Process method for producing ectoine through efficient fermentation

Abstract

The invention relates to the technical field of biochemical engineering, in particular to a process method for producing ectoine through efficient fermentation. According to the method, a culture medium formula of a base material culture medium and trace elements is developed by optimizing the culture medium, and the base material culture medium comprises glucose, ammonium sulfate, ammonium dihydrogen phosphate, citric acid, potassium sulfate, magnesium sulfate, ferrous sulfate, manganese sulfate, ampicillin and streptomycin; the microelements comprise vitamin B1, vitamin B6, NiCl2, CuSO4, CoCl2 and NaMoO4, and the culture medium disclosed by the invention can be used for remarkably improving the metabolic activity of thalli and has a positive effect on improving the yield and the conversion rate of ectoine; according to the method, the yield of ectoine is high through the ectoine synthesis strain, the fermentation dissolved oxygen and the rotating speed are controlled in a segmented mode in the fermentation process, the yield of the ectoine synthesis strain reaches 134.29 g / L and is successfully amplified to the industrial scale, the fermentation content of a 5m < 3 > tank reaches 90.13 g / L, and the method has great industrialization value.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical engineering, and in particular to a process for producing ectoine by efficient fermentation. Background Art

[0002] Ectoine (also known as ectoine and ectoine carboxylic acid) is a high-value-added cyclic amino acid and an important biologically active substance with multiple functional properties, such as anti-adversity protection, molecular chaperone, radiation protection and moisturizing effects. It is widely used in cosmetics, medicine, enzyme preparations and other fields.

[0003] At present, the fermentation production process of tetrahydropyrimidine has some shortcomings: 1. High culture medium cost: Existing processes mostly use complex organic nitrogen sources such as yeast powder and peptone, and some processes use mixed carbon source systems, resulting in high raw material costs. At the same time, the complex culture medium components also cause great difficulties in extraction and purification; 2. Low fermentation efficiency: Traditional processes have long fermentation cycles, low conversion rates, and extensive control of parameters such as dissolved oxygen and pH, which limits the increase in production; 3. Difficulty in process scale-up: Laboratory small-scale processes are difficult to effectively scale up to industrial scale. There are problems such as fermentation parameter mismatch and reduced product synthesis efficiency, which restrict the industrialization process.

[0004] With the continuous growth of market demand, the development of efficient and low-cost fermentation production processes for ectoine is of great significance. To address the above problems, the present invention proposes an innovative solution that integrates culture medium optimization, segmented control and scale-up process, realizes efficient fermentation production of ectoine by recombinant Escherichia coli, and achieves high-yield and large-scale industrial production of ectoine, providing new ideas and methods for the industrial production of ectoine. Summary of the Invention

[0005] The purpose of the present invention is to provide a process for producing tetrahydropyrimidine by efficient fermentation, so as to solve the problems of high cost of culture medium, low fermentation efficiency and difficulty in process scale-up in existing fermentation processes.

[0006] To achieve the above object, the present invention adopts the following technical solution: a process for producing tetrahydropyrimidine by efficient fermentation, comprising the following steps:

[0007] S1. Culture of ectoine

[0008] The ectoine synthesis strain is cultured on a plate, cultured as a first-level seed, and cultured as a second-level seed to obtain a second-level seed solution;

[0009] S2. Fermentation

[0010] The S1 secondary seed solution was inoculated into a fermentation tank containing fermentation medium, and the fermentation dissolved oxygen and rotation speed were controlled in stages to achieve efficient fermentation and production of ectoine.

[0011] The fermentation medium comprises a base medium and trace elements; the base medium comprises glucose, ammonium sulfate, ammonium dihydrogen phosphate, citric acid, potassium sulfate, magnesium sulfate, ferrous sulfate, manganese sulfate, ampicillin and streptomycin; the trace elements comprise vitamin B1, vitamin B6, NiCl2, CuSO4, CoCl2 and NaMoO4;

[0012] In the initial fermentation period of 0 to 12 hours, the dissolved oxygen is controlled to be ≥40%, and the rotation speed is maintained at a low level of 30 to 200 rpm. In the middle fermentation period after 12 hours, when the dissolved oxygen drops below 40%, the ventilation volume is gradually increased to 3 to 4 VVM, and then the stirring speed is linked to maintain the dissolved oxygen at 18 to 23%.

[0013] Furthermore, in the S1, the plate culture is to inoculate the strain into LB solid culture medium by streaking, and culture it at 32-40°C for 16-24 hours; the first-level seed culture is to pick the strain cultured on the plate, inoculate it into liquid LB culture medium, and culture it at 32-40°C for 12-20 hours to obtain the first-level seed liquid; the second-level seed culture is to inoculate the first-level seed liquid into the seed tank culture medium with an inoculation amount of 2-10%, and ferment and culture it at 32-40°C for 6-10 hours to obtain the second-level seed liquid.

