolve, and use HPLC for detection.

[0043] (2) HPLC detection conditions of ectoine

[0044] Chromatographic column: ZORBAX StableBond aq (4.6×250mm, 5μm);

[0045] Mobile phase: 100% 0.02 M potassium dihydrogen phosphate;

[0046] Flow rate: 0.5 mL / min;

[0047] Sample volume: 10 μL;

[0048] Column temperature: 25°C;

[0049] Detection wavelength: 210nm;

[0050] Elution mode: isocratic elution.

[0051] (3) Ecdoin standard curve

[0052] Dissolve 500 mg of ectoine standard in aqueous solution and dilute to 5 mL to prepare 100 mg / mL ectoine stock solution, and store at 4°C in the dark.

[0053] The 100 mg / mL ectoine mother solution was diluted with water to prepare a series of standard working solutions of 1 mg / mL, 3 mg / mL, 5 mg / mL, 10 mg / mL, 30 mg / mL, and 50 mg / mL, respectively, filtered through a 0.22 μm filter membrane, and tested by HPLC. A standard curve was drawn with the concentration of the ectoine standard working solution as the horizontal axis and the HPLC peak area of ​​ectoine as the vertical axis (see Figure 1 ).

[0054] In this detection method, the concentration range of ectoine was 5-100 μg / mL, and the obtained standard curve equation was y=46.696*x-1.3824, with a correlation coefficient R 2 =0.9999, indicating that the concentration of ectoine in the sample can be quantified using the standard curve.

[0055] Example 1: Fermentation medium of an engineered strain of Escherichia coli producing ectoine

[0056] 1. Preparation of culture medium

[0057] Preparation of chloramphenicol mother solution: Dissolve 0.34 g chloramphenicol in 10 mL anhydrous ethanol, filter using a sterile organic filter membrane, divide into portions, and store at -20°C.

[0058] LB plate: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, 16 g / L agar. pH 7.0 ± 0.1, sterilized at 121°C for 20 min;

[0059] LB broth: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride. pH 7.0 ± 0.1, sterilized at 121°C for 20 min;

[0060] Seed culture medium: glucose 20g / L, yeast extract 5g / L, tryptone 10g / L, sodium chloride 10g / L. pH 7.2±0.1, sterilized at 121℃ for 20min.

[0061] Basic fermentation medium: 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 vitamin B complex, sterilized at 121℃ for 20min;

[0062] Before use, chloramphenicol was added to the above culture medium to a final concentration of 34 μg / mL.

[0063] 2. Optimization experiment of components of Ecdoin culture medium

[0064] Strain activation: The preserved ectoine-producing engineered strain Escherichia coli was inoculated on an LB plate for activation, cultured at 37°C for 24 h, and single colonies were transferred to a 5 mL LB test tube and cultured at 37°C, 200 rpm for 12 h.

[0065] Seed liquid culture: transfer 1% activated bacterial liquid to seed culture medium with a volume of 50mL / 250mL, culture at 37℃, 200rpm for 16h.

[0066] The seed liquid was inoculated into a 500mL shaking flask containing 200mL of basic fermentation medium at a 10% inoculation rate, and the initial pH was adjusted to 7.2 with NaOH. The fermentation temperature was set at 37°C and 200rpm in a shaking incubator for 43h. Based on the basic fermentation medium, the effects of the following factors on the yield of ectoine were investigated.

[0067] Screening of carbon source types: The experiment was set up with 10g / L yeast extract powder as the nitrogen source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, citric acid monohydrate, trace elements, and vitamin B complex as the same basic fermentation medium; 5 kinds of culture media were prepared with 20g / L soluble corn starch, maltodextrin, glucose, sucrose, and glycerol as carbon sources, and the pH was adjusted to 7.2.

[0068] The results of the effects of different carbon sources are shown in Table 1. Glucose is most conducive to the synthesis of ectoine by the engineered strain, with a yield of 16.52 g / L. Therefore, glucose was selected as the carbon source.

[0069] Table 1 Effects of different carbon sources on edodene production

[0070]

[0071] Screening of organic nitrogen sources: The experiment was set up with 20 g / L glucose as the carbon source, and potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, citric acid monohydrate, trace elements, and vitamin B complex as the same basic fermentation medium; five culture media were prepared with 10 g / L yeast extract powder, corn steep liquor powder, peptone, beef extract, and trypsin as organic nitrogen sources, and the pH was adjusted to 7.2.

