A fermentation method of an escherichia coli engineering strain for producing o-acetylhomoserine

Abstract

The application discloses a fermentation method of an engineered Escherichia coli strain for producing O-acetylhomoserine and relates to the technical field of biotechnology.The fermentation method comprises the following steps: fermenting and culturing the Escherichia coli YA-11 by using a fermentation medium; and the fermentation medium comprises the following components: 10-20 g / L of glucose, 0.5-2.5 g / L of magnesium sulfate heptahydrate, 5-10 g / L of sugar beet molasses, 5-10 g / L of ammonium sulfate, 0.5-4 g / L of yeast extract, 2-14 g / L of corn syrup dry powder, 1-3 g / L of potassium dihydrogen phosphate, 0.5-1 g / L of threonine and 1-3 mL / L of trace element solution; and the trace element solution contains 8.35 g / L of manganese sulfate and 8.35 g / L of ferrous sulfate heptahydrate.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a fermentation method of an engineered Escherichia coli strain for producing O-acetylhomoserine. BACKGROUND

[0002] O-acetyl-L-homoserine (OAH) is an acylated amino acid, which does not directly participate in the synthesis of proteins, and is an important precursor for the synthesis of sulfur-containing compounds such as methionine and S-adenosylmethionine in cells. OAH can generate methionine and acetic acid by reacting with methyl mercaptan or sodium methyl mercaptan under the action of acetylhomoserine thiolase, which is an important process route for the synthesis of methionine by coupling fermentation-enzyme method. At present, the level of OAH synthesized by fermentation method in China is limited and has not reached the level of industrial production.

[0003] Escherichia coli is an important industrial strain, which has been used for the fermentation production of L-aspartate family amino acids such as threonine and isoleucine. The growth and production conditions of the engineered bacteria with different metabolic modifications will change compared with the previous ones, so optimizing the fermentation conditions suitable for the specific strain is an important basis for the research and development of the strain. SUMMARY

[0004] The purpose of the present application is to provide a fermentation method of an engineered Escherichia coli strain for producing O-acetylhomoserine, so as to solve the problems existing in the prior art. The present application develops a fermentation medium suitable for the application of OAH engineered bacteria, Escherichia coli YA-11, in the fermentation production of OAH. The fermentation yield of OAH can reach 13.9 g / L by using the fermentation medium to carry out fermentation culture of Escherichia coli YA-11, and the highest yield in a fermenter can reach 111 g / L.

[0005] To achieve the above purpose, the present application provides the following solutions:

[0006] The present application provides a fermentation medium of Escherichia coli YA-11 for producing O-acetylhomoserine, which comprises the following components: glucose 10-20 g / L, magnesium sulfate heptahydrate 0.5-2.5 g / L, sugar beet molasses 5-10 g / L, ammonium sulfate 5-10 g / L, yeast extract 0.5-4 g / L, corn syrup dry powder 2-14 g / L, potassium dihydrogen phosphate 1-3 g / L, threonine 0.5-1 g / L, and trace element solution 1-3 mL / L.

[0007] The trace element solution contains manganese sulfate 8.35 g / L and ferrous sulfate heptahydrate 8.35 g / L.

[0008] The Escherichia coli YA-11 is preserved in the China General Microbiological Culture Collection Center on September 3, 2024, the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 31852.

[0009] Preferably, the fermentation medium comprises the following components: glucose 20 g / L, magnesium sulfate heptahydrate 2 g / L, sugar beet molasses 6.719 g / L, ammonium sulfate 8.072 g / L, yeast extract 1 g / L, corn syrup dry powder 6.711 g / L, potassium dihydrogen phosphate 2 g / L, threonine 0.75 g / L, and the trace element solution 2 mL / L.

[0010] The application also provides the use of the above-mentioned fermentation medium in the fermentation production of O-acetylhomoserine by using the Escherichia coli YA-11.

[0011] The application also provides a fermentation method of the Escherichia coli engineering strain for producing O-acetylhomoserine, comprising the step of fermenting and culturing the Escherichia coli YA-11 by using the above-mentioned fermentation medium.

[0012] The Escherichia coli YA-11 is preserved in the China General Microbiological Culture Collection Center on September 3, 2024, the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 31852.

[0013] Further, in the process of the fermentation culture, the initial aeration amount is 2 L / min, the stirring rate is 300 rpm, the temperature is 37 DEG C, and the pH is controlled to be 6.5+ / -0.1.

[0014] Further, the pH is controlled to be 6.5+ / -0.1 by adding 25% ammonia water.

[0015] Further, in the process of the fermentation culture, the dissolved oxygen is controlled to be more than 25% by adjusting the stirring, tank pressure and aeration.

[0016] Further, in the process of the fermentation culture, after the bottom sugar concentration is reduced to below 1 g / L, a glucose solution with a mass fraction of 50% is added, the initial addition rate is 4 g / L / h, and the glucose in the fermentation broth is used as an index to increase the sugar supplement amount by 1 g / L / h each time.

[0017] Further, in the process of the fermentation culture, the OUR is maintained at 100-160 mmol / L / h by adding a mixed solution of methionine, lysine, citric acid and sugar beet molasses.

[0018] Further, in the mixed solution, the adding amount of the methionine, the lysine, the citric acid and the sugar beet molasses is 1 g / L, 1 g / L, 3 g / L and 10 g / L respectively.

[0019] The present application discloses the following technical effects:

[0020] The present application screens the adding amount of the optimal nitrogen source, phosphorus source and other components through the previous single factor experiment, screens the ratio of yeast extract and corn syrup dry powder, ammonium sulfate and sugar beet molasses three important influencing factors by using the Plackett-buman method, obtains the optimal adding amount of the three factors through the Box-Behnken method regression analysis, and thus obtains the fermentation medium suitable for the application of the OAH engineering bacterium, i.e. Escherichia coli YA-11 in the fermentation production of OAH. The fermentation yield of OAH can reach 13.9 g / L by using the optimized fermentation medium of the present application for the fermentation culture of Escherichia coli YA-11, which is about 1.62 times higher than that before optimization.

[0021] The present application further optimizes the process on the fermenter, in the fermentation in the 5L fermenter, designs and utilizes the real-time monitoring tail gas mass spectrum to collect data, optimizes the feeding and oxygen consumption rate control relationship, and proposes the optimal OUR level control feeding flow rate fermentation process. By optimizing the feeding through the control of the oxygen consumption rate, the oxygen consumption rate in the fermentation process can be improved to 160 mmol / L / h compared with the original fermentation culture, and the OAH yield can be improved by 1.9 times, reaching 111 g / L. The present application provides important technical support for the industrial production of OAH by Escherichia coli. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0023] Figure 1 The 3D model diagram of the influence of the sugar beet molasses concentration (A) and the ratio of yeast extract and corn syrup dry powder (B) on the product of Escherichia coli;

[0024] Figure 2 The 3D model diagram of the influence of the sugar beet molasses concentration (A) and the ammonium sulfate concentration (C) on the product of Escherichia coli;

[0025] Figure 3 The 3D model diagram of the influence of the ratio of yeast extract and corn syrup dry powder (B) and the ammonium sulfate concentration (C) on the product of Escherichia coli;

[0026] Figure 4 Statistical plots of physiological metabolism parameters during the fermentation process of Example 2;

[0027] Figure 5 OD variation curve plot under different feeding modes; 600

[0028] Figure 6 OUR variation curve plot under different feeding modes;

[0029] Figure 7 OAH yield variation curve plot under different feeding modes. DETAILED DESCRIPTION

[0030] Various example embodiments of the present application will now be described in detail with reference to the figures. Such description, however, is to be considered in

[0031] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, where particular ranges of values are given, understand that each intervening value, to the upper or lower limit of the ranges is also specifically included. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is also specifically encompassed. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and are also encompassed within the application, subject to any specifically excluded limit in the stated

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in detail the methods and / or materials which are related to the present application. In the case of conflict between the present specification and any document incorporated herein by reference, the present specification will control.

[0033] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0034] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed material or step.

[0035] Example 1

[0036] 1. Experimental materials​

[0037] 1.1 Production strain

[0038] Escherichia coli YA-11, deposited on September 3, 2024 at the China General Microbiological Culture Collection Center, located at No. 1, Yihuan West Road, Beijing, China, and assigned accession number CGMCC No. 31852.

[0039] 1.2 Preparation of culture medium

[0040] Preparation of LB solid medium: sodium chloride 10 g / L, yeast extract 5 g / L, peptone 10 g / L, agar 15 g / L, sterilized at 121°C for 30 min, and then cooled to about 55°C before pouring into plates.

[0041] Preparation of LB liquid medium: sodium chloride 10 g / L, yeast extract 5 g / L, peptone 10 g / L, aliquoted into 500 mL shake flasks, 100 mL per flask, sterilized at 121°C for 20 min, and used after the temperature is reduced to room temperature.

[0042] 2. Methods

[0043] 2.1 Biomass OD 600

[0044] The fermentation broth was diluted 100-fold, and the OD was measured at 600 nm on a spectrophotometer. 600 .

[0045] 2.2 Product OAH determination

[0046] Analysis conditions: column temperature 40°C, flow rate 1 mL / min, detection wavelength 308 nm, injection volume 20 μL.

[0047] Derivation method: centrifuge the sample at 12000 rpm for 3 min, take the supernatant, pass through a 0.22 μm water filter membrane, dilute 100-fold, and then analyze using OPA automatic pre-column derivation.

[0048] 2.3 Determination of glucose concentration in fermentation broth

[0049] Determined using an SBA-40E biological sensor analyzer. After centrifugation, the supernatant of the fermentation broth was diluted appropriately, and 25 μL of the diluted solution was taken with a micro-syringe for determination.

[0050] 2.4 Seed plate activation

[0051] A disposable inoculation loop was used to take the bacteria from the glycerol tube and streaked on LB solid medium plates, which were then incubated at 37°C in an inverted incubator for 20 h.

[0052] 2.5 Seed shake flask culture

[0053] Scratch fresh 3-5 single colonies into 500 mL shake flask with 100 mL LB medium, incubate in 37℃, 220 rpm for 12-16 h.

[0054] 2.6 Shake flask fermentation

[0055] 500 mL shake flask with 50 mL medium, pH adjusted to 7.0±0.2 with 2 mol / L HCl solution, 0.5 g CaCO3 was added before sterilization. Inoculation amount was 5%, incubated in 37℃, 220 rpm for 24 h.

[0056] 2.7 5 L fermenter cultivation

[0057] Initial aeration rate was 2 L / min, stirring rate was 300 rpm, temperature was 37℃, 25% ammonia was added to control pH at 6.5±0.1 during the process, stirring rate, tank pressure and aeration rate were adjusted to control dissolved oxygen above 25%. When the concentration of the substrate was below 1 g / L, 50% glucose solution was added at an initial rate of 4 g / L / h, and the rate was increased by 1 g / L / h each time until the glucose concentration in the fermentation broth was below 1 g / L. The tail gas was detected by mass spectrometry to calculate physiological parameters such as OUR, CER and RQ.

[0058] 2.8 Medium optimization

[0059] The initial fermentation medium formula range was: glucose 10-20 g / L, magnesium sulfate heptahydrate 0.5-2.5 g / L, sugar beet molasses 5-10 g / L, ammonium sulfate 5-10 g / L, yeast extract 0.5-4 g / L, corn syrup dry powder 2-14 g / L, potassium dihydrogen phosphate 1-3 g / L, threonine 0.5-1 g / L and trace element solution 1-3 mL / L; the trace element solution contained manganese sulfate 8.35 g / L and ferrous sulfate heptahydrate 8.35 g / L.

[0060] Based on the initial fermentation medium formula range, the fermentation medium was optimized.

[0061] (1) Significant influencing factor screening

[0062] Plackeet-burman experiment was used to determine the important influencing components in the medium during OAH synthesis, the matrix design of the experiment was designed by Design Expert software, and the factors and levels are shown in Table 1. The influence of each component of the medium on the response value was analyzed by analysis software, and the statistical results are shown in Tables 2 and 3. The P value of the ratio of yeast extract and corn syrup dry powder and the P value of sugar beet molasses were both less than 0.05, the P value of ammonium sulfate was 0.0538, which was close to the required range, and was a significant influencing factor for the synthesis of OAH by the engineering bacteria.

[0063] Table 1 Factors and levels of Plackett-Burman experimental design

[0064]

[0065] Table 2 Fermentation results of shake flask of Plackett-Burman experimental design

[0066]

[0067]

[0068] Table 3 Analysis of variance of Plackett-Burman experimental design

[0069]

[0070] (2) Box-Behnken method to determine the optimal value of important factors

[0071] Box-Behnken experiment was used to investigate the three factors that significantly affect the production of OAH: beet molasses, ammonium sulfate, and the ratio of yeast extract and corn syrup dry powder addition. With OAH production as the index, the effects of these three factors on the yield were investigated. Each group was set up in triplicate. The experimental design and results of each factor and level are shown in Table 4, and the variance analysis is shown in Table 5.

[0072] Table 4 Box-Behnken design and results

[0073] Group number A: sugar beet molasses B: yeast extract - corn steep liquor dry powder ratio C: ammonium sulfate Yield (g / L) 1 10 0.25 7.5 13.55 2 7.5 0.1665 7.5 13.68 3 7.5 0.1665 7.5 13.69 4 5 0.1665 10 13.59 5 10 0.1665 10 13.5 6 7.5 0.25 5 13.38 7 7.5 0.083 5 13.44 8 7.5 0.083 10 13.57 9 10 0.083 7.5 13.5 10 10 0.1665 5 13.33 11 5 0.1665 5 13.42 12 7.5 0.1665 7.5 13.68 13 7.5 0.1665 7.5 13.7 14 7.5 0.1665 7.5 13.71 15 7.5 0.25 10 13.51 16 5 0.083 7.5 13.64 17 5 0.25 7.5 13.57

[0074] Table 5 Analysis of variance of the experiment

[0075]

[0076]

[0077] The results were fitted by multiple quadratic regression using Design Expert software, and the regression equation was obtained: OAH yield = 13.692 - 0.0425*A - 0.0175*B + 0.075C + 0.03*AB - 0.071*A 2 -0.056*B 2 -0.161*C 2 .

[0078] Where the predicted OAH yield, A is the concentration of beet molasses, B is the ratio of yeast extract and corn syrup dry powder, and C is the concentration of ammonium sulfate. The coefficients in front of A, B, and C are linear correlation coefficients, A 2 , B2 , C 2 The former coefficient is a square coefficient, and the AB former coefficient is an interaction coefficient. An analysis graph is generated according to the regression equation, and the response surface shape is investigated. The response contour maps of the factors are shown in Figures 1-3 The contour centers of the three response surfaces are all within the set range, indicating that the optimal conditions exist within the factor levels designed in the application.

[0079] Optimization analysis of the three factors shows that the optimal concentration of beet molasses is 6.719 g / L, the optimal concentration of ammonium sulfate is 8.072 g / L, and the optimal ratio of yeast extract and corn syrup dry powder is 0.149.

[0080] The optimal fermentation medium formula is as follows: glucose 20 g / L, magnesium sulfate heptahydrate 2 g / L, beet molasses 6.719 g / L, ammonium sulfate 8.072 g / L, yeast extract 1 g / L, corn syrup dry powder 6.711 g / L, potassium dihydrogen phosphate 2 g / L, threonine 0.75 g / L, and trace element solution 2 mL / L; the trace element solution contains manganese sulfate 8.35 g / L and ferrous sulfate heptahydrate 8.35 g / L.

[0081] After shake flask verification, the OAH yield is 13.74 g / L under the use of the predicted optimal medium, proving that the model is reasonable and providing a good basic medium for further research.

[0082] Example 2

[0083] According to the method of Example 1, the E. coli YA-11 seed is prepared, and a 5% inoculation amount is inoculated into a fermenter containing 1.8 L of fermentation medium (the optimal fermentation medium obtained in Example 1). The fermentation verification of E. coli on the optimized medium is carried out, and the fermentation culture conditions are the same as those in Example 1, Part 2.7. The physiological metabolism parameters in the fermentation process are shown in Figure 4 During the fermentation process, the glucose concentration in the fermentation broth is controlled not to exceed 1 g / L, and the rotation speed, aeration, and pressure are adjusted to control the dissolved oxygen to be about 25%.