[0014] Furthermore, the seed tank culture medium is 25 g / L glucose, 5 g / L yeast extract powder, 4 g / L peptone, 2 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, 0.01 g / L ferrous sulfate, and 0.01 g / L manganese sulfate, and ampicillin with a final concentration of 100 mg / L and streptomycin with a final concentration of 50 mg / L are added at the beginning of fermentation.

[0015] Furthermore, the secondary seed culture is heavy, the inoculation amount of the primary seed liquid is 10% (v / v), the ventilation volume is 1VVM, the stirring speed is 300-600rpm, the dissolved oxygen is controlled at 20%, the fermentation culture is carried out at 37°C, and the pH is controlled at 7.0±0.2 throughout the process with ammonia water, and the culture is carried out for 6-8h.

[0016] Furthermore, in the S2, the contents of each component in the fermentation medium are glucose 20-50 g / L, ammonium sulfate 6-12 g / L, ammonium dihydrogen phosphate 3-8 g / L, citric acid 2-5 g / L, potassium sulfate 3-8 g / L, magnesium sulfate 2-5 g / L, ferrous sulfate 0.2-1 g / L, manganese sulfate 0.2-0.8 g / L, vitamin B1 2-10 mg / L, vitamin B6 1-5 mg / L, NiCl2 0.5-3 mg / L, CuSO4 0.5-3 mg / L, CoCl2 0.01-0.1 mg / L, and NaMoO4 0.01-0.1 mg / L, and ampicillin at a final concentration of 50-100 mg / L and streptomycin 50-100 mg / L are added at the beginning of fermentation.

[0017] Furthermore, the contents of each component in the fermentation medium are 20 g / L glucose, 9 g / L ammonium sulfate, 4 g / L ammonium dihydrogen phosphate, 3 g / L citric acid, 4 g / L potassium sulfate, 2 g / L magnesium sulfate, 0.6 g / L ferrous sulfate, 0.3 g / L manganese sulfate, 3.2 mg / L vitamin B1, 3 mg / L vitamin B6, 2 mg / L NiCl2, 2 mg / L CuSO4, 0.04 mg / L CoCl2, and 0.04 mg / L NaMoO4. At the beginning of fermentation, ampicillin at a final concentration of 100 mg / L and streptomycin at 50 mg / L are added.

[0018] Furthermore, in the S2, during the fermentation process, the inoculation amount of the secondary seed liquid is 2-10% (v / v), the fermentation temperature is 32-40°C, and the pH is controlled at 7.0±0.2 throughout the process with ammonia water. When the sugar consumption in the fermentation tank is below 2 g / L, glucose is added to maintain the glucose concentration in the fermentation tank at 0-5 g / L, and IPTG is added when OD600 is ≥7-9.

[0019] Furthermore, the S2 also includes a process amplification step, during which the process is amplified according to the matching principle of volume oxygen transfer coefficient and stirring shear force.

[0020] Furthermore, in a 5L fermentation tank, the stirring speed is 200-300rpm at the initial stage of fermentation. In the middle stage of fermentation, the ventilation volume is gradually increased to 4VVM, and the stirring is set at 300-800rpm in association with dissolved oxygen, and the dissolved oxygen is controlled to be ≥20%; in a 50L fermentation tank, the stirring speed is 150-200rpm at the initial stage of fermentation. In the middle stage of fermentation, the ventilation volume is gradually increased to 3.5VVM, and the stirring is set at 200-600rpm in association with dissolved oxygen, and the dissolved oxygen is controlled to be ≥20%; in a 500L fermentation tank, the stirring speed is 100-150rpm at the initial stage of fermentation. In the middle stage of fermentation, the ventilation volume is gradually increased to 3.5VVM, and the stirring is set at 150-350rpm in association with dissolved oxygen, and the dissolved oxygen is controlled to be ≥20%; in a 5m 3 In the fermentation tank, the stirring speed is 30-60 rpm at the initial stage of fermentation. In the middle stage of fermentation, the ventilation volume is gradually increased to 3VVM, and the stirring speed is set to 60-180 rpm in conjunction with the dissolved oxygen, and the dissolved oxygen is controlled to be ≥20%.

[0021] Beneficial effects of the present invention:

[0022] 1. Compared with traditional culture media, the present invention optimizes the concentrations of inorganic salts such as magnesium sulfate, ferrous sulfate, and manganese sulfate, and introduces a combination of trace elements to achieve synergistic optimