Method for producing Ectoine through salt impact-glutamine synergistic material supplement

Abstract

The invention provides a method for producing Ectoine through salt impact-glutamine synergistic feeding, and relates to the field of fermentation engineering. The invention particularly relates to a metabolism regulation strategy of salt impact in cooperation with glutamine supplement. According to the method, after salt impact is applied in the logarithmic phase of halophilic bacteria, glutamine is accurately supplemented to activate a thallus stress synthesis pathway. Through the method, the yield of Ectoin can be remarkably increased, the fermentation period is shortened, and the metabolic activity of thalli is enhanced. The method is simple and convenient to operate and controllable in cost, and an innovative process solution is provided for efficient production of industrial fermentation of the halophilic bacteria.

Description

Technical Field

[0001] The present invention belongs to the field of microbial fermentation engineering, and particularly relates to a method for significantly improving the yield and efficiency of ectoine fermentation by Halomonas elongata in a high-salt environment through optimizing the timing strategy of salt shock and glutamine feeding. Background Art

[0002] Due to their unique salt tolerance characteristics, halophilic microorganisms exhibit important application potential in extreme industrial environments, especially in the field of biomanufacturing, where they can significantly reduce the energy consumption for sterilization and freshwater consumption.

[0003] Among them, Halomonas elongata DSM 2581 has become the core strain for industrial production of ectoine due to its unique metabolic adaptation mechanism. This strain can rapidly synthesize and accumulate ectoine in a high-salt environment, and at the same time, efficiently release intracellular products through low-salt shock. The characteristic of a single intracellular solute composition significantly simplifies the product separation process, providing ideal conditions for continuous production.

[0004] However, the existing fermentation processes based on Halomonas elongata DSM 2581 still face multiple challenges. First, although high-salt stress can induce ectoine synthesis, long-term high osmotic pressure will inhibit the growth rate of the bacteria, resulting in an extended fermentation cycle.

[0005] Secondly, traditional feeding strategies mostly rely on the supplementation of a single carbon and nitrogen source, failing to combine the stage effects of salt shock on the metabolic activity of the bacteria, leading to the disconnection between precursor supply and the regulation of the synthesis pathway (such as the ectABC gene cluster), which limits the product synthesis efficiency. In addition, although the ectoine synthesis pathway of this strain has been analyzed, its genetic operation system is still imperfect, and there is a lack of precise means for the expression regulation of key enzymes (such as EctB and EctC), making it difficult to further increase the yield through metabolic engineering.

[0006] In the prior art, although attempts have been made to alleviate osmotic inhibition by adding compatible solute precursors (such as aspartic acid) or optimizing the salt concentration gradient, there are still problems such as high cost and complex processes.

[0007] For example, the exogenous addition of precursors easily interferes with downstream purification, while intermittent salt shock easily causes metabolic fluctuations, resulting in unstable product release. Therefore, there is an urgent need to develop a coordinated regulation strategy for Halomonas elongata DSM 2581, through precisely timed salt shock and key precursor feeding, to activate the stress metabolic pathway of the bacteria, and while maintaining a high growth rate, directionally strengthen ectoine synthesis, thereby breaking through the existing production bottleneck and achieving efficient and low-cost industrial applications. Summary of the Invention

[0008] To solve the problems of limited fermentation performance of halophilic bacteria and low ectoine synthesis efficiency in a high-salt environment, the present invention provides a method for producing ectoine by salt shock-glutamine co-feeding, which significantly improves the ectoine yield and production efficiency of Halomonas elongata DSM 2581. The present invention provides a theoretical basis and technical solution for the directional regulation of halophilic bacteria under high-salt stress.

[0009] To achieve the above technical objectives, the technical solution of the present invention is as follows:

[0010] The present invention first provides a method for improving the fermentation performance of Halomonas elongata under salt stress by glutamine. Further, the application is to improve the fermentation performance of Halomonas elongata under the stress condition of a salt concentration of 13% (w / v) after salt shock; the concentration of glutamine is 3 g / L.

[0011] The present invention also provides a method for improving the fermentation performance of Halomonas elongata under salt stress, which is to add glutamine to the fermentation broth at an appropriate time.

[0012] Further, when adding glutamine in the present invention, the concentration of glutamine is 3 g / L.

[0013] Further, the addition time of glutamine in the present invention is 1 hour after salt shock.

[0014] Further, the components and their concentrations of the fermentation medium are as follows: glucose 30.00, sodium chloride 80.00, sodium citrate dihydrate 3.00, ammonium chloride 3.00, sodium sulfate 2.50, magnesium sulfate heptahydrate 3.08, zinc sulfate 0.25, manganese sulfate 0.09, disodium hydrogen phosphate dodecahydrate 7.50, sodium dihydrogen phosphate dihydrate 2.00, potassium chloride 3.00, PTM1 1.00 mL / L, antifoaming agent 0.5 mL / L, pH 7.00 ± 0.10, and the volume is supplemented with deionized water, antifoaming agent 0.5 mL / L, pH 7.0.

[0015] Further, the improved performance may include improving the product synthesis rate, shortening the fermentation cycle, reducing the fermentation cost, etc.

[0016] Further, the number of the Halomonas elongata is Halomonas elongata DSM 2581.

[0017] The beneficial effects of the present invention:

[0018] (1) The present invention provides the application of glutamine in improving the fermentation performance of Halomonas elongata under salt stress. Experiments show that after a 1-hour salt shock treatment with the salt concentration increased from 8% to 13% (w / v), adding 3 g / L of glutamine can increase the ectoine yield by 14% compared to the control group without addition, significantly reducing the production cost.

[0019] (2) The glutamine used in the present invention is a safe and healthy natural amino acid, a conditionally essential amino acid widely present in human muscles, blood, and foods such as dairy products and meats. It is commonly used in sports nutrition supplements and the field of medical nutrition, and has the characteristics of promoting muscle repair and enhancing immune function. Its biocompatibility is excellent and the metabolic process is mild, meeting the concept of green environmental protection.

[0020] (3) The operation process of the present invention is efficient and convenient. It only needs to be added regularly and quantitatively at the preset fermentation time node, and there is no need to precisely control process parameters or support by professional equipment throughout the process. Description of the Drawings

[0021] Figure 1 Shows the effects of different addition factors on the yield of ectoine in the shake-flask fermentation of Halomonas elongata.

[0022] Figure 2 Is a comparison chart of the ectoine content produced by Halomonas elongata under the conditions of adding different concentrations of glutamine, sodium glutamate, sodium malate, thiamine, niacin and the control group.

[0023] Figure 3 Is a comparison curve of the ectoine yield between different addition times of glutamine and the control group. Detailed Embodiments

[0025] Through experimental research, the inventor of the present invention has revealed the synergistic regulation strategy method of salt environment stimulation and sequential feeding of glutamine. The inventor has found that by adding glutamine at specific nodes during the fermentation process, the fermentation performance of Halomonas elongata can be extremely effectively improved. The present invention provides a method with simple operation and easy process control.

[0026] As used in the invention, the improvement of the "fermentation performance" includes the increase in the product synthesis rate and the shortening of the fermentation cycle.

[0027] As used in the present invention, the "salt shock" refers to adding sodium chloride during the fed-batch fermentation process when the OD of the fermentation broth 600 reaches 30 - 35, so that the salt concentration of the fermentation broth is increased from 8% to 13%.

[0028] As used in the present invention, the "specific node" refers to 1 hour after the salt shock.

[0029] As used in the present invention, the "glutamine" refers to an aqueous solution of glutamine with a concentration of 95% and a concentration of 3 g / L.

[0030] In the present invention, the meanings of the terms "comprising", "having" or "covering" cover the expressions such as "containing", "mainly composed of...", "substantially consisting of..." and "completely composed of..."; among them, "mainly composed of...", "substantially consisting of..." and "completely composed of..." belong to the specific limiting forms of "comprising", "having" or "covering".

[0031] The specific embodiments of the present invention will be elaborated in detail in conjunction with the accompanying drawings. The listed embodiments are intended to further clarify the details of the technical solution, but should not be regarded as a limitation on the scope of patent protection. It should be particularly noted that unless otherwise clearly defined, the professional terms and scientific expressions involved in this document shall be subject to the general interpretations in this field.

[0032] In the method of the present invention, the fermentation medium comprises: ammonium chloride 3.00 ± 0.5 g / L, sodium sulfate 2.50 ± 0.2 g / L, magnesium sulfate heptahydrate 3.08 ± 0.1 g / L, zinc sulfate 0.25 ± 0.05 g / L, manganese sulfate 0.09 ± 0.01 g / L, disodium hydrogen phosphate dodecahydrate 7.50 ± 0.5 g / L, sodium dihydrogen phosphate dihydrate 2.00 ± 0.1 g / L, potassium chloride 3.00 ± 0.3 g / L, PTM1 1.00 ± 0.1 mL / L, antifoaming agent 0.5 ± 0.05 mL / L, pH 7.00 ± 0.10.

[0033] The culture medium system of the present invention provides the necessary nutritional support for the synthesis of halophilic bacteria and target products through synergistic action with the fermentation process. It should be clear that during the actual application process, it is allowed to make adaptive adjustments to the culture medium components (for example, implementing equivalent replacement with other carbon and nitrogen sources that can be assimilated by the strain), or directly selecting the same type of culture medium commonly used in this field. Such adjusted culture systems need to be used in combination with the chronological feeding strategy of glutamine.

[0034] Subsequently, the technical solution will be elaborated in detail through specific implementation cases. It should be particularly noted that the said embodiments are only used to illustrate the technical features of the present invention and do not constitute any limitation to the scope of the claims.

[0035] Materials and Methods

[0036] 1. Starting Strain

[0037] Halomonas elongata DSM 2581, a Gram-negative bacterium, was provided by the Institute of Microbiology, Chinese Academy of Sciences.

[0038] 2. Culture Medium and Culture Conditions

[0039] Plate medium (g / L): Peptone 10.00, Yeast extract 5.00, Sodium chloride 80.00, Agar 16.00, pH 7.0 ± 0.1.

[0040] Strain activation medium (g / L): Peptone 10.00, Yeast extract 5.00, Sodium chloride 80.00, Agar 16.00, pH 7.00 ± 0.10.

[0041] Primary seed medium (g / L): Yeast extract 5.00, Peptone 10.00, Sodium chloride 80.00, pH 7.00 ± 0.10.

[0042] Secondary seed medium (g / L) and fermentation medium HE9: Glucose 10.00 - 20.00, Sodium chloride 80.00, Sodium citrate dihydrate 3.00, Ammonium chloride 3.00, Sodium sulfate 2.50, Magnesium sulfate heptahydrate 3.08, Zinc sulfate 0.25, Manganese sulfate 0.09, Disodium hydrogen phosphate dodecahydrate 7.50, Sodium dihydrogen phosphate dihydrate 2.00, Potassium chloride 3.00, PTM1 1.00 mL / L, Antifoaming agent 0.5 mL / L, pH 7.00 ± 0.10, volume supplemented with deionized water.

[0043] 5L fermenter fermentation medium (g / L): Glucose 30.00, Sodium chloride 80.00, Sodium citrate dihydrate 3.00, Ammonium chloride 3.00, Sodium sulfate 2.50, Magnesium sulfate heptahydrate 3.08, Zinc sulfate 0.25, Manganese sulfate 0.09, Disodium hydrogen phosphate dodecahydrate 7.50, Sodium dihydrogen phosphate dihydrate 2.00, Potassium chloride 3.00, PTM1 1.00 mL / L, Antifoaming agent 0.5 mL / L, pH 7.00 ± 0.10, volume supplemented with deionized water, Antifoaming agent 0.5 mL / L, pH 7.0. Inoculation ratio is 10% (v / v), culture temperature is 37 °C.

[0044] 3. Determination of cell concentration and biomass

[0045] After the fermentation broth is serially diluted, the absorbance value at 600 nm wavelength is measured using a microplate reader in static mode, OD 600 is calculated as the absorbance value multiplied by the dilution factor. Take a quantitative sample, centrifuge at 12000 rpm for 10 min at 4 °C, discard the supernatant, wash twice with isotonic NaCl solution, centrifuge to collect the cell precipitate, dry at 90 °C to constant weight, and weigh the dry weight.

[0046] 4. Quantitative analysis of ectoine

[0047] Take 0.5 mL of the fermentation broth and add it to an EP tube containing grinding beads. Inject 1 mL of ultrapure water, and disrupt it at 75 Hz for 1800 s in a cryogenic grinder. After centrifuging at 10000 rpm for 10 min, collect the supernatant, and detect it by HPLC after treatment with a 0.22 μm filter membrane. Chromatographic conditions: C18 reversed-phase column, mobile phase: water-acetonitrile (96:4, v / v), flow rate 0.4 mL / min, column temperature 30 °C, detection wavelength 210 nm. Glucose concentration was determined by the GOD-POD microplate method.