[0084] The results show that the cell growth reaches the maximum OD 600 (about 60) at 16 h, and then maintains this level, and the oxygen consumption rate OUR of the fermentation reaches 80 mmol / L / h. Increasing the glucose feeding rate cannot increase the OUR, and the OUR decreases after 28 h. The product content does not increase after 32 h, and the yield reaches 58.41 g / L after 36 h of fermentation.

[0085] Example 3

[0086] In order to improve the oxygen uptake rate of the bacteria and maintain at a higher level, the present embodiment carries out experiments of different feeding modes, i.e. respectively adding mixed solution 1, mixed solution 2 and mixed solution 3. The fermentation medium is the same as that in embodiment 2. Except for the feeding control, other fermentation culture conditions are the same as those in part 2.7 of embodiment 1. The fermentation experiment without the above-mentioned feeding is used as a control.

[0087] Mixed solution 1: 1 g / L lysine and 1 g / L methionine;

[0088] Mixed solution 2: 1 g / L lysine, 1 g / L methionine and 10 g / L sugar beet molasses.

[0089] Mixed solution 3: 1 g / L lysine, 1 g / L methionine, 3 g / L citric acid and 10 g / L sugar beet molasses.

[0090] The experimental results are shown in Table 1. Figures 5-7 The experimental groups with additional lysine and methionine can improve the oxygen consumption rate to a higher level and maintain well, which promotes the growth of the bacteria and the synthesis of OAH. On this basis, the experimental groups with further additional sugar beet molasses have significantly improved OAH yield and conversion rate. In the case of simultaneously adding citric acid, lysine and methionine, and sugar beet molasses, the OUR can reach a higher level and maintain at 160 mmol / L / h well, and the OD 600 The OUR is improved from 60 to 90, and the highest OAH yield reaches 111 g / L after 36 h of fermentation.

[0091] The above-described embodiments only describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A fermentation method for an engineered strain of *Escherichia coli* that produces O-acetylhomoserine, characterized in that, This includes the step of fermenting Escherichia coli YA-11 using a fermentation medium; The OUR was maintained at 100-160 mmol / L / h by feeding a mixture of methionine, lysine, citric acid and beet molasses. The Escherichia coli YA-11 was deposited on September 3, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 31852. The fermentation medium comprises the following components: glucose 10-20 g / L, magnesium sulfate heptahydrate 0.5-2.5 g / L, beet molasses 5-10 g / L, ammonium sulfate 5-10 g / L, yeast extract 0.5-4 g / L, corn steep liquor powder 2-14 g / L, potassium dihydrogen phosphate 1-3 g / L, threonine 0.5-1 g / L, and trace element solution 1-3 mL / L; The trace element solution contains 8.35 g / L manganese sulfate and 8.35 g / L ferrous sulfate heptahydrate; During the fermentation process, after the base sugar concentration was reduced to below 1 g / L, a 50% glucose solution was added. The initial flow rate was 4 g / L / h. With the glucose concentration in the fermentation broth below 1 g / L as an indicator, the amount of glucose added was increased by 1 g / L / h each time.

2. The fermentation method according to claim 1, characterized in that, The fermentation medium comprises the following components: 20 g / L glucose, 2 g / L magnesium sulfate heptahydrate, 6.719 g / L beet molasses, 8.072 g / L ammonium sulfate, 1 g / L yeast extract, 6.711 g / L corn steep liquor powder, 2 g / L potassium dihydrogen phosphate, 0.75 g / L threonine, and 2 mL / L of the trace element solution.

3. The fermentation method according to claim 1, characterized in that, During the fermentation process, the initial aeration rate was 2 L / min, the stirring rate was 300 rpm, the temperature was 37℃, and the pH was controlled at 6.5±0.

1.

4. The fermentation method according to claim 3, characterized in that, The pH was controlled to be 6.5 ± 0.1 by adding 25% ammonia water.

5. The fermentation method according to claim 3, characterized in that, During the fermentation process, dissolved oxygen is controlled to be above 25% by adjusting stirring, tank pressure, and aeration.

6. The fermentation method according to claim 1, characterized in that, In the mixture, the amounts of methionine, lysine, citric acid, and beet molasses added are 1 g / L, 1 g / L, 3 g / L, and 10 g / L, respectively.

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