A method for high-yield production of l-methionine by whole fermentation

Abstract

The application discloses a method for high-yield L-methionine by full fermentation, and recombinant Escherichia coli for producing L-methionine is used as a production strain; after fermentation to the initial glucose concentration of the fermentation liquor being lower than 2 g / L, a dissolved oxygen feedback mode is used to flow feed a culture medium to maintain the dissolved oxygen value at 20-30%; when OD 600 reaches 10, 30 and 45, the constant flow rate of 10-20, 20-30 and 25-40 mL / h is respectively used to flow feed the culture medium to maintain the dissolved oxygen value at 20-30%; when the dissolved oxygen value rises to 50% and above, the flow feeding is stopped; according to the cell growth condition, the feeding rate in the fermentation process is changed, the problems such as substrate inhibition, product feedback inhibition and high acetic acid concentration in the fermentation process are successfully solved, the L-methionine yield is increased from 18.2 g / L to 31.71 g / L, the sugar acid conversion rate is increased to 12.2%, and meanwhile, the fermentation time is shortened to 68 h.

Description

(I) Technical Field

[0001] This invention relates to a total fermentation method for increasing L-methionine production. (II) Background Technology

[0002] The molecular formula of L-methionine (L-Met) is C5H 11 NO2S, also known as L-methionine, or 2-amino-4-methylthiobutyric acid, is a sulfur-containing nonpolar α-amino acid belonging to the aspartic acid family, along with lysine, threonine, and isoleucine. Its CAS number is 63-68-3. Based on different configurations, methionine can be divided into D-form and L-form, with only the L-form possessing biological activity. L-methionine is the only essential amino acid containing sulfur in organisms and plays a crucial role. It was first isolated from casein by Mueller in 1922, and subsequently, Barger and Coyne determined its structural formula and formally named it L-methionine. Extensive research has since been conducted on L-methionine. It is now known that L-methionine serves as a precursor to S-adenosylmethionine (SAM), acting as a methyl donor and participating in the synthesis of important metabolic intermediates such as lipoic acid and polyamines. Fluctuations in L-methionine concentration alter the concentration of SAM, which plays a crucial role in the synthesis of biomolecules such as DNA and RNA. L-methionine also plays an indispensable role in cellular activities, including regulating many post-translational modifications of proteins. Therefore, L-methionine is widely used in the feed, food, and pharmaceutical industries.

[0003] Currently, the main method for producing methionine is chemical synthesis. There are several chemical synthesis methods for L-methionine, which can be categorized by raw material route, including the acrolein method, aminolactone method, malonic ester method, and tyrosine hydrolysis method. Among these, the acrolein method is the earliest and still the most important production process for L-methionine producers worldwide. This method was invented by the German company Degussa AG in the early 1960s. Its main principle is to use acrolein and methanethiol as raw materials to generate methylthiopropional, which is then condensed and hydrolyzed to produce L-methionine. Currently, companies such as Rhône-Plunkett, Degussa, and Monsanto have their own unique hydrolysis and acidification methods, resulting in various technical patents based on the acrolein method, leading to continuous advancements in L-methionine production processes with technological progress. Chemical synthesis reduces reaction steps, lowers production costs, and increases product yield. However, it still inevitably uses highly volatile and toxic substrates such as hydrogen cyanide and methanethiol, and suffers from harsh reaction conditions, large amounts of waste that are difficult to treat, and significant environmental pollution. Enzymatic methods are also important for producing methionine. The enzymatic hydrolysis of L-methionine mainly involves two approaches: eliminating D-methionine from racemic mixtures and converting D-methionine to L-methionine. Enzymatic hydrolysis primarily utilizes enzymes to produce L-methionine in vitro, but its raw material prices are generally high, and the production process is cumbersome, making it unsuitable for large-scale fermentation production. Compared to these two methods, microbial fermentation has gained increasing attention due to its relatively green process and low cost. However, the low yield of L-methionine produced by fermentation remains the biggest obstacle to its industrial production.

[0004] Compared to chemical and enzymatic methods, microbial fermentation offers milder reaction conditions and less environmental impact, making it a superior method for synthesizing target products. Since the 20th century, researchers have explored biological methods for producing L-methionine. However, due to the complex synthetic pathway, numerous regulatory nodes, and the toxic side effects of L-methionine production on the bacteria themselves, no high-yield L-methionine strain suitable for large-scale production has yet been identified. Currently, publicly known L-methionine-producing strains include *Escherichia coli*, *Corynebacterium glutamicum*, *Bacillus subtilis*, and *Corynebacterium lilium*. Existing research on microbial fermentation primarily focuses on strain construction and metabolic studies, but significant obstacles remain in *E. coli* production of L-methionine. For example, although many feasible engineered bacterial construction schemes have been proposed, the conversion rate of glucose to L-methionine remains low.

[0005] Microorganisms may have different environmental requirements at different stages of growth and at different times of producing their intended metabolites. Therefore, improving the conversion rate of glucose to L-methionine is an urgent problem to be solved. To this end, examining the changes in metabolic flux of methionine-producing strains under different fermentation regulation processes can further clarify their synthetic biological processes and lay the foundation for improving fermentation yield and sugar-acid conversion rate. (III) Summary of the Invention

[0006] The purpose of this invention is to provide a method for high-yield L-methionine production through full fermentation. This method improves L-methionine yield, sugar-acid conversion rate, and shortens the fermentation cycle by rationally changing the composition of the fermentation medium and using different fermentation processes and feeding strategies. It solves the problems of low L-methionine yield, low sugar-acid conversion rate, and long fermentation cycle in existing processes.

[0007] The technical solution adopted in this invention is:

[0008] This invention provides a method for high-yield L-methionine production via total fermentation. The method includes the following steps: using L-methionine-producing recombinant *Escherichia coli* as the production strain, inoculating it into a fermentation medium containing 50 mg / L kanamycin resistance, culturing at 37°C, and stirring at 300-1000 r / min during fermentation; controlling the dissolved oxygen value at 20-30% through stirring and aeration, and controlling the pH at 6.75-6.85 by adding ammonia; fermenting to OD... 600 When the OD value reaches 0.6-0.8, add IPTG to a final concentration of 24 mg / L and induce fermentation at 30℃ and 300-1000 r / min. During fermentation, samples are taken every 4 hours to detect OD. 600 The content of residual sugar and L-methionine; after fermentation to the point where the initial glucose concentration of the fermentation broth is below 2 g / L, the fed-batch medium is maintained at a dissolved oxygen (DO) value of 20-30% using a dissolved oxygen feedback method (preferably at a flow rate of 40 mL / h when the dissolved oxygen value is above 30%); when OD 600 When the dissolved oxygen level reaches 10, feed culture medium is added at a constant flow rate of 10-20 mL / h to maintain the dissolved oxygen level at 20-30%; when the OD... 600 When the dissolved oxygen level reaches 30%, feed culture medium is added at a constant flow rate of 20-30 mL / h to maintain the dissolved oxygen level at 20-30%; when the OD... 600 When the dissolved oxygen level reaches 45, feed medium is added at a constant flow rate of 25-40 mL / h to maintain the dissolved oxygen level at 20-30%. In the later stage of fermentation, the cells gradually die and the dissolved oxygen level rises. Feeding is stopped when the dissolved oxygen level rises to 50% or above. Fermentation ends when the dissolved oxygen level reaches 100%.