[0072] The results of the effect of organic nitrogen sources are shown in Table 2. Peptone is most conducive to the engineering strain to synthesize ectoine, with a yield of 20.85 g / L, followed by yeast extract powder, with a yield of 18.26 g / L.

[0073] Table 2 Effects of different nitrogen sources on edodene yield

[0074]

[0075] Screening of single nitrogen source and combined nitrogen source: The experiment was set up with 20 g / L glucose as the carbon source, and potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, citric acid monohydrate, trace elements, and vitamin B complex as the basic fermentation medium; three culture media were prepared with 10 g / L yeast extract powder, 10 g / L peptone, and 10 g / L yeast extract powder + 10 g / L peptone as organic nitrogen sources, and the pH was adjusted to 7.2.

[0076] Further screening and comparison of the effects of peptone, yeast extract powder, and peptone + yeast extract powder revealed that the combination of peptone + yeast extract powder was better than that of a single nitrogen source.

[0077] Table 3 Effects of single nitrogen source and combined nitrogen source on edodene yield

[0078]

[0079]

[0080] Screening of inorganic nitrogen sources: The experiment was set up with 20 g / L glucose as the carbon source, 10 g / L peptone and 10 g / L yeast extract powder as organic nitrogen sources, and potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, citric acid monohydrate, trace elements, and vitamin B complex as the basic fermentation medium; 5 kinds of culture media were prepared with 5 g / L urea, ammonium sulfate, ammonium bicarbonate, ammonium citrate and sodium nitrite as inorganic nitrogen sources, and the pH was adjusted to 7.2.

[0081] The results of the effect of inorganic nitrogen sources are shown in Table 4. Ammonium sulfate is beneficial to the engineering strain to synthesize ectoine, with a yield of 24.75 g / L.

[0082] Table 4 Effect of inorganic nitrogen sources on edodene production

[0083]

[0084] Substrate screening: The experiment was set up with 20 g / L glucose as the carbon source, 10 g / L peptone, 10 g / L yeast extract powder and 5 g / L ammonium sulfate as the composite nitrogen source, and potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, citric acid monohydrate, trace elements, and complex vitamin B as the basic fermentation medium; three culture media with 50 mM sodium aspartate, 50 mM sodium fumarate and 50 mM diaminobutyric acid as substrates were prepared respectively, and the pH was adjusted to 7.2.

[0085] The results of the influence of the substrate are shown in Table 5. Sodium aspartate as the substrate is beneficial to the engineering strain to synthesize ectoine, with a yield of 25.86 g / L.

[0086] Table 5 Effect of substrate on ectoine production

[0087]

[0088] 3. Single factor optimization experiment of Yikeduoyin

[0089] Screening of glucose addition amount: The experimental settings of glucose content were 5g / L, 10g / L, 15g / L, 20g / L, 25g / L, 30g / L and 35g / L, respectively, the nitrogen source was 10g / L peptone, 10g / L yeast extract powder, 5g / L ammonium sulfate, 50mM sodium aspartate; potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, citric acid monohydrate, trace elements, and vitamin B complex were the same as the basic fermentation medium, and the pH was 7.2;

[0090] The results of the effects of different glucose contents are shown in Table 6. When the glucose concentration is 25 g / L, the maximum yield of ectoine is 28.64 g / L.

[0091] Table 6 Effect of different glucose concentrations on ectoine yield

[0092]

[0093]

[0094] Screening of peptone addition: The peptone contents were set to 5g / L, 10g / L, 15g / L, 20g / L, 25g / L and 30g / L, respectively, glucose was 25g / L, yeast extract was 10g / L, ammonium sulfate was 5g / L, sodium aspartate was 50mM; potassium dihydrogen phosphate, potassium hydrogen phosphate, magnesium sulfate, citric acid monohydrate, trace elements, and vitamin B complex were the same as the basic fermentation medium, and the pH was 7.2;

[0095] The results of the effects of different peptone contents are shown in Table 7. When the peptone concentration was 20 g / L, the maximum yield of ectoine was 31.83 g / L.

[0096] Table 7 Effect of different peptone concentrations on the yield of ectoine

[0097]

[0098] Screening of yeast extract powder addition amount: The experimental settings of yeast extract powder content were 5g / L, 10g / L, 15g / L, 20g / L, 25g / L and 30g / L, glucose was 25g / L, peptone was 20g / L, 5g / L ammonium sulfate, 50mM sodium aspartate; potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, citric acid monohydrate, trace elements, and vitamin B complex were the same as the basic fermentation medium, and the pH was 7.2;

[0099] The results of the effects of different yeast extract powder contents are shown in Table 8. When the yeast extract powder concentration is 10 g / L, the maximum ectoine yield is 33.26 g / L.

[0100] Table 8 Effect of different yeast extract powder concentrations on ectoine yield

[0101]

[0102] Screening of ammonium sulfate addition: The experimental settings of ammonium sulfate were 2g / L, 4g / L, 6g / L, 8g / L, 10g / L and 12g / L, glucose was 25g / L, peptone content was 20g / L, yeast extract powder was 10g / L, 50mM sodium aspartate; potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, citric acid monohydrate, trace elements, and vitamin B complex were the same as the basic fermentation medium, and the pH was 7.2;

[0103] The results of the effects of different ammonium sulfate contents are shown in Table 9. When the ammonium sulfate concentration is 6 g / L, the maximum yield of ectoine is 34.98 g / L.

[0104] Table 9 Effect of different ammonium sulfate concentrations on ectoine production

[0105]

[0106]

[0107] Screening of sodium aspartate addition: The sodium aspartate content was set to 20mM, 50mM, 100mM, 150mM, 200mM and 250mM, glucose was 25g / L, peptone content was 10g / L, yeast extract powder was 10g / L, and ammonium sulfate was 6g / L; potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, citric acid monohydrate, trace elements, and vitamin B complex were the same as the basic fermentation medium, and the pH was 7.2;

[0108] The results of the effects of different sodium aspartate contents are shown in Table 10. When the sodium aspartate concentration was 100 mM, the maximum ectoine yield was 36.28 g / L.

[0109] Table 10 Effect of different sodium aspartate concentrations on ectoine production

[0110]

[0111] Inoculum size screening: The experimental inoculum size was set at 1%, 2%, 4%, 6%, 8% and 10%, glucose was 25g / L, peptone content was 20g / L, yeast extract powder was 10g / L, ammonium sulfate was 6g / L, sodium aspartate content was 100mM; potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, citric acid monohydrate, trace elements, and vitamin B complex were the same as the basic fermentation medium, and the pH was 7.2;

[0112] The results of the effects of different inoculation amounts are shown in Table 11. When the inoculation amount is 6%, the maximum yield of ectoine is 38.63 g / L.

[0113] Table 11 Effect of different inoculation amounts on edodene yield

[0114]

[0115] Initial pH screening: The experimental pH values ​​were set to 6.0, 6.6, 7.2, 7.8, 8.4 and 9.0, with glucose at 25 g / L, peptone at 20 g / L, yeast extract at 10 g / L, ammonium sulfate at 6 g / L, and sodium aspartate at 100 mM; potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, citric acid monohydrate, trace elements, and vitamin B complex were the same as the basic fermentation medium, and the inoculation amount was 6%;

[0116] The results of the effects of different pH values ​​are shown in Table 12. When the pH value is 7.2, the maximum yield of ectoine is 37.51 g / L.

[0117] Table 12 Effect of different pH values ​​on the yield of edodene

[0118]

[0119]

[0120] Fermentation time: The experimental fermentation time was set to 0h, 18h, 24h, 28h, 43h, 48h, 52h and 68h, respectively, with glucose at 25g / L, peptone content at 10g / L, yeast extract powder at 10g / L, ammonium sulfate at 6g / L, and sodium aspartate content at 100mM; potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, citric acid monohydrate, trace elements, and vitamin B complex were the same as the basic fermentation medium, with an inoculation volume of 6% and a pH value of 7.2;

[0121] The results of the effect of fermentation time are shown in Table 13. Since 68h is 12h longer than 52h, the yield of ectoine is only increased by 1.07g, and the production cost and economic benefits of fermentation production are unbalanced. The optimal fermentation time of ectoine engineering strain is 52h, and the yield of ectoine is 45.76g / L.

[0122] Table 13 Effect of fermentation time on edodein yield

[0123]

[0124] Finally, an optimized culture medium and fermentation process were obtained, that is, the seed liquid of the Ecdoin engineering strain was inoculated into the preferred fermentation medium at an inoculation rate of 6% and cultured for 52 hours.

[0125] The preferred fermentation medium formula is: 25 g / L glucose, 20 g / L peptone, 10 g / L yeast extract, 6 g / L ammonium sulfate, 100 mM sodium aspartate, 7 g / L potassium dihydrogen phosphate, 14 g / L dipotassium hydrogen phosphate, 1 g / L magnesium sulfate, 1.1 g / L citric acid monohydrate, 0.1 g / L vitamin B complex, 2 mL / L trace elements (1.5 g / L manganese sulfate, 7 g / L ferrous sulfate, 0.8 g / L cobalt chloride, 4 g / L zinc chloride, 0.1 g / L sodium molybdate, 1.1 g / L calcium chloride, dissolved in 5 mol / L HCI solution), initial pH value 7.2, culture 52 hours.

[0126] 4. Ecdoin response surface optimization experiment

[0127] Based on the results of the single factor experiment, on the basis of the basic fermentation medium, glucose (A), peptone (B), yeast extract powder (C) and initial pH value (D) which have a greater impact on the results were selected as the investigation factors, and the ectoine content (Y) of the ectoine-producing engineering strain was selected as the response value. The Box-Behnken design of 4 factors and 3 levels of response surface experiment was carried out using Design-Expert 8.0.6 software. The experimental factor levels and experimental design schemes are shown in Tables 14 and 15:

[0128] Table 14 Response surface experimental factor level design

[0129]

[0130]

[0131] Table 15 Response surface experimental design scheme

[0132]

[0133] The Design-Expert 12 software was used to perform multivariate quadratic regression fitting on the test results in Table 16, and the multivariate quadratic regression equation was obtained:

[0134] Y=

[0135] -1312.86+5.69A+8.636B+1.902C+329.72D-0.055AB-0.022AC+0.54AD-0.047BC-0.148BD+0.253CD-0.164A 2 -0.133B 2 -0.1C 2 -23.806D 2 .

[0136] Table 16 Response surface design test results

[0137]

[0138] Table 17 Response surface variance analysis

[0139]

[0140]

[0141] Note: “*” indicates a significant effect on the results (P < 0.05); “**” indicates an extremely significant effect on the results (P < 0.01).

[0142] As shown in Table 17, the model is extremely significant (P < 0.0001), the P value of the lack of fit term is 0.08, and the lack of fit term is not significant (P > 0.05), indicating that the model has a good degree of fit and high reliability. 2 The value of P is 0.9670, indicating that the functional relationship between the independent variable and the dependent variable of the model is extremely significant, and there is almost no lack of fit phenomenon, indicating that the regression equation and the surface have a good fit and can be used to analyze and predict the production of edodein. 2 , B 2 , C 2 , D 2 The effect on the yield of ectoine was extremely significant (P < 0.01), the interaction terms AB and BC had a significant effect on the yield of ectoine (P < 0.05), and the other terms had no significant effect on the results (P > 0.05).

[0143] Design-Expert 12 software was used to draw the response surface and contour map of the interaction between various factors on the yield of edodein. The results are shown in Figure 2-7 As can be seen from the figure, the interactions between glucose and yeast extract powder, peptone and initial pH value, and yeast extract powder and initial pH value are strong, the response surface is convex, the parabola is steeper, and the contour lines are elliptical, which is consistent with the variance analysis results in Table 16.

[0144] 5. Response surface optimization verification

[0145] The regression equation was optimally solved using Design-Expert 12 software, and the optimal fermentation conditions were 25 g / L glucose, 21.37 g / L peptone, 10.83 g / L yeast extract powder, and an initial pH of 7.2. For the convenience of actual operation, the optimal fermentation conditions were adjusted to 25 g / L glucose, 21.5 g / L peptone, 11 g / L yeast extract powder, and an initial pH of 7.2. Four verification tests were carried out under these conditions, and the average yield of Ecdoin was 48.36 g / L, with a relative error of 1.73, which was close to the predicted value, proving that the model is reliable and effective.

[0146] In order to compare it with the unoptimized fermentation medium, the unoptimized basic fermentation medium and fermentation process were used as the control group (the seed liquid of the Ekdoin engineering strain was inoculated into the preferred fermentation medium at an inoculation rate of 10% and cultured for 43 hours. The formula of the basic fermentation medium was: glucose 20g / L, yeast extract 10g / L, potassium dihydrogen phosphate 7g / L, potassium 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 HCl solution), 0.1g / L vitamin B complex. The initial pH value was 7.2. ), and the culture medium and fermentation process optimized by response surface were used as the test group (the seed liquid of the Ekedoin engineering strain was inoculated into the preferred fermentation medium at an inoculation rate of 6% and cultured for 52 hours. The preferred fermentation medium formula was: glucose 25g / L, peptone 21.5g / L, yeast extract powder 11g / L, ammonium sulfate 6g / L, sodium aspartate 100mM, potassium dihydrogen phosphate 7g / L, potassium hydrogen phosphate 14g / L, magnesium sulfate 1g / L, citric acid monohydrate 1.1g / L, 0.1g / L vitamin B complex, 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 HCl solution), initial pH value 7.2. ) for experiment and comparison. The results showed that the ectoine production in the test group was 48.36 g / L, which was 54.12% higher than that in the control group (22.19 g / L), indicating that the optimization effect was significant.

[0147] Example 2: Fermentation production of ectoine-producing engineered strains in a 200L fermenter

[0148] 1. Experimental Group 1

[0149] Bacterial activation: streak the glycerol tube seeds of the Ekedoin engineering strain on the LB plate for activation, culture at 37°C for 24 h, pick a single colony into 10 mL of LB medium, and culture at 37°C, 200 rpm for 12 h.

[0150] Seed culture: transfer 1% of the first-level seed solution to 800 mL of LB medium and culture at 37°C, 200 rpm for 16 hours. Transfer 10% of the second-level seed solution to a 20 L fermenter filled with 10 L of sterilized seed medium and culture at 37°C, 200 rpm, and 100 L / min air volume for 6 hours.

[0151] Fermentation culture: transfer 6% seed liquid to a 200L volume fermentation tank filled with 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, 0.1g / L vitamin B complex, 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), initial pH 7.2). Fermentation culture conditions are: temperature control at 37°C, initial rotation speed 200rpm, initial air volume 100L / min. During the whole fermentation process, the system automatically added ammonia water to control the pH at 7.2, and maintained the dissolved oxygen at not less than 20% by stirring and changing the ventilation volume. The rotation speed was 200rpm-800rpm, and the fermentation was carried out for 52h. The OD was tested by sampling every hour. 600 , wet weight of bacteria, residual sugar content, when the dissolved oxygen is lower than 70%, 40% concentration (mass percentage) of glucose is added, and no peptone is added. The control feedback is used to adjust the feeding rate throughout the process to control the residual sugar in the fermentation liquid to 2g / L.

[0152] 2. Experimental Group 2

[0153] When the dissolved oxygen was lower than 70%, 40% glucose was added to control the residual sugar to 5 g / L throughout the process. No peptone was added. The other steps were referred to Experimental Group 1.

[0154] 3. Experimental Group 3

[0155] When the dissolved oxygen was lower than 70%, 40% glucose was added to control the residual sugar to 1 g / L throughout the process. No peptone was added. The other steps were referred to Experimental Group 1.

[0156] 4. Experimental Group 4

[0157] When the dissolved oxygen was lower than 70%, 40% glucose was added to control the residual sugar at 2 g / L throughout the process, and 20% peptone was added at a constant rate of 80 mL / h. The other steps were referred to Experimental Group 1.

[0158] 5. Experimental Group 5

[0159] When the dissolved oxygen was lower than 70%, 40% glucose was added to control the residual sugar at 2 g / L throughout the process, and 20% peptone was added at a constant rate of 160 mL / h. The other steps were referred to Experimental Group 1.

[0160] 6. Experimental Group 6

[0161] When the dissolved oxygen was lower than 70%, 40% glucose was added to control the residual sugar at 2 g / L, and 20% peptone was added at a constant rate of 240 mL / h. The other steps were referred to Experimental Group 1.

[0162] OD of the fermentation broth of each experimental group 600 The comparison of edodene yield and wet weight of fungus is shown in Table 18.

[0163] Table 18 OD of each experimental group 600 Comparison of edodene yield and wet weight of fungus

[0164]

[0165] Through the comparative analysis of experimental groups 1, 2 and 3, it can be concluded that the growth performance and production performance of the engineered strain of Ekdoin are closely related to the glucose content. When the glucose content is low, the lack of carbon source leads to a decrease in the activity of the bacteria. On the contrary, excessive residual sugar will lead to high osmotic pressure and thus affect the growth and production performance of the bacteria. In experimental group 1, when the dissolved oxygen was lower than 70%, 40% glucose was added to control the residual sugar in the fermentation liquid to 2g / L. The fermentation OD of the engineered strain was 600 The value reached 119, the ectoine yield was 136.68 g / L, and the fermentation performance was the best.

[0166] Analysis of experimental groups 1, 4, 5 and 6 showed that the carbon and nitrogen sources were supplemented by double flow addition, and the carbon-nitrogen ratio had a positive effect on the yield of ectoin. In experimental group 6, the excessive proportion of nitrogen source would make the bacteria grow too vigorously, the pH would be high, which was not conducive to the accumulation of metabolites. Insufficient nitrogen source would result in low bacterial reproduction, affecting the yield of ectoin. In experimental group 4, the excessive proportion of carbon source would easily form a lower pH. If the carbon source was insufficient, it would easily cause bacterial aging and autolysis. In experimental group 5, when the dissolved oxygen was lower than 70%, 40% glucose concentration was added to control the residual sugar at 2g / L throughout the process and 20% peptone was added at a constant rate of 160mL / h. The yield of ectoin was 176.32g / L, and the fermentation performance was the best. In addition, compared with experimental group 1, the yield of ectoin in experimental group 5 increased by 32.89%.

[0167] In the fermentation process of the present invention, there is no need to use an inducer such as IPTG to induce the strain to express ectoine synthesis, thereby avoiding the problem that IPTG is costly and toxic and is not suitable for industrial fermentation production of ectoine. The fermentation process of the present invention greatly reduces the cost per liter of culture medium and effectively shortens the fermentation time, providing technical support for realizing industrial production.

[0168] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A fermentation medium for producing an engineered strain of ectoine, characterized in that: Includes the following components: Glucose 5g / L-35g / L, peptone 5g / L-30g / L, yeast extract powder 5g / L-30g / L, sodium aspartate 20mM-250mM, ammonium sulfate 2g / -12g / L, potassium dihydrogen phosphate 5g / L-10g / L, dipotassium hydrogen phosphate 10g / L-18g / L, magnesium sulfate 0.5g / L-2g / L, citric acid monohydrate 0.5g / L-2g / L, vitamin B complex 0.05g / L-0.5g / L.

2. The fermentation medium according to claim 1, characterized in that The fermentation medium also includes trace elements; the trace elements include the following components: Manganese sulfate 0.5g / L-2g / L, ferrous sulfate 5g / L-10g / L, cobalt chloride 0.5g / L-1.5g / L, zinc chloride 2g / L-5g / L, sodium molybdate 0.05g / L-0.5g / L, calcium chloride 0.5g / L-1.5g / L, 2mol / L-8mol / L HCI solution.

3. The fermentation medium according to claim 1, characterized in that The pH value of the fermentation medium is 6-7.

8.

4. The fermentation medium according to claim 1, characterized in that Includes the following components: Glucose 20g / L-25g / L, peptone 15g / L-21.5g / L, yeast extract powder 10g / L-11g / L, sodium aspartate 100mM-200mM, ammonium sulfate 2g / -6g / L, potassium dihydrogen phosphate 5g / L-7g / L, dipotassium hydrogen phosphate 10g / L-14g / L, magnesium sulfate 0.5g / L-1g / L, citric acid monohydrate 0.5g / L-1.1g / L, vitamin B complex 0.05g / L-0.1g / L.

5. A fermentation method for producing an engineered strain of ectoine, characterized in that: The following steps are involved: (1) activating and culturing the ectoine-producing engineered strain to obtain an activated bacterial solution; (2) transferring the activated bacterial solution to a seed culture medium for culturing to obtain a seed solution; (3) taking the seed liquid and inoculating it into the fermentation medium according to any one of claims 1 to 4 for fermentation.

6. The fermentation method according to claim 5, characterized in that In step (3): the fermentation conditions are: temperature 35°C-40°C, initial rotation speed 150rpm-250rpm, initial air volume 50L / min-150L / min; pH value controlled at 6.5-7.5; associated rotation speed 200rpm-800rpm.

7. The fermentation method according to claim 5, characterized in that In step (3): the fermentation time is 36h-72h.

8. The fermentation method according to claim 5, characterized in that In step (3): the dissolved oxygen content during the fermentation is ≥ 20%.

9. The fermentation method according to claim 8, characterized in that When the dissolved oxygen content is lower than 70%, glucose with a concentration of 30%-40% is added to control the residual sugar in the fermentation liquid to be 1g / L-5g / L, and peptone with a concentration of 15%-25% is added at 0-160mL / h.

10. Use of the fermentation medium according to any one of claims 1 to 4 and the fermentation method according to any one of claims 5 to 9 in the industrial-scale production of ectoine.

Images