Method for producing amino acids and nucleosides by microbial enhanced fermentation

Abstract

The present application provides a method for producing amino acid and nucleoside by microbial reinforced fermentation, which is achieved by analyzing lysine fermentation process, screening excellent fermentation strains, and adding the preferred E. coli (or adding the preferred C. glutamicum) in a timely and quantitative manner in the fermentation process based on C. glutamicum (or in the fermentation process based on E. coli), so as to obviously improve the yield and conversion rate of lysine. The present application can effectively solve the problems of unstable product quality and difficult effective control of production process in traditional lysine fermentation, and has important significance for improving fermentation efficiency and conversion rate.

Description

Technical Field

[0001] This invention relates to microbial fermentation technology, and more specifically, to a method for producing amino acids and nucleosides using microbial enhanced fermentation. Background Technology

[0002] Lysine is one of the eight essential amino acids for the human body and is the world's second largest amino acid industry after glutamic acid. Currently, about 90% of lysine products are used as feed additives, and about 10% are used in the food and medical industries. With social development, the demand for lysine products continues to increase. Researchers have continuously improved the conversion rate and yield of lysine production by optimizing fermentation strains and fermentation conditions, such as optimizing the sugar intake system of the strains, blocking redundant systems in microbial metabolism, and optimizing fermentation substrate conditions.

[0003] Currently, Corynebacterium glutamicum and Escherichia coli are the most common fermentation strains in lysine fermentation. During the middle and later stages of lysine fermentation, nutrients are continuously consumed in the culture medium, and the product concentration continuously increases, leading to a slowdown in microbial growth and reproduction, a decrease in cell activity, and a decline in lysine production rate. Summary of the Invention

[0004] The purpose of this invention is to provide a method for producing amino acids and nucleosides using microbial enhanced fermentation, particularly a method for producing lysine (L-lysine) using microbial enhanced fermentation.

[0005] To achieve the objective of this invention, a method for producing amino acids and nucleosides using microbial enhanced fermentation is provided, comprising the following steps: S1. Analyze the amino acid and nucleoside fermentation processes of microorganisms capable of producing amino acids and nucleosides, and screen out strains M1, M2...M1 with good fermentation performance and osmotic pressure tolerance. n n is an integer; and strains M1, M2...M n They have different taxonomic characteristics; S2. During the fermentation process of strain M1 (preferably in the middle and late stages of fermentation), inoculate strains M2...M n One or more of these can be used to increase the fermentation yield and conversion rate of amino acids and nucleosides.

[0006] The amino acids described in this invention include, but are not limited to, lysine, glutamic acid, and threonine; the nucleosides include, but are not limited to, inosine, guanosine, and adenosine.

[0007] When the amino acid is lysine, the method includes the following steps: A. Analyze the lysine fermentation process of Corynebacterium glutamicum and Escherichia coli respectively, and screen out Corynebacterium glutamicum strain M1 and Escherichia coli strain M2 with good fermentation performance and osmotic pressure tolerance. B. Inoculate strain M2 after strain M1 has fermented for 4-36 hours; or inoculate strain M1 after strain M2 has fermented for 4-36 hours, in order to improve the fermentation yield and conversion rate of lysine.

[0008] In step A, molasses, corn steep liquor, corn steep liquor hydrolysate, glucose, and water are used as the main components. The above substances are mixed in a mass ratio of 1-2:1-2:1-2:0.2-0.5:4-6. The temperature is set at 35℃ and the pH is set at 6.7. The osmotic pressure of the mixture is adjusted to 200-2000 mOsm with sodium chloride. The lysine-producing strains are then screened.

[0009] Preferably, strain M1 is *Corynebacterium glutamicum* Cgl-8 with accession number CGMCC NO. 11942, and strain M2 is *Escherichia coli* with accession number CGMCC NO. 22648. Escherichia coli MHZ-0914 (i.e. E.coli-10).

[0010] Corynebacterium glutamicum ( Corynebacterium glutamicum Cgl-8 is currently deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, with accession number CGMCC NO.11942 and deposit date of December 25, 2015. Corynebacterium glutamicum with accession number CGMCC NO.11942 can be found in ZL 201610119394.1.

[0011] Escherichia coli ( Escherichia coli MHZ-0914 is currently deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, accession number CGMCC NO.22648, deposited on June 1, 2021.

[0012] The aforementioned method involves inoculating strain M2, which has been cultured to the logarithmic phase, with the inoculation amount being 15% of the total mass of the fermentation system of strain M1 after 16 hours of fermentation by strain M1.

[0013] The aforementioned method involves inoculating strain M1, which has been cultured to the logarithmic phase, with the inoculation amount being 20% ​​of the total mass of the fermentation system of strain M2 after 12 hours of fermentation.

[0014] The aforementioned method, step B includes: activating the lysine-producing strain stored at -80℃ by inoculating it onto an agar slant culture medium; inoculating the bacterial cells from the agar slant culture medium into a seed shake flask for primary seed culture; transferring the primary seed culture into a 10 L secondary fermenter for secondary seed culture; and transferring the secondary seed culture into a fermentation medium for fermentation culture in a 50 L fermenter.

[0015] Preferably, the culture medium used for primary seed culture is: 30 g / L sucrose, 1.2 g / L potassium dihydrogen phosphate, 1.2 g / L magnesium sulfate, 10 g / L sodium glutamate, 15 g / L yeast extract, 20 g / L ammonium sulfate, 1 g / L threonine, 0.005 g / L ferrous sulfate and 0.005 g / L manganese sulfate.

[0016] Preferably, the culture medium used for secondary seed culture is: corn steep liquor 30 g / L, corn steep liquor hydrolysate 20 g / L, ammonium sulfate 8 g / L, magnesium sulfate 2 g / L, threonine 0.5 g / L, ferrous sulfate 0.015 g / L, and manganese sulfate 0.015 g / L.

[0017] Preferably, the fermentation medium consists of: molasses 150 g / L, corn steep liquor 150 g / L, corn steep liquor hydrolysate 150 g / L, ammonium sulfate 2 g / L, and glucose 50 g / L.

[0018] Step B includes the following sub-steps: B1. The primary seed culture temperature is 35℃, 300 rpm, and cultured until OD... 562 =10, then transfer 200 mL of the primary seed culture to the secondary fermenter for secondary seed culture; B2. Secondary seed culture temperature is 35℃, dissolved oxygen is coupled with rotation speed to ensure dissolved oxygen is not lower than 30%, pH is adjusted with ammonia to maintain 6.7, and culture is carried out until OD reaches... 562 After 40 minutes, 3 kg of secondary seed culture was transferred to the fermenter for fermentation culture. B3. The fermentation temperature is 35℃. The pH is adjusted to 6.7 with ammonia water. During the fermentation process, ammonium sulfate solution is added to maintain the ammonia nitrogen level in the culture medium at 1-1.5 g / L. During the fermentation process, glucose solution is added to maintain the glucose concentration in the culture medium at 0.2-2 g / L.

[0019] Preferably, the concentration of the added ammonium sulfate solution is 30-50% (more preferably 37%), and the concentration of the glucose solution is 40-80% (more preferably 60.5%).

[0020] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: (i) Based on the lysine fermentation process, this invention rationally designs microbial screening conditions and uses a specific culture medium based on molasses, corn steep liquor and corn steep liquor hydrolysate to restore the fermentation environment of the functional microorganisms. This overcomes the shortcomings of traditional constituent or semi-constituent culture media that are not suitable for the growth of the selected functional microorganisms, and screens functional strains from Corynebacterium glutamicum and Escherichia coli.

[0021] (ii) During the screening of superior functional microorganisms, one strain of Corynebacterium glutamicum and one strain of Escherichia coli were selected. These two strains are able to withstand high osmotic pressure and produce high levels of lysine.