[0009] The fermentation medium consisted of: glucose 10 g / L, CaCl₂·2H₂O 0.08 g / L, MgSO₄·7H₂O 5 g / L, citric acid 2.5 g / L, KH₂PO₄ 2.5 g / L, K₂HPO₄·3H₂O 1.38 g / L, FeCl₃·6H₂O 0.12 g / L, (NH₄)₂SO₄ 3.3 g / L, Na₂S₂O₃ 3.95 g / L, and VB 0.01 g / L. 12 0.01 g / L of VB1, 0.05 g / L of L-lysine, 2 mL / L of SSA, and 1 mL / L of SSB;

[0010] Feeding medium: glucose 500 g / L, MgSO4·7H2O 5 g / L, FeCl3·6H2O 0.06 g / L, (NH4)2SO4 33 g / L, Na2S2O3 39.5 g / L, betaine 0.5 g / L, SSA 1.6 mL / L, SSB 0.8 mL / L, VB 0.01 g / L 12 0.01 g / L of vitamin B1 and 0.05 g / L of L-lysine, in water;

[0011] The SSA composition is: ZnSO4·7H2O 8.5g / L, MnCl2·2H2O 7.5g / L, CuCl2·2H2O 0.75g / L, CoCl2·6H2O 1.25g / L, EDTA 4g / L, and water as the solvent.

[0012] The SSB composition is: H3BO3 3g / L, Na2MoO4·2H2O 2.5g / L, and the solvent is water.

[0013] Preferably, the production strain is Escherichia coli ZJBSSC362, deposited at the China Center for Type Culture Collection (CCTCC), date of deposit: December 4, 2020, accession number: CCTCC NO: M2020846, address: Wuhan University, China, postcode: 430072, and has been published in patent application CN 112779200A.

[0014] Preferably, when OD 600 When the dissolved oxygen level reaches 10, feed culture medium is added at a constant flow rate of 15 mL / h to maintain the dissolved oxygen level at 20-30%; when the OD... 600 When the dissolved oxygen level reaches 30, feed culture medium is added at a constant flow rate of 20 mL / h to maintain the dissolved oxygen level at 20-30%; when the OD... 600When the dissolved oxygen level reaches 45%, feed medium is added at a constant flow rate of 25 mL / h to maintain the dissolved oxygen level at 20-30%. In the later stage of fermentation, the cells gradually die and the dissolved oxygen level rises. Feeding is stopped when the dissolved oxygen level rises to 50% or above. Fermentation ends when the dissolved oxygen level reaches 100%.

[0015] Preferably, the production strain is activated on an agar slant and cultured on a seed culture medium before inoculation, and the seed culture is inoculated into the fermentation medium at a volume concentration of 10-15%; the seed culture is prepared according to the following steps:

[0016] (1) Activation culture: The production strain was inoculated onto LB agar medium and cultured overnight at 37°C to obtain activated bacteria; the composition of the LB agar medium was: 10 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl, 20 g / L agar powder, water as solvent, pH = 6.8-7.0;

[0017] (2) Primary seed culture: The activated bacteria obtained in step (1) were inoculated into LB liquid medium and cultured at 37℃ and 200rpm for 12h to obtain primary seed culture; LB liquid medium composition: 10g / L peptone, 5g / L yeast extract, 5g / L NaCl, solvent is water, pH=6.8-7.0;

[0018] (3) Secondary seed culture: The primary seed culture obtained in step (2) is inoculated into LB liquid medium at a volume concentration of 1-3% and cultured at 37℃ and 200rpm for 12-16h to obtain the secondary seed culture; the composition of the LB liquid medium is the same as in step (2).

[0019] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0020] L-methionine synthesis is complex and influenced by many factors. Metabolic engineering techniques used to modify *E. coli* accumulation often lead to growth problems. This invention optimizes the initial sugar concentration of the fermentation medium to promote rapid and uninhibited cell growth in the early stages, thereby enhancing L-methionine accumulation. The experimental strain is a lysine auxotroph, requiring the addition of lysine to the fermentation broth to ensure that lysine deficiency does not negatively impact fermentation. However, based on previous research on L-methionine fermentation, appropriate "lysine starvation" during fermentation directs metabolic flux towards L-methionine synthesis. Therefore, a suitable feeding method is needed to control lysine concentration during fermentation. Furthermore, to address premature cell senescence and low acid production efficiency, and to improve cell viability in the mid-to-late stages, various glucose feeding strategies were explored based on the different environmental requirements for cell growth and product synthesis at different fermentation stages. This improved the cell's acid production capacity in the later stages, further increasing L-methionine yield. The final solution employed a strategy of first using dissolved oxygen feedback feed to achieve a certain cell density in the culture medium, and then using a staged variable-rate feeding method to successfully address issues such as substrate inhibition, product feedback inhibition, and excessively high acetic acid concentration during fermentation by the metabolically engineered bacteria. This resulted in an increase in L-methionine yield from 18.2 g / L to 31.71 g / L, a peak sugar-acid conversion rate of 12.2%, and a reduction in fermentation time to 68 hours. Therefore, this method has significant industrial application value and provides guidance for the fermentation production of other amino acids and related compounds. (iv) Description of the attached drawings

[0021] Figure 1 Example 1: Initial fermentation process diagram.

[0022] Figure 2 Example 2: Fermentation process diagram with different initial sugar concentrations.

[0023] Figure 3 Example 3: Fermentation process diagram of dissolved oxygen feedback feeding.

[0024] Figure 4 Example 4: Fermentation process diagram of pH feedback feeding.

[0025] Figure 5 Example 5: Fermentation process diagram of a feed with a constant residual sugar concentration of 1-5 g / L.

[0026] Figure 6 Example 6: Fermentation process diagram of fed feed with a constant residual sugar concentration of 5-10 g / L.

[0027] Figure 7 Example 7: Fermentation process diagram with constant feed rate of 10 mL / h.

[0028] Figure 8Example 8: Fermentation process diagram with constant feed rate of 20 mL / h.

[0029] Figure 9 Example 9: Fermentation process diagram of the novel feeding method (1).

[0030] Figure 10 Example 10: Fermentation process diagram of the novel feeding method (2).

[0031] Figure 11 Example 11 Fermentation process diagram of the novel feeding method (3). (V) Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0033] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0034] The experimental strain was Escherichia coli ZJBSSC362, deposited at the China Center for Type Culture Collection (CCTCC) on December 4, 2020, with accession number CCTCC NO: M 2020846, address: Wuhan University, China, postcode: 430072, and published in patent application CN 112779200 A.

[0035] The LB plate medium consists of: 10 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl, 20 g / L agar powder, water as solvent, and pH = 6.8-7.0.

[0036] The composition of LB liquid medium is: 10 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl, with water as the solvent, and pH = 6.8-7.0.

[0037] Fermentation medium: glucose 10 g / L, CaCl2·2H2O 0.08 g / L, MgSO4·7H2O 5 g / L, citric acid 2.5 g / L, KH2PO4 2.5 g / L, K2HPO4·3H2O 1.38 g / L, FeCl3·6H2O 0.12 g / L, (NH4)2SO4 3.3 g / L, Na2S2O3 3.95 g / L, VB 0.01 g / L 12 0.01 g / L of VB1, 0.05 g / L of L-lysine, 2 mL / L of SSA, and 1 mL / L of SSB, in water as the solvent;

[0038] Feeding medium: glucose 500 g / L, MgSO4·7H2O 5 g / L, FeCl3·6H2O 0.06 g / L, (NH4)2SO4 33 g / L, Na2S2O3 39.5 g / L, betaine 0.5 g / L, SSA 1.6 mL / L, SSB 0.8 mL / L, VB 0.01 g / L 12 0.01 g / L of vitamin B1 and 0.05 g / L of L-lysine, in water;

[0039] The SSA composition is: ZnSO4·7H2O 8.5g / L, MnCl2·2H2O 7.5g / L, CuCl2·2H2O 0.75g / L, CoCl2·6H2O 1.25g / L, EDTA 4g / L, and water as the solvent.

[0040] The SSB composition is: H3BO3 3g / L, Na2MoO4·2H2O 2.5g / L, and the solvent is water.

[0041] Among them, VB 12 With 10g / L of VB 12 The following solutions were added: 1 mL / L aqueous solution; VB1 was added as a 5 g / L aqueous solution, at a volume of 2 mL / L; L-lysine was added as a 200 g / L aqueous solution, at a volume of 0.25 mL / L; IPTG was added as a 120 g / L aqueous solution, at a volume of 0.2 mL / L.

[0042] Example 1: Method for producing L-methionine by fermentation

[0043] (1) Activation culture: Escherichia coli ZJBSSC362 was streaked onto LB agar plates containing 50 mg / L kan resistance and incubated overnight at 37°C to obtain activated bacteria;

[0044] (2) Primary seed culture: Pick a single colony from step (1) and inoculate it into a test tube containing 10 mL of LB liquid medium with 50 mg / L Kan resistance. Incubate at 37°C and 200 rpm for 12 h to obtain primary seed culture.

[0045] (3) Secondary seed culture: Take 1 mL of the primary seed culture from step (2) and inoculate it into 3 500 mL shake flasks containing 100 mL of LB liquid medium containing 50 mg / L kan resistance. Culture at 37℃ and 200 rpm for 12-16 h to obtain secondary seed culture.

[0046] (4) Fermentation culture: At an inoculation rate of 10% by volume, the secondary seed culture obtained in step (3) was inoculated into a 5L fermenter containing 50mg / L Kan resistance fermentation medium, with a liquid volume of 2L / 5L. The culture temperature was 37℃, the initial stirring speed was 300rpm, the dissolved oxygen value was controlled at 20-30% by stirring and ventilation, and the pH was controlled at 6.75-6.85 by adding ammonia water. Fermentation was carried out until the OD reached 10 ... 600 When the OD value reached 0.6-0.8, IPTG was added to a final concentration of 24 mg / L, and induction culture was carried out at 30℃ and 300 rpm. During fermentation, OD was measured every 4 hours. 600 The residual sugar content and L-methionine content; after 16 hours of fermentation, when the initial sugar is basically consumed and the initial glucose concentration is below 2 g / L, fed-batch culture medium is started; that is, from 16 hours, fed-batch culture medium is added at a constant flow rate of 30 mL / h to maintain the DO (dissolved oxygen) value at 30%; when OD 600 When the DO value reaches 30, the feed medium is added at a constant flow rate of 40 mL / h to maintain the DO value at 30%; when the OD value reaches 30%, the feed medium is added at a constant flow rate of 40 mL / h to maintain the DO value at 30%. 600 When the dissolved oxygen (DO) level reaches 50%, fed medium is added at a constant flow rate of 50 mL / h to maintain a DO level of 30%. During the later stages of fermentation, the cells gradually die, and the DO level rises. Feeding is stopped when the DO level reaches 50% or higher. Fermentation ends when the DO level reaches 100%. OD levels during fermentation are... 600 The curves showing the changes in residual sugar content and L-methionine content are shown below. Figure 1 As shown, from Figure 1 It can be seen that the yield of L-methionine was the highest at 72 h after fermentation, which was 18.20 g / L, and the highest sugar-acid conversion rate was 7.0%.

[0047] Sugar-acid conversion rate = (total mass of L-methionine produced ÷ total mass of glucose consumed) × 100%.

[0048] Determination of biomass OD by ultraviolet spectrophotometer 600 After obtaining the fermentation broth sample through the sampling port, dilute it appropriately and take 2 mL into a colorimetric tube. Measure the absorbance (OD) at 600 nm using a UV spectrophotometer. 600 ).

[0049] The DNS method is used to determine the residual sugar (i.e., glucose) content: DNS reagent: Dissolve 6.3g of 3,5-dinitrosalicylic acid in 400mL of distilled water, gradually add 21g of sodium hydroxide, then add 182g of potassium sodium tartrate tetrahydrate, 5.0g of phenol, and 5.0g of anhydrous sodium sulfite. Heat in a warm water bath, stirring constantly until the solution is clear and transparent. Dilute to 1000mL with distilled water and store in a brown bottle, isolated from carbon dioxide, for 5-7 days before use. Take five 1.5mL centrifuge tubes and add 0.2mL, 0.4mL, 0.6mL, 0.8mL, and 1mL of 1g / L glucose aqueous solution to each tube sequentially. Add water to each tube to bring the volume to 1.0mL, and vortex to mix. These yield glucose aqueous solutions of 0.2, 0.4, 0.6, 0.8, and 1.0g / L, respectively. Take five 1.5 mL centrifuge tubes and pipette 500 μL of glucose aqueous solutions of different concentrations into each tube, then add 500 μL of DNS reagent. Heat in a water bath for 5 min, then take 600 μL of the reaction solution and add 2.4 mL of distilled water. Detect at 540 nm using a spectrophotometer. Plot a standard curve with the measured value at 540 nm as the ordinate and glucose concentration as the abscissa. The equation of the standard curve is: y = 0.1133x + 0.0004(R² + π / 4)². 2 =0.9954). Under the same conditions, the absorbance of the sample was measured, and the glucose content in the sample was obtained according to the standard curve.

[0050] High-performance liquid chromatography (HPLC) for L-methionine content determination: Centrifuge 1 mL of fermentation broth at 12000 rpm for 1 min at room temperature. Dilute the supernatant appropriately (fermentation broth before 24 h of fermentation is not diluted; fermentation broth from 24-48 h is diluted 5 times; fermentation broth after 48 h is diluted 10 times) to ensure the L-methionine concentration is within the range of 0.5-3 g / L of the standard curve. Then, determine the L-methionine yield using a Thermo Fisher UPLC. Mobile phase A: Pure acetonitrile filtered through a 0.22 μm filter membrane, degassed by ultrasonication, and prepared fresh for each use. Mobile phase B: Pure water:acetonitrile:triethylamine = 84.8:15:0.2 (volume ratio), pH adjusted to 4.9 with acetic acid; filtered through a 0.22 μm filter membrane, degassed by ultrasonication, and prepared fresh for each use. Sample derivatization method: Add 300 μL of derivatization reagent I and 500 μL of buffer II to 100 μL of sample. The mixture was placed in a mixer and reacted at 60℃ and 400 rpm in the dark for 1 h. After the reaction, it was filtered through a 0.22 μm syringe-type organic filter membrane and stored for detection. Derivatization reagent I: 0.2700 g CNBF (3,5-dinitro-4-chloro-trifluoromethylbenzene) / 10 mL acetonitrile; Buffer II: pH 9.0 H3BO3-Na2B4O7 buffer (a solution of 0.2 mol / L boric acid (H3BO3) and 0.05 mol / L borax (Na2B4O7)). The prepared solution should be stored in the dark. Detection conditions: Column: J&K C18-H Column (4.6 × 250 mm, 5 μm); Mobile phase: gradient elution using the proportions in Table 1; Flow rate: 0.80 mL / min; Column temperature: 30℃; Injection volume: 10 μL; Detection wavelength: 260 nm.

[0051] Table 1 Gradient elution settings

[0052]

[0053] Example 2: Effect of different initial sugar concentrations on the fermentation production of L-methionine

[0054] (1) Activation culture: Escherichia coli ZJBSSC362 was streaked onto LB agar containing 50 mg / L kan resistance and incubated overnight at 37°C to obtain activated bacteria;

[0055] (2) Primary seed culture: Pick a single colony from step (1) and inoculate it into a test tube containing 10 mL of LB liquid medium with 50 mg / L kan resistance. Incubate at 37°C and 200 rpm for 12 h to obtain primary seed culture.

[0056] (3) Secondary seed culture: Take 1 mL of the primary seed culture from step (2) and inoculate it into 3 500 mL shake flasks containing 100 mL of LB liquid medium containing 50 mg / L kan resistance. Culture at 37℃ and 200 rpm for 12-16 h to obtain secondary seed culture.

[0057] (4) Fermentation culture: Using an inoculum concentration of 15% (v / v), take the secondary seed culture obtained in step (3) and inoculate it into a 5L fermenter containing 2L of fermentation medium with 50mg / L kan resistance. The filling volume is 2L / 5L. The culture temperature is 37℃, the initial stirring speed is 300rpm, and the dissolved oxygen value is controlled at 20-30% through stirring and ventilation. The pH is controlled at 6.75-6.85 by adding ammonia. Fermentation continues until OD... 600 When the OD reached 0.6-0.8, IPTG was added to a final concentration of 24 mg / L, and induction culture was carried out at 30℃ and 300 rpm. During fermentation, samples were taken every 4 hours, and OD was detected using the method in Example 1. 600 The residual sugar content and L-methionine content were measured. In the early stage of fermentation, the cells were allowed to grow under natural dissolved oxygen (DO) conditions. The DO value of the fermentation system would continuously decrease until it was below 10%. At this point, the initial sugar was basically consumed, and the DO value would rise rapidly. When it rose to 30%, the dissolved oxygen feedback method was used to feed the culture medium at a rate of 40 mL / h to stabilize the DO value at 30%. In the later stage of fermentation, the cells gradually died, and the DO value rose. When the DO value rose to 50% or above, the feeding was stopped. When the DO value reached 100%, the fermentation ended.

[0058] Under the same conditions, when the glucose concentration in the fermentation medium was changed to 5 g / L, 15 g / L, and 20 g / L, the yield of L-methionine was as follows: Figure 2 As shown.

[0059] When the concentration of nutrients, such as carbon source, in the culture system exceeds a certain critical value, microorganisms will form an ion gradient to resist high osmotic pressure and maintain the intracellular environment. This process requires energy and will significantly inhibit cell growth. Conversely, if the initial glucose concentration is too low, the cells may be forced to stop growing before reaching their optimal state due to carbon source depletion. Therefore, selecting an appropriate initial carbon source concentration is necessary. Four gradients were set for the initial glucose: 5 g / L, 10 g / L, 15 g / L, and 20 g / L. Results Figure 2 As shown, the L-methionine yields after 84 hours of fermentation at four different initial sugar concentrations were 23.71 g / L, 24.26 g / L, 21.12 g / L, and 20.72 g / L, respectively. The optimal initial sugar concentration of 10 g / L yielded the highest fermentation yield, but the fermentation period was 84 hours, which was 12 hours longer than before optimization, and the sugar-acid conversion rate was only 9.33%.

[0060] Example 3: L-Methionine Production by Dissolved Oxygen Feedback Fermentation under Optimized Initial Sugar Concentration

[0061] (1) Activation culture: Escherichia coli ZJBSSC362 was streaked onto LB agar containing 50 mg / L kan resistance and incubated overnight at 37°C to obtain activated bacteria;

[0062] (2) Primary seed culture: Pick a single colony and inoculate it into a test tube containing 10 mL of LB liquid medium with 50 mg / L kan resistance, and incubate at 37℃ and 200 rpm for 12 h to obtain primary seed culture;

[0063] (3) Secondary seed culture: Take 1 mL of the primary seed culture from step (2) and inoculate it into 3 500 mL shake flasks containing 100 mL of LB liquid medium containing 50 mg / L kan resistance. Culture at 37℃ and 200 rpm for 12-16 h to obtain secondary seed culture.

[0064] (4) Fermentation culture: Using an inoculum concentration of 15% (v / v), take the secondary seed culture obtained in step (3) and inoculate it into a 5L fermenter containing 50 mg / L Kan resistance-resistant fermentation medium. The filling volume is 2L / 5L. The culture temperature is 37℃, the initial stirring speed is 300 rpm, and the dissolved oxygen value is controlled at 20-30% through stirring and ventilation. The pH is controlled at 6.75-6.85 by adding ammonia. Fermentation continues until OD... 600 When the OD reached 0.6-0.8, IPTG was added to a final concentration of 24 mg / L, and induction culture was carried out at 30℃ and 300 rpm. During fermentation, samples were taken every 4 hours, and OD was detected using the method in Example 1. 600 The residual sugar content and L-methionine content were measured. In the early stage of fermentation, the cells were allowed to grow under natural DO conditions. The DO value of the fermentation system would continuously decrease to below 10%, at which point the initial sugar was basically consumed, and the DO value would rapidly increase. When it rose to 30%, dissolved oxygen feedback was used to feed the culture medium at 40 mL / h to stabilize the DO value at 30%. In the later stage of fermentation, the cells gradually died, and the DO value rose. When the DO value rose to 50% or above, the feeding was stopped. When the DO value reached 100%, the fermentation ended.

[0065] See results Figure 3 As shown, using dissolved oxygen feedback feeding fermentation, the highest L-methionine yield was 24.26 g / L after 84 hours of fermentation, and the highest sugar-acid conversion rate was 9.33%. However, the fermentation time was extended to 84 hours, which is extremely unfavorable for the cost of amino acid fermentation.

[0066] Example 4: L-Methionine Production by pH Feedback Fermentation under Optimized Initial Sugar Concentration

[0067] (1) Activation culture: Escherichia coli ZJBSSC362 was streaked onto LB agar containing 50 mg / L kan resistance and incubated overnight at 37°C to obtain activated bacteria;

[0068] (2) Primary seed culture: Pick a single colony and inoculate it into a test tube containing 10 mL of LB liquid medium with 50 mg / L kan resistance, and incubate at 37℃ and 200 rpm for 12 h to obtain primary seed culture;

[0069] (3) Secondary seed culture: Take 1 mL of the primary seed culture from step (2) and inoculate it into 3 500 mL shake flasks containing 100 mL of LB liquid medium containing 50 mg / L kan resistance. Culture at 37℃ and 200 rpm for 12-16 h to obtain secondary seed culture.

[0070] (4) Fermentation culture: Using an inoculum concentration of 15% (v / v), take the secondary seed culture obtained in step (3) and inoculate it into a 5L fermenter containing 50 mg / L Kan resistant fermentation medium. The filling volume is 2L / 5L. The culture temperature is 37℃, the initial stirring speed is 300 rpm, and the dissolved oxygen value is controlled at 20-30% through stirring and ventilation. The pH is controlled at 6.75-6.85 by adding ammonia. Fermentation culture is continued until OD... 600 When the OD reached 0.6-0.8, IPTG was added to a final concentration of 24 mg / L, and induction culture was carried out at 30℃ and 300 rpm. During fermentation, samples were taken every 4 hours, and OD was detected using the method in Example 1. 600 The residual sugar content and L-methionine content were monitored. After the initial glucose was consumed, the pH spiked. When the pH rose above 6.82, a pH feedback method was used to feed the culture medium at a rate of 40 mL / h to stabilize the pH at 6.8. In the later stages of fermentation, the cells gradually died, and the dissolved oxygen (DO) value increased. Feeding was stopped when the DO value reached 50% or above, and fermentation ended when the DO value reached 100%.

[0071] See results Figure 4 As shown in the figure, fed-batch fermentation with pH feedback was used. The highest yield of L-methionine was observed at 84 hours after fermentation, with a yield of only 20.19 g / L and a maximum sugar-acid conversion rate of 7.77%.

[0072] Example 5: The effect of low residual sugar control on the fermentation production of L-methionine

[0073] (1) Activation culture: Escherichia coli ZJBSSC362 was streaked onto LB agar containing 50 mg / L kan resistance and incubated overnight at 37°C to obtain activated bacteria;

[0074] (2) Primary seed culture: Pick a single colony and inoculate it into a test tube containing 10 mL of LB liquid medium with 50 mg / L kan resistance. Incubate at 37°C and 200 rpm for 12 h to obtain primary seed culture;

[0075] (3) Secondary seed culture: Take 1 mL of the primary seed culture from step (2) and inoculate it into 3 500 mL shake flasks containing 100 mL of LB liquid medium containing 50 mg / L kan resistance. Culture at 37℃ and 200 rpm for 12-16 h to obtain secondary seed culture.

[0076] (4) Fermentation culture: Using an inoculum concentration of 15% (v / v), take the secondary seed culture obtained in step (3) and inoculate it into a 5L fermenter containing 50 mg / L Kan resistant fermentation medium. The filling volume is 2L / 5L. The culture temperature is 37℃, the initial stirring speed is 300 rpm, and the dissolved oxygen value is controlled at 20-30% through stirring and ventilation. The pH is controlled at 6.75-6.85 by adding ammonia. Fermentation continues until OD... 600 When the OD reached 0.6-0.8, IPTG was added to a final concentration of 24 mg / L, and induction culture was carried out at 30℃ and 300 rpm. During fermentation, samples were taken every 4 hours to detect OD using the method in Example 1. 600 The residual sugar and L-methionine content were monitored. After fermentation until the initial glucose concentration in the fermentation broth was below 2 g / L, fed-batch culture medium was started at a flow rate of 10 mL / h. The feeding rate was then adjusted based on the measured residual sugar concentration (increasing the feeding rate when the residual sugar concentration was below 1 g / L and decreasing the feeding rate when the residual sugar concentration was above 5 g / L) to maintain the residual sugar concentration between 1 and 5 g / L. During the later stages of fermentation, the cells gradually died, and dissolved oxygen (DO) increased. Feeding was stopped when the DO value reached 50% or higher, and fermentation ended when the DO reached 100%.

[0077] See results Figure 5 As shown in the figure, the highest L-methionine yield was 22.26 g / L at the end of fermentation, with a maximum sugar-acid conversion rate of 8.56%. However, the fermentation process was prolonged to 92 hours.

[0078] Example 6: The effect of high residual sugar control on the fermentation production of L-methionine

[0079] (1) Activation culture: Escherichia coli ZJBSSC362 was streaked onto LB agar containing 50 mg / L kan resistance and incubated overnight at 37°C to obtain activated bacteria;

[0080] (2) Primary seed culture: Pick a single colony and inoculate it into a test tube containing 10 mL of LB liquid medium with 50 mg / L kan resistance. Incubate at 37°C and 200 rpm for 12 h to obtain primary seed culture;

[0081] (3) Secondary seed culture: Take 1 mL of the primary seed culture from step (2) and inoculate it into 3 500 mL shake flasks containing 100 mL of LB liquid medium containing 50 mg / L kan resistance. Culture at 37℃ and 200 rpm for 12-16 h to obtain secondary seed culture.