[0048] 5.0 Strain activation and monoclonal isolation

[0049] Take the bacteria from the glycerol preservation tube and inoculate them into an LB liquid medium containing 8% NaCl, and shake culture at 37 °C and 220 rpm for 20 h for activation. Take the activated bacterial liquid for 10 -7 gradient dilutions, coat them on a solid medium plate, and pick single colonies for secondary purification culture after culturing at 37 °C for 20 h.

[0050] 7.0 Shake flask scale-up culture

[0051] Rinse the bacterial lawn on the surface of the solid medium with 5 mL of sterile normal saline, transfer it to a 50 mL centrifuge tube containing glass beads, and vortex for 30 s to prepare a homogeneous bacterial suspension. Inoculate the bacterial suspension into the seed medium (500 mL conical flask) at an inoculation amount of 10%, and shake culture at 37 °C and 220 rpm for 18 h.

[0052] Example 1. Effects of different feeding factors on the product synthesis of Halomonas

[0053] In order to explore what factors can promote the production of ectoine by halophilic bacteria, a shake flask screening experiment was carried out. Specifically, amino acids and their derivatives, including glutamine, aspartic acid, asparagine, glutamic acid; vitamin B group, including thiamine, niacin, pyridoxol, folic acid; organic acids, including malic acid, acetic acid; methyl donors and osmoregulatory substances, including betaine.

[0054] The addition concentrations of amino acids and their derivatives are all 1 g / L, the addition concentration of vitamin B group is 0.5 g / L, the addition concentration of organic acids is 1 g / L, and the concentration of methyl donors and osmoregulatory substances is 1 g / L. In the experiment, each substance was added to the fermentation system separately, and a control group without adding any substance was set. All experimental conditions were kept consistent. After shaking flask fermentation for 14 h, samples were taken to determine the ectoine yield by liquid phase to evaluate the effects of each additive on product synthesis.

[0055] As Figure 1 shown, compared with the control group, glutamine, thiamine, glutamic acid, malic acid, and niacin have a more significant promoting effect on product synthesis (see Table 1).

[0056] Table 1. Results of the yield of Halomonas elongata in shake flask fermentation under different additive conditions

[0057] To further explore what factors added in the pilot-plant experiment can produce better effects and screen the appropriate addition concentrations, subsequent batch fermentation pilot-plant experiments were carried out. Respectively, on the basis of the original synthetic medium, 1 g / L sodium malate, 3 g / L sodium malate, 5 g / L sodium malate, 1 g / L glutamic acid, 3 g / L glutamic acid, 5 g / L glutamic acid, 1 g / L glutamine, 3 g / L glutamine, and 5 g / L glutamine were added to the medium. Since the yield of the group with VB1 and VB3 added to the medium increased relatively less in the shaking experiment, only the experimental group with 0.5 g / L of VB1 and 0.5 g / L of VB3 added to the original synthetic medium was set, and samples were taken after about 10 h of fermentation to measure the product. Due to the differences in the blank groups of each batch of pilot-plant experiments, the data were preprocessed, that is, irrelevant factors were eliminated by the proportional scaling method, and Gaussian Process Interpolation was used for fitting interpolation to achieve the effect of time series alignment. Among them, 3 g / L glutamine had a more obvious effect on the product improvement (see Table 2).

[0058] Table 2. Results of the yield of Halomonas elongata in fed-batch fermentation in pilot-plant under different additive conditions (after preprocessing)

[0059] To further explore how to add glutamine in the fed-batch fermentation experiment to further improve the yield of ectoine, an iterative Bayesian optimization model was used to screen the optimal conditions. After two iterations, the best conditions were obtained as follows: in the fed-batch fermentation, when the OD 600 of the fermentation broth reached 30–35, sodium chloride was added for salt shock operation, and 3 g / L glutamine was added 1 hour after salt shock.

[0060] Example 2. Experiment on the improvement of yield and verification of different feeding times of glutamine

[0061] Furthermore, a fed-batch fermentation verification experiment was set up in a 5 L tank. Based on an addition amount of 3 g / L, this experiment set the addition of glutamine before salt shock and the addition of glutamine 1 hour after salt shock. During the fermentation process, samples were taken at regular intervals to measure the product content. The results are shown in Table 3. Adding glutamine after salt shock can promote product synthesis more effectively.

[0062] Table 3. Comparison of the yields between the experimental group with glutamine added after salt shock and the blank group

[0063] Start the batch fermentation of halophilic bacteria in a basal medium containing 8 ± 1% (w / v) sodium chloride. When the OD600 of the fermentation broth reaches 30 - 35, apply a salt shock to increase the sodium chloride concentration to 13 ± 1% (w / v), and add 3 ± 0.3 g / L glutamine 1 hour after the salt shock. Continuously monitor and adjust the fermentation parameters until the ectoine yield reaches the peak.

[0064] The salt shock is achieved by adding solid sodium chloride or a high - concentration sodium chloride solution, and the process of increasing the salt concentration is controlled to be completed within 5 minutes. Glutamine is dissolved in a small amount of sterilized physiological saline and added to the fermentation system by pulsed feeding.

[0065] The basal medium includes 30 ± 3 g / L glucose, 8 ± 1% (w / v) sodium chloride, pH 7.0 ± 0.1, and may also contain auxiliary components such as 3.00 ± 0.5 g / L ammonium chloride, 2.50 ± 0.2 g / L sodium sulfate, 3.08 ± 0.1 g / L magnesium sulfate heptahydrate, etc. to support high - density cell growth. Through the sequential coordination of salt shock and glutamine feeding, the ectoine yield is increased by 14% and the fermentation cycle is shortened.

[0066] The beneficial effects of the present invention include: through the precise cooperation of salt shock and glutamine feeding, the ectoine yield is increased from 16.17 ± 0.3 g / L to 18.46 ± 0.4 g / L, with an increase of 14%. As a natural amino acid precursor, glutamine is safe and non - toxic, and its feeding strategy can alleviate the inhibition of high - osmotic stress on cell growth. The process operation is simple, only requiring a single salt shock and feeding, without the need for complex equipment or strict sterilization conditions, and is applicable to open - type continuous fermentation on a scale of 5 L to 50 L, reducing production costs.

[0067] Example verification shows that in a 5 - L fermenter, when OD 600 = 30 - 35, apply a salt shock (8% → 13% NaCl), and add 3 g / L glutamine 1 hour later. The ectoine yield reaches 18.46 ± 0.4 g / L, fully demonstrating the advantages of synergistic regulation

[0068] The present invention proposes a method for producing ectoine by salt - shock - glutamine synergistic feeding. The method of the present invention is simple and easy to implement. Just add 3 g / L of glutamine to the fermentation broth 1 hour after the salt shock, which can significantly improve the product synthesis efficiency and shorten the fermentation cycle.

[0069] The above - described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for producing ectoine by salt shock-glutamine co-feeding, characterized in that, The method includes supplementing glutamine after salt shock.

2. The method according to claim 1, wherein The salt shock time is when the biomass concentration (OD 600 ) of the halophilic bacteria is between 30 and 35.

3. The method according to claim 1, characterized in that, The time point for supplementing glutamine is after salt shock.

4. The method according to claim 1, characterized in that, The optimal time point for supplementing glutamine is 1 h after salt shock.

5. The method according to claim 1, characterized in that, The added concentration of the glutamine is 1-6 g / L.

6. The method according to claim 5, characterized in that, The optimal added concentration of the glutamine is 3 g / L.

7. The method according to claim 1, characterized in that, The fermentation medium includes: ammonium chloride 3.00 ± 0.5 g / L, sodium sulfate 2.50 ± 0.2 g / L, magnesium sulfate heptahydrate 3.08 ± 0.1 g / L, zinc sulfate 0.25 ± 0.05 g / L, manganese sulfate 0.09 ± 0.01 g / L, disodium hydrogen phosphate dodecahydrate 7.50 ± 0.5 g / L, sodium dihydrogen phosphate dihydrate 2.00 ± 0.1 g / L, potassium chloride 3.00 ± 0.3 g / L, PTM1 1.00 ± 0.1 mL / L, antifoaming agent 0.5 ± 0.05 mL / L, pH 7.00 ± 0.

10.

8. The method according to claim 7, wherein The sodium chloride concentration in the basic medium is 8 ± 1% (w / v).

9. The method according to claim 1, characterized in that The improved fermentation performance includes increasing the yield, increasing the product yield, shortening the fermentation cycle, reducing the fermentation cost, etc.

10. The method according to any one of claims 1-8, characterized in that, The halophilic bacterium is Halomonas elongata DSM 2581.

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