[0022] (III) By strengthening *Corynebacterium glutamicum* during fermentation, lysine yield increased by 39.98% and conversion rate increased by 8.31%. By strengthening *Corynebacterium glutamicum* during *E. coli* fermentation, lysine yield increased by 46.49% and conversion rate increased by 8.70%. This invention effectively solves the problems faced by traditional lysine fermentation, such as unstable product quality and difficulty in effectively controlling the production process, and is of great significance for improving fermentation efficiency and conversion rate. Attached Figure Description

[0023] Figure 1 The following is a preferred embodiment of the present invention showing the changes in fermentation conditions during the fermentation of Corynebacterium glutamicum and Escherichia coli; wherein, A: Corynebacterium glutamicum; B: Escherichia coli.

[0024] Figure 2 This invention relates to the screening of lysine-producing strains of Corynebacterium glutamicum and Escherichia coli for fermentation in a preferred embodiment of the present invention; wherein, A: Corynebacterium glutamicum; B: Escherichia coli.

[0025] Figure 3 In a preferred embodiment of the present invention, the amount and duration of Escherichia coli enhancement during the fermentation of Corynebacterium glutamicum are specified; wherein, A: amount of Escherichia coli enhancement; B: duration of Escherichia coli enhancement.

[0026] Figure 4 In a preferred embodiment of the present invention, the amount and duration of Corynebacterium glutamicum enhancement during Escherichia coli fermentation are specified; wherein, A: Corynebacterium glutamicum enhancement amount; B: Corynebacterium glutamicum enhancement time.

[0027] Figure 5 This is a preferred embodiment of the enhanced fermentation process during glutamic acid fermentation; wherein, A: the amount of E. coli-10 used to enhance the fermentation of Cgl-8; B: the enhancement time of E. coli-10 during the fermentation of Cgl-8; C: the amount of Cgl-8 used to enhance the fermentation of E. coli-10; D: the enhancement time of Cgl-8 during the fermentation of E. coli-10.

[0028] Figure 6This is a preferred embodiment of the threonine fermentation process of the present invention; wherein, A: the amount of E. coli-10 used to enhance the fermentation of Cgl-8; B: the enhancement time of E. coli-10 during the fermentation of Cgl-8; C: the amount of Cgl-8 used to enhance the fermentation of E. coli-10; D: the enhancement time of Cgl-8 during the fermentation of E. coli-10. Detailed Implementation

[0029] This invention provides a method for enhancing lysine fermentation using microorganisms. This method analyzes the lysine fermentation process, screens superior fermentation strains, and, during the fermentation process based on Corynebacterium glutamicum, introduces selected Escherichia coli at regular intervals and in specific quantities (or, during the fermentation process based on Escherichia coli, introduces selected Corynebacterium glutamicum at regular intervals and in specific quantities), which can significantly improve the yield and conversion rate of lysine.

[0030] The technical solution of this invention is as follows: (1) Material analysis of lysine fermentation process The changes in various substances and states during lysine fermentation were analyzed using instrumental methods such as ultraviolet-visible spectrophotometer, high performance liquid chromatography (HPLC), and freezing point osmotic pressure meter.

[0031] (2) Screening of superior functional microorganisms Based on the lysine fermentation process, reasonable microbial screening conditions were designed, and functional strains of microorganisms were screened from Corynebacterium glutamicum and Escherichia coli preserved in the laboratory, using specific screening media as a basis.

[0032] Osmotic screening conditions: Adjust the osmotic pressure of the culture medium to 200-2000 mOsm with NaCl, set the temperature to 35℃, set the pH to 6.7, and culture on a shaker at 300 rpm.

[0033] (3) Lysine-enhanced fermentation process Based on the changes in lysine fermentation cycle and material content, the selected superior strains are added to the lysine fermentation process in a timely and quantitative manner to improve the theoretical acid, raw material utilization rate and conversion rate of lysine fermentation.