[0082] (4) Fermentation culture: Using an inoculum concentration of 15% (v / v), take the secondary seed culture obtained in step (3) and inoculate it into a 5L fermenter containing fermentation medium. The filling volume is 2L / 5L. The culture temperature is 37℃, and the initial stirring speed is 300 rpm. The dissolved oxygen value is controlled at 20-30% through stirring and ventilation, and the pH is controlled at 6.75-6.85 by adding ammonia. Fermentation continues until the OD reaches 1000%. 600 When the OD reached 0.6-0.8, IPTG was added to a final concentration of 24 mg / L, and induction culture was carried out at 30℃ and 300 rpm. During fermentation, samples were taken every 4 hours, and OD was detected using the method in Example 1. 600 The residual sugar and L-methionine content were monitored. After fermentation reached an initial glucose concentration below 2 g / L, fed-batch culture medium was started at a flow rate of 15 mL / h. The feeding rate was then adjusted based on the measured residual sugar concentration (increasing the feeding rate when the residual sugar concentration was below 5 g / L and decreasing the feeding rate when the residual sugar concentration was above 10 g / L) to maintain the residual sugar concentration at 5-10 g / L. During the later stages of fermentation, the cells gradually died, and the dissolved oxygen (DO) value increased. Feeding was stopped when the DO value reached 50% or higher, and fermentation ended when the DO value reached 100%.

[0083] See results Figure 6 As shown, the highest yield of L-methionine, 21.32 g / L, was still observed at 92 h after fermentation, with a maximum sugar-acid conversion rate of 8.2%.

[0084] Example 7: Effect of constant low-rate feed on fermentation production of L-methionine

[0085] (1) Activation culture: Escherichia coli ZJBSSC362 was streaked onto LB agar containing 50 mg / L kan resistance and incubated overnight at 37°C to obtain activated bacteria;

[0086] (2) Primary seed culture: Pick a single colony and inoculate it into a test tube containing 10 mL of LB liquid medium with 50 mg / L kan resistance. Incubate at 37°C and 200 rpm for 12 h to obtain primary seed culture;

[0087] (3) Secondary seed culture: Take 1 mL of the primary seed culture from step (2) and inoculate it into 3 500 mL shake flasks containing 100 mL of LB liquid medium containing 50 mg / L kan resistance. Culture at 37℃ and 200 rpm for 12-16 h to obtain secondary seed culture.

[0088] (4) Fermentation culture: Using an inoculum concentration of 15% (v / v), take the secondary seed culture obtained in step (3) and inoculate it into a 5L fermenter containing 50 mg / L Kan resistant fermentation medium. The filling volume is 2L / 5L. The culture temperature is 37℃, the initial stirring speed is 300 rpm, and the dissolved oxygen value is controlled at 20-30% through stirring and ventilation. The pH is controlled at 6.75-6.85 by adding ammonia. Fermentation continues until OD... 600 When the OD reached 0.6-0.8, IPTG was added to a final concentration of 24 mg / L, and induction culture was carried out at 30℃ and 300 rpm. During fermentation, samples were taken every 4 hours, and OD was detected using the method in Example 1. 600 The residual sugar content and L-methionine content were measured. After 12 hours of fermentation, the initial sugar was basically consumed, and the initial glucose concentration of the fermentation broth was below 2 g / L. Feeding culture medium was then added at a rate of 10 mL / h until the fermentation was complete. During the later stages of fermentation, the cells gradually died, and the dissolved oxygen (DO) value increased. Feeding was stopped when the DO value reached 50% or higher, and fermentation ended when the DO value reached 100%.

[0089] See results Figure 7 As shown, the highest yield of L-methionine was observed at 76 hours after fermentation, at only 15.94 g / L, with a maximum sugar-acid conversion rate of 7.97%.

[0090] Example 8: Effect of constant high-speed feedstock on fermentation production of L-methionine

[0091] (1) Activation culture: Escherichia coli ZJBSSC362 was streaked onto LB agar containing 50 mg / L kan resistance and incubated overnight at 37°C to obtain activated bacteria;

[0092] (2) Primary seed culture: Pick a single colony and inoculate it into a test tube containing 10 mL of LB liquid medium with 50 mg / L kan resistance. Incubate at 37°C and 200 rpm for 12 h to obtain primary seed culture;

[0093] (3) Secondary seed culture: Take 1 mL of the primary seed culture from step (2) and inoculate it into 3 500 mL shake flasks containing 100 mL of LB liquid medium containing 50 mg / L kan resistance. Culture at 37℃ and 200 rpm for 12-16 h to obtain secondary seed culture.

[0094] (4) Fermentation culture: Using an inoculum concentration of 15% (v / v), take the secondary seed culture obtained in step (3) and inoculate it into a 5L fermenter containing 50 mg / L Kan resistant fermentation medium. The filling volume is 2L / 5L. The culture temperature is 37℃, the initial stirring speed is 300 rpm, and the dissolved oxygen value is controlled at 20-30% through stirring and ventilation. The pH is controlled at 6.75-6.85 by adding ammonia. Fermentation continues until OD... 600 When the OD reached 0.6-0.8, IPTG was added to a final concentration of 24 mg / L, and induction culture was carried out at 30℃ and 300 rpm. During fermentation, samples were taken every 4 hours, and OD was detected using the method in Example 1. 600 The residual sugar content and L-methionine content were measured. After 12 hours of fermentation, the initial sugar was basically consumed, and the initial glucose concentration of the fermentation broth was below 2 g / L. Feeding medium was then added at a rate of 20 mL / h until the end of fermentation. During the later stages of fermentation, the cells gradually died, and the dissolved oxygen (DO) value increased. Feeding was stopped when the DO value reached 50% or higher, and fermentation ended when the DO value reached 100%.

[0095] See results Figure 8 As shown, the highest yield of L-methionine (17.35 g / L) was observed at 92 h after fermentation, with a maximum sugar-acid conversion rate of 6.67%.

[0096] Through the above examples 1-8, it was found that conventional fermentation optimization operations can increase fermentation yield to a certain extent, but at the same time, it will prolong the fermentation cycle and cause huge waste of fermentation industrial costs. Meanwhile, through the two constant-rate feeding examples 7 and 8, it was found that although constant-rate feeding simplifies feeding control, the fermentation process does not meet the needs of the cells themselves, resulting in insufficient or excessive glucose during fermentation, which has a negative impact on cell growth and product synthesis. Therefore, based on the analysis of the results of the above examples, this invention further proposes a multi-stage variable-rate feeding strategy in order to increase fermentation yield while shortening fermentation time.

[0097] Example 9: A novel fed-batch fermentation method for producing L-methionine (1)

[0098] (1) Activation culture: Escherichia coli ZJBSSC362 was streaked onto LB agar containing 50 mg / L kan resistance and incubated overnight at 37°C to obtain activated bacteria;

[0099] (2) Primary seed culture: Pick a single colony and inoculate it into a test tube containing 10 mL of LB liquid medium with 50 mg / L kan resistance. Incubate at 37°C and 200 rpm for 12 h to obtain primary seed culture;

[0100] (3) Secondary seed culture: Take 1 mL of the primary seed culture from step (2) and inoculate it into 3 500 mL shake flasks containing 100 mL of LB liquid medium containing 50 mg / L kan resistance. Culture at 37℃ and 200 rpm for 12-16 h to obtain secondary seed culture.

[0101] (4) Fermentation culture: Using an inoculum concentration of 15% (v / v), take the secondary seed culture obtained in step (3) and inoculate it into a 5L fermenter containing 50 mg / L Kan resistant fermentation medium. The filling volume is 2L / 5L. The culture temperature is 37℃, the initial stirring speed is 300 rpm, and the dissolved oxygen value is controlled at 20-30% through stirring and ventilation. The pH is controlled at 6.75-6.85 by adding ammonia. Fermentation continues until OD... 600 When the OD reached 0.6-0.8, IPTG was added to a final concentration of 24 mg / L, and induction culture was carried out at 30℃ and 300 rpm. During fermentation, samples were taken every 4 hours, and OD was detected using the method in Example 1. 600 The content of residual sugar and L-methionine; after 12 hours of fermentation, the initial sugar is basically consumed, and the initial glucose concentration of the fermentation broth is below 2 g / L, so fed medium is started; that is, from 12 hours, dissolved oxygen feedback is used to maintain the dissolved oxygen value at 30% by feeding medium at a rate of 40 mL / h; when OD 600 When the dissolved oxygen level reaches 10, the culture medium is fed at a constant flow rate of 10 mL / h to maintain the dissolved oxygen level at 30%; when the OD... 600 When the dissolved oxygen level reaches 30, the culture medium is fed at a constant flow rate of 20 mL / h to maintain the dissolved oxygen level at 30%; when the OD... 600 When the dissolved oxygen level reaches 45%, the culture medium is fed at a constant flow rate of 30 mL / h to maintain a dissolved oxygen level of 30%. During the later stages of fermentation, the cells gradually die, and the dissolved oxygen (DO) level rises. Feeding is stopped when the DO level rises to 50% or higher; fermentation ends when the DO level reaches 100%.

[0102] See results Figure 9 As shown, the highest yield of L-methionine (29.09 g / L) was observed at 84 h after fermentation, with a maximum sugar-acid conversion rate of 11.19%.

[0103] Example 10: A method for producing L-methionine by fermentation using a novel fed-batch (2) method

[0104] (1) Activation culture: Escherichia coli ZJBSSC362 was streaked onto LB agar containing 50 mg / L kan resistance and incubated overnight at 37°C to obtain activated bacteria;

[0105] (2) Primary seed culture: Pick a single colony and inoculate it into a test tube containing 10 mL of LB liquid medium with 50 mg / L kan resistance. Incubate at 37°C and 200 rpm for 12 h to obtain primary seed culture;

[0106] (3) Secondary seed culture: Take 1 mL of the primary seed culture from step (2) and inoculate it into 3 500 mL shake flasks containing 100 mL of LB liquid medium containing 50 mg / L kan resistance. Culture at 37℃ and 200 rpm for 12-16 h to obtain secondary seed culture.

[0107] (4) Fermentation culture: Using an inoculum concentration of 15% (v / v), take the secondary seed culture obtained in step (3) and inoculate it into a 5L fermenter containing 50 mg / L Kan resistant fermentation medium. The filling volume is 2L / 5L. The culture temperature is 37℃, the initial stirring speed is 300 rpm, and the dissolved oxygen value is controlled at 20-30% through stirring and ventilation. The pH is controlled at 6.75-6.85 by adding ammonia. Fermentation continues until OD... 600 When the OD reached 0.6-0.8, IPTG was added to a final concentration of 24 mg / L, and induction culture was carried out at 30℃ and 300 rpm. During fermentation, samples were taken every 4 hours, and OD was detected using the method in Example 1. 600 The content of residual sugar and L-methionine; when the initial sugar is basically consumed after 12 hours of fermentation and the initial glucose concentration of the fermentation broth is below 2 g / L, fed-batch culture medium is started; that is, from 12 hours, dissolved oxygen feedback is used to feed the culture medium at a rate of 40 mL / h to maintain the dissolved oxygen value at 30%; when OD 600 When the dissolved oxygen level reaches 10, the culture medium is fed at a constant flow rate of 20 mL / h to maintain the dissolved oxygen level at 30%; when the OD... 600 When the dissolved oxygen level reaches 30, feed culture medium is added at a constant flow rate of 30 mL / h to maintain the dissolved oxygen level at 30%; when the OD... 600 When the dissolved oxygen (DO) level reaches 45%, the culture medium is fed at a constant flow rate of 40 mL / h to maintain a dissolved oxygen level of 30%. During the later stages of fermentation, the cells gradually die, and the DO level rises. Feeding is stopped when the DO level rises to 50% or higher; fermentation ends when the DO level reaches 100%.

[0108] See results Figure 10 As shown, the highest yield of L-methionine (30.11 g / L) was observed at 88 h after fermentation, with a maximum sugar-acid conversion rate of 11.58%.

[0109] Example 11: A method for producing L-methionine by fermentation using a novel fed-batch (3) method

[0110] (1) Activation culture: Escherichia coli ZJBSSC362 was streaked onto LB agar containing 50 mg / L kan resistance and incubated overnight at 37°C to obtain activated bacteria;

[0111] (2) Primary seed culture: Pick a single colony and inoculate it into a test tube containing 10 mL of LB liquid medium with 50 mg / L kan resistance. Incubate at 37°C and 200 rpm for 12 h to obtain primary seed culture;

[0112] (3) Secondary seed culture: Take 1 mL of the primary seed culture from step (2) and inoculate it into 3 500 mL shake flasks containing 100 mL of LB liquid medium containing 50 mg / L kan resistance. Culture at 37℃ and 200 rpm for 12-16 h to obtain secondary seed culture.

[0113] (4) Fermentation culture: Using an inoculum concentration of 15% (v / v), take the secondary seed culture obtained in step (3) and inoculate it into a 5L fermenter containing 50 mg / L Kan resistant fermentation medium. The filling volume is 2L / 5L. The culture temperature is 37℃, the initial stirring speed is 300 rpm, and the dissolved oxygen value is controlled at 20-30% through stirring and ventilation. The pH is controlled at 6.75-6.85 by adding ammonia. Fermentation continues until OD... 600 When the OD reached 0.6-0.8, IPTG was added to a final concentration of 24 mg / L, and induction culture was carried out at 30℃ and 300 rpm. During fermentation, samples were taken every 4 hours, and OD was detected using the method in Example 1. 600 The content of residual sugar and L-methionine; after 12 hours of fermentation, the initial sugar is basically consumed, and the initial glucose concentration of the fermentation broth is below 2 g / L, so fed medium is started; that is, from 12 hours, dissolved oxygen feedback is used to maintain the dissolved oxygen value at 30% by feeding medium at a rate of 40 mL / h; when OD 600 When the dissolved oxygen level reaches 10, the culture medium is fed at a constant flow rate of 15 mL / h to maintain the dissolved oxygen level at 30%; when the OD... 600 When the dissolved oxygen level reaches 30, the culture medium is fed at a constant flow rate of 20 mL / h to maintain the dissolved oxygen level at 30%; when the OD... 600 When the DO value reaches 45%, fed culture medium is added at a constant flow rate of 25 mL / h. During the later stages of fermentation, the cells gradually die, and the DO value increases. Feeding is stopped when the DO value rises to 50% or higher; fermentation ends when the DO value reaches 100%.

[0114] See results Figure 11 As shown in the figure. The fermentation results show that by adopting the above-mentioned variable-rate feeding strategy, the fermentation process can be completed at 68 hours, which is 4 hours shorter than the initial conditions; at this time, the yield of L-methionine is the highest, at 31.71 g / L, which is 74.23% higher than the initial conditions; the highest sugar-acid conversion rate is 12.2%, which is 74.29% higher than the initial conditions.

[0115] As can be seen from the above three examples of novel feeding methods, changing the feeding rate during fermentation according to cell growth can significantly increase the fermentation yield of L-methionine, and controlling an appropriate feeding rate can shorten the fermentation time, thus meeting the needs of industrial production.

Claims

1. A method for high-yield L-methionine production via total fermentation, characterized in that, The method includes the following steps: using L-methionine-producing recombinant *Escherichia coli* as the production strain, inoculating it into a fermentation medium containing 50 mg / L kanamycin resistance, culturing at 37°C, and stirring at 300-1000 r / min during fermentation; controlling the dissolved oxygen value at 20-30% through stirring and aeration, and controlling the pH at 6.75-6.85 by adding ammonia water; fermenting to OD... 600 When the OD value reaches 0.6-0.8, add IPTG to a final concentration of 24 mg / L and induce fermentation at 30℃ and 300-1000 r / min. During fermentation, samples are taken every 4 hours to detect OD. 600 Residual sugar content and L-methionine content; after fermentation until the residual sugar concentration in the fermentation broth is below 2 g / L, a fed-batch culture medium with dissolved oxygen feedback is used to maintain the dissolved oxygen value at 20-30%; when OD 600 When the dissolved oxygen level reaches 10, feed culture medium is added at a constant flow rate of 10-20 mL / h to maintain the dissolved oxygen level at 20-30%; when the OD... 600 When the dissolved oxygen level reaches 30%, feed culture medium is added at a constant flow rate of 20-30 mL / h to maintain the dissolved oxygen level at 20-30%; when the OD... 600 When the dissolved oxygen level reaches 45%, feed medium is added at a constant flow rate of 25-40 mL / h to maintain the dissolved oxygen level at 20-30%. During the later stages of fermentation, the cells gradually die and the dissolved oxygen level rises. Feeding is stopped when the dissolved oxygen level rises to 50% or above. Fermentation ends when the dissolved oxygen level reaches 100%. The fermentation medium consisted of: glucose 10 g / L, CaCl₂·2H₂O 0.08 g / L, MgSO₄·7H₂O 5 g / L, citric acid 2.5 g / L, KH₂PO₄ 2.5 g / L, K₂HPO₄·3H₂O 1.38 g / L, FeCl₃·6H₂O 0.12 g / L, (NH₄)₂SO₄ 3.3 g / L, Na₂S₂O₃ 3.95 g / L, and VB 0.01 g / L. 12 0.01 g / L of VB1, 0.05 g / L of L-lysine, 2 mL / L of SSA, and 1 mL / L of SSB; Feeding medium: glucose 500 g / L, MgSO4·7H2O 5 g / L, FeCl3·6H2O 0.06 g / L, (NH4)2SO4 33 g / L, Na2S2O3 39.5 g / L, betaine 0.5 g / L, SSA 1.6 mL / L, SSB 0.8 mL / L, VB 0.01 g / L 12 0.01 g / L of VB1 and 0.05 g / L of L-lysine, in water; The SSA composition is: ZnSO4·7H2O 8.5 g / L, MnCl2·2H2O 7.5 g / L, CuCl2·2H2O 0.75 g / L, CoCl2·6H2O 1.25 g / L, EDTA 4 g / L, with water as the solvent; The SSB composition is: H3BO3 3 g / L, Na2MoO4·2H2O 2.5 g / L, and the solvent is water.

2. The method for high-yield L-methionine production via total fermentation as described in claim 1, characterized in that, The production strain is Escherichia coli (Escherichia coli) Escherichia coli CCTCC NO: M 2020846.

3. The method for high-yield L-methionine production via total fermentation as described in claim 1, characterized in that, When OD 600 When the dissolved oxygen level reaches 10, feed culture medium is added at a constant flow rate of 15 mL / h to maintain the dissolved oxygen level at 20-30%; when the OD... 600 When the dissolved oxygen level reaches 30%, feed culture medium is added at a constant flow rate of 20 mL / h to maintain the dissolved oxygen level at 20-30%; when the OD... 600 When the dissolved oxygen level reaches 45%, the culture medium is fed at a constant flow rate of 25 mL / h to maintain the dissolved oxygen level at 20-30%. In the later stage of fermentation, the cells gradually die and the dissolved oxygen level rises. When the dissolved oxygen level rises to 50% or above, the feeding is stopped. Fermentation ends when the dissolved oxygen level reaches 100%.

4. The method for high-yield L-methionine production via total fermentation as described in claim 1, characterized in that, The production strain was activated by slant culture and seed culture was performed before inoculation. The seed culture was inoculated into the fermentation medium at a volume concentration of 10-15%. The seed culture was prepared according to the following steps: (1) Activation culture: The production strain was inoculated onto LB agar medium and cultured overnight at 37°C to obtain activated bacteria; the composition of the LB agar medium was: 10 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl, 20 g / L agar powder, water as solvent, pH=6.8-7.0; (2) Primary seed culture: The activated bacteria obtained in step (1) were inoculated into LB liquid medium and cultured at 37℃ and 200 rpm for 12h to obtain primary seed culture; LB liquid medium composition: 10 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl, solvent is water, pH= 6.8-7.0; (3) Secondary seed culture: The primary seed culture obtained in step (2) is inoculated into LB liquid medium at a volume concentration of 1-3% and cultured at 37℃ and 200 rpm for 12-16h to obtain the secondary seed culture; the composition of the LB liquid medium is the same as in step (2).

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