[0034] The specific technical approach for implementation is as follows: 1. Material analysis of lysine fermentation process Microbial concentration determination: Take 1 mL of the fermentation broth during fermentation and add it to a 25 mL volumetric flask, then dilute to volume with deionized water. The microbial concentration is determined using an ultraviolet spectrophotometer (OD).562 The lysine concentration was measured at a wavelength of 562 nm. The lysine concentration was determined according to GB 8245-1987. For osmotic pressure measurement, 2 mL of fermentation broth was centrifuged at 1200 rpm for 2 min, and the supernatant was collected. The osmotic pressure was measured using a freezing point osmoremeter to determine the lysine concentration.

[0035] 2. Screening of superior functional microorganisms A screening culture medium for superior functional microorganisms was prepared by mixing molasses, corn steep liquor, corn steep liquor hydrolysate, glucose, and water as the main components after sterilization in a mass ratio of 1:1:1:0.2:5. Fermentation and tolerance (osmotic pressure) tests were conducted on laboratory-preserved strains, and microorganisms with better fermentation and tolerance performance were selected as dominant microorganisms.

[0036] 3. Lysine-enhanced fermentation process ① The L-lysine-producing strain stored at -80℃ in a preservation tube was inoculated onto an agar slant for activation; ② The bacterial cells on the agar slant were inoculated into a primary seed shake flask for culture; ③ The primary seed culture was transferred into a 10 L secondary fermenter for secondary seed culture; ④ The secondary seed culture was transferred into a fermentation medium and fermented in a 50 L fermenter.

[0037] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0038] Unless otherwise specified, the percentage sign "%" used in this invention refers to mass percentage. The percentage of a solution refers to the number of grams of solute contained in 100 mL. The percentage between liquids refers to the volume ratio of the solution at 25°C.

[0039] In the following examples, molasses was purchased from Shandong Xingqi Chemical Technology Co., Ltd., and corn steep liquor and corn steep liquor hydrolysate were purchased from Meihua Biotechnology Group Co., Ltd.

[0040] Example 1: Physicochemical property analysis of the fermentation process of Corynebacterium glutamicum and Escherichia coli The changes in temperature, pH, and osmotic pressure during the fermentation of Corynebacterium glutamicum Cgl-1 and Escherichia coli E. coli-1 (provided by Meihua Biotechnology Group Co., Ltd.) were analyzed, such as... Figure 1 As shown.

[0041] During the fermentation of both Corynebacterium glutamicum and Escherichia coli, the temperature was consistently maintained at 35℃ and the pH at 6.7. Therefore, for the screening of superior microorganisms, the temperature and pH should be set at 35℃ and 6.7. The osmotic pressure of the fermentation broth consistently increased during the fermentation process of both strains. The lowest osmotic pressure during the fermentation of Corynebacterium glutamicum was 151.7 mOsm, and the highest was 2079.2 mOsm, while for Escherichia coli, the values ​​were 154.6 and 1980.3 mOsm, respectively. Therefore, adjusting the osmotic pressure to 200-2000 mOsm using NaCl was used to screen for superior fermentation strains.

[0042] Example 2: Screening of superior fermentation strains (Corynebacterium glutamicum and Escherichia coli) Superior strains were screened using a culture medium primarily composed of molasses, corn steep liquor, corn steep liquor hydrolysate, glucose, and water (mass ratio 1:1:1:0.2:5). During lysine fermentation, the osmotic pressure of the culture medium continuously increases due to microbial growth and metabolism; therefore, strains capable of tolerating higher osmotic pressure and producing high levels of lysine were screened. From laboratory-preserved Corynebacterium glutamicum and Escherichia coli, 15 strains were selected that exhibited good growth under higher osmotic pressure.

[0043] Lysine production was analyzed in 15 strains of Corynebacterium glutamicum and 15 strains of Escherichia coli, respectively. Figure 2 As shown in Figure A, the average lysine concentration after fermentation by Corynebacterium glutamicum was 123.70 g / L. The strain producing the highest lysine was Cgl-8, with a lysine concentration of 152.15 g / L (conversion rate of 59.80%), which was 23.00% higher than the average. Figure 2 As shown in Figure B, the average lysine concentration after E. coli fermentation was 125.66 g / L. The strain producing the highest lysine was E. coli-10 (MHZ-0914), with a maximum lysine concentration of 148.89 g / L (conversion rate of 60.10%), which was 15.60% higher than the average. Therefore, Cgl-8 (CGMCC accession number CGMCC NO. 11942) and E. coli-10 (CGMCC accession number CGMCC NO. 22648) were selected as the strains for fermentation and enhancement.

[0044] Example 3: Enhancement during Corynebacterium glutamicum fermentation Previous studies have shown that using Corynebacterium glutamicum to enhance fermentation during the Corynebacterium glutamicum fermentation process did not significantly improve lysine yield and conversion rate after fermentation. Therefore, Escherichia coli was used to enhance fermentation during the Corynebacterium glutamicum fermentation process. Experiments were conducted to determine the amount and duration of E. coli enhancement during the fermentation process.

[0045] Fermentation strains (Corynebacterium glutamicum and Escherichia coli) preserved at -80℃ were activated and then inoculated into primary seed culture medium. When the OD of the primary seed culture medium... 562 When the OD value is 10, 200 mL of each culture medium is inoculated into the secondary seed culture medium for secondary seed culture. When the OD value of the secondary seed culture medium is... 562 When the concentration reaches 40, take 10 kg of each for later use.

[0046] After sterilizing the fermentation medium, pour it into a 50 L fermenter and add the cultured Corynebacterium glutamicum seed (OD). 562 =40) After 3 kg, the volume was adjusted to 15 L. Dissolved oxygen was maintained at 50% by adjusting the rotation speed and ventilation. pH was maintained at 6.7 by adding ammonia. Free nitrogen concentration was maintained at 2.5 g / L by adding 37% ammonium sulfate solution. Glucose concentration in the fermentation broth was maintained at 1 g / L by adding 60.5% glucose solution. When the tank weight reached 25 kg, it was reduced to 24 kg, and this process was repeated continuously. After a period of fermentation, the cultured E. coli secondary seed (OD) was... 562 =40) was added to the fermentation tank. The total fermentation time was 36 hours.

[0047] The fermentation of different amounts (based on 15 L at constant volume) of *E. coli* was enhanced as follows: Figure 3 As shown in Figure A. The experimental groups with 5% and 10% vaccination not only had the largest OD... 562 The lysine concentration in the culture medium was lower than that of the 15%, 20%, and 25% groups after fermentation. The maximum OD values ​​were observed in the 15% and 20% and 25% inoculated groups. 562 Value and reaching maximum OD 562 Since the fermentation time and lysine concentration in the culture medium were consistent, the inoculum size for *E. coli* enhancement was determined to be 15%. Subsequently, *E. coli* was enhanced at different stages of *Corynebacterium glutamicum* fermentation, and the results are as follows: Figure 3 As shown in Figure B. The results showed that the highest lysine concentration, reaching 222.96 g / L, was achieved after 16 h of fermentation with *Corynebacterium glutamicum* and followed by *Escherichia coli*. At this point, the conversion rate reached 65.6%, and the lysine yield and conversion rate were 46.54% and 9.70% higher than those of the control group (lysine concentration of 152.15 g / L and conversion rate of 59.8%), respectively.

[0048] Example 4: Enhancement during E. coli fermentation Similar to Example 3, previous studies have shown that using *E. coli* to enhance fermentation during *E. coli* fermentation did not significantly improve lysine concentration and conversion rate. Therefore, *Corynebacterium glutamicum* was used to enhance fermentation during *E. coli* fermentation. The amount and duration of *Corynebacterium glutamicum* enhancement during fermentation were tested.

[0049] Fermentation strains (Corynebacterium glutamicum and Escherichia coli) preserved at -80℃ were activated and then inoculated into primary seed culture medium. When the OD of the primary seed culture medium... 562 When the OD value is 10, 200 mL of each culture medium is inoculated into the secondary seed culture medium for secondary seed culture. When the OD value of the secondary seed culture medium is... 562 When the concentration reaches 40, take 10 kg of each for later use.

[0050] After sterilization, the fermentation medium was poured into a 50 L fermenter. 3 kg of cultured *E. coli* secondary seed culture was added, and the volume was adjusted to 15 L. Dissolved oxygen was maintained at 50% by adjusting the turbine speed and aeration. The pH was maintained at 6.7 by adding ammonia, the free nitrogen concentration was maintained at 2.5 g / L by adding 37% ammonium sulfate solution, and the glucose concentration in the fermentation broth was maintained at 1 g / L by adding 60.5% glucose dissolved oxygen. When the fermenter reached 25 kg, it was reduced to 24 kg, and this process was repeated continuously. After a period of fermentation, the cultured *Corynebacterium glutamicum* seed culture (OD) was... 562 =40) was added to the fermentation tank. The total fermentation time was 36 hours.

[0051] The fermentation results of different inoculation amounts (based on 15 L at constant volume) of Corynebacterium glutamicum are as follows: Figure 4 As shown in Figure A. The experimental groups with 5% and 10% vaccination not only had the largest OD... 562 The lysine concentration in the culture medium was lower than that of the 15%, 20%, and 25% inoculation groups after fermentation. The maximum OD values ​​were found in the 15% inoculation group and the 20% and 25% inoculation groups. 562 Value and reaching maximum OD 562 The fermentation time was approximately 8 hours, and the lysine concentration in the culture medium was slightly lower after fermentation. The microbial growth was similar in the 20% inoculation group and the 25% inoculation group, and the lysine concentration was also consistent after fermentation. Therefore, the inoculation amount of *Corynebacterium glutamicum* was determined to be 20%. Subsequently, *Corynebacterium glutamicum* was inoculated at different stages of *E. coli* fermentation, and the results are as follows... Figure 4 As shown in Figure B. The results showed that the highest lysine concentration, reaching 231.35 g / L, was achieved after 12 h of Escherichia coli fermentation with enhanced inoculation of Corynebacterium glutamicum and fermentation. At this point, the conversion rate reached 66.20%, and the lysine yield and conversion rate were increased by 55.38% and 10.15%, respectively, compared with the control group (lysine concentration of 148.89 g / L and conversion rate of 60.10%).

[0052] Example 5 Biofortification of Cgl-8 and E. coli-10 in glutamic acid fermentation Fermentation strains (Corynebacterium glutamicum and Escherichia coli) preserved at -80℃ were activated and then inoculated into primary seed culture medium. When the OD of the primary seed culture medium... 562 When the OD value is 10, 200 mL of each culture medium is inoculated into the secondary seed culture medium for secondary seed culture. When the OD value of the secondary seed culture medium is... 562 When the concentration reaches 40°C, 10 kg of each culture medium is collected for later use. After sterilization, the fermentation medium is poured into a 50 L fermenter. 3 kg of the cultured *Corynebacterium glutamicum* (or *Escherichia coli*) secondary seed culture is added, and the volume is adjusted to 10 L. Dissolved oxygen is maintained at 50% by adjusting the turbine speed and aeration rate. The pH is maintained at 6.7 by adding ammonia, and the glucose concentration in the fermentation broth is maintained at 1 g / L by adding 60.5% glucose dissolved oxygen. After fermentation for a period of time, the cultured *E. coli* (or *Corynebacterium glutamicum*) secondary seed culture is inoculated into the fermenter. Ammonium sulfate is not added during the glutamate fermentation process, and continuous addition is not required. The total fermentation time is 36 hours.

[0053] During the fermentation process of Corynebacterium glutamicum Cgl-8, it was enhanced with Escherichia coli E. coli-10, and the results were as follows: Figure 5 As shown in Figures A and B. The amount of E. coli-10 used for enhanced inoculation was 15% of the fermentation broth mass, and the inoculation time was 16 h of fermentation. The results showed that during the fermentation process of Cgl-8 for glutamic acid production, the maximum glutamic acid concentration reached by enhanced inoculation with E. coli-10 was 105.47 g / L, at which point the conversion rate reached 50.86%. The glutamic acid yield and conversion rate were increased by 19.92% and 26.05%, respectively, compared to the control group (glutamic acid concentration 87.95 g / L, conversion rate 40.35%).

[0054] During the fermentation of E. coli-10, Cgl-8 was used for enhancement, and the results were as follows: Figure 5 As shown in C and D. The amount of Cgl-8 inoculated for enhanced fermentation was 20% of the fermentation broth mass, and the inoculation time was 12 h of fermentation. The results showed that in the fermentation process of E. coli-10 to produce glutamic acid, the maximum glutamic acid concentration reached was 98.45 g / L with enhanced inoculation of Cgl-8, at which point the conversion rate reached 50.12%. The glutamic acid yield and conversion rate were increased by 31.25% and 27.92% respectively compared with the control group (glutamic acid concentration was 75.01 g / L, and the conversion rate was 39.18%).

[0055] Example 6 Biofortification of Cgl-8 and E. coli-10 in threonine fermentation Fermentation strains (Corynebacterium glutamicum and Escherichia coli) preserved at -80℃ were activated and then inoculated into primary seed culture medium. When the OD of the primary seed culture medium... 562When the OD value is 10, 200 mL of each culture medium is inoculated into the secondary seed culture medium for secondary seed culture. When the OD value of the secondary seed culture medium is... 562 When the concentration reaches 40°C, 10 kg of each culture medium is taken for later use. After sterilizing the fermentation medium, it is poured into a 50 L fermenter. 3 kg of cultured Corynebacterium glutamicum (or Escherichia coli) secondary seed solution is added, and the volume is adjusted to 10 L. Dissolved oxygen is maintained at 50% by adjusting the turbine speed and aeration rate. The pH is maintained at 6.7 by adding ammonia, and the glucose concentration in the fermentation broth is maintained at 1 g / L by adding 60.5% glucose dissolved oxygen. After fermentation for a period of time, the cultured Escherichia coli (or Corynebacterium glutamicum) secondary seed solution is inoculated into the fermenter. No ammonium sulfate is added during threonine fermentation, and continuous addition is not required. The total fermentation time is 36 hours.

[0056] During the fermentation process of Corynebacterium glutamicum Cgl-8, it was enhanced with Escherichia coli E. coli-10, and the results were as follows: Figure 6 As shown in Figures A and B. The amount of E. coli-10 used for enhanced inoculation was 15% of the fermentation broth mass, and the inoculation time was 16 h of fermentation. The results showed that during the fermentation process of Cgl-8 for threonine production, the maximum glutamate concentration reached by enhanced inoculation with E. coli-10 was 76.88 g / L, at which point the conversion rate reached 40.79%. The threonine yield and conversion rate were 32.67% and 28.87% higher than the control group (glutamate concentration 57.95 g / L, conversion rate 31.65%), respectively.

[0057] During the fermentation of E. coli-10, Cgl-8 was used for enhancement, and the results were as follows: Figure 6 As shown in C and D. The amount of Cgl-8 inoculated for enhanced fermentation was 20% of the fermentation broth mass, and the inoculation time was 12 h of fermentation. The results showed that during the fermentation process of E. coli-10 to produce threonine, the maximum glutamate concentration reached by Cgl-8 inoculation was 68.45 g / L, at which point the conversion rate reached 40.85%. The glutamate yield and conversion rate were increased by 51.74% and 46.47% respectively compared with the control group (glutamate concentration of 45.11 g / L, conversion rate of 27.89%).

[0058] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for producing amino acids using microbial enhanced fermentation, characterized in that, Includes the following steps: The amino acid is lysine; the method includes the following steps: A. Fermentation was carried out using Corynebacterium glutamicum strain M1 and Escherichia coli strain M2; B. Inoculate strain M2 after strain M1 has fermented for 4-36 hours; or inoculate strain M1 after strain M2 has fermented for 4-36 hours, in order to improve the fermentation yield and conversion rate of lysine. The strain M1 is Corynebacterium glutamicum with accession number CGMCC NO. 11942. Corynebacterium glutamicum The strain M2 is *Escherichia coli* with accession number CGMCC NO.22648. Escherichia coli ).

2. The method according to claim 1, characterized in that, After strain M1 has fermented for 16 h, strain M2, which has been cultured to the logarithmic phase, is inoculated at a rate of 15% of the total mass of the fermentation system of strain M1.

3. The method according to claim 1, characterized in that, After strain M2 has fermented for 12 h, strain M1, which has been cultured to the logarithmic phase, is inoculated, with the inoculation amount being 20% ​​of the total mass of the fermentation system of strain M2.

4. The method according to claim 2 or 3, characterized in that, Step B includes: activating the lysine-producing strain stored at -80℃ by inoculating it onto an agar slant medium; inoculating the bacterial cells from the agar slant medium into a seed shake flask for primary seed culture; transferring the primary seed culture into a 10 L secondary fermenter for secondary seed culture; and transferring the secondary seed culture into a fermentation medium for fermentation culture in a 50 L fermenter.

5. The method according to claim 4, characterized in that, The culture medium used for primary seed culture was: sucrose 30 g / L, potassium dihydrogen phosphate 1.2 g / L, magnesium sulfate 1.2 g / L, monosodium glutamate 10 g / L, yeast extract 15 g / L, ammonium sulfate 20 g / L, threonine 1 g / L, ferrous sulfate 0.005 g / L and manganese sulfate 0.005 g / L. The culture medium used for secondary seed culture was: corn steep liquor 30 g / L, corn steep liquor hydrolysate 20 g / L, ammonium sulfate 8 g / L, magnesium sulfate 2 g / L, threonine 0.5 g / L, ferrous sulfate 0.015 g / L, and manganese sulfate 0.015 g / L. The fermentation medium consisted of molasses 150 g / L, corn steep liquor 150 g / L, corn steep liquor hydrolysate 150 g / L, ammonium sulfate 2 g / L, and glucose 50 g / L.

6. The method according to claim 4, characterized in that, Step B includes the following sub-steps: B1. The primary seed culture temperature is 35℃, 300 rpm, and cultured until OD... 562 =10, then transfer 200 mL of the primary seed culture to the secondary fermenter for secondary seed culture; B2. Secondary seed culture temperature is 35℃, dissolved oxygen is coupled with rotation speed to ensure dissolved oxygen is not lower than 30%, pH is adjusted with ammonia to maintain 6.7, and culture is carried out until OD reaches... 562 After 40 minutes, 3 kg of secondary seed culture was transferred to the fermenter for fermentation culture. B3. The fermentation temperature is 35℃. The pH is adjusted to 6.7 with ammonia water. During the fermentation process, ammonium sulfate solution is added to maintain the ammonia nitrogen level in the culture medium at 1-1.5 g / L. During the fermentation process, glucose solution is added to maintain the glucose concentration in the culture medium at 0.2-2 g / L.

7. The method according to claim 6, characterized in that, The concentration of the added ammonium sulfate solution is 30%-50%, and the concentration of the glucose solution is 40%-80%.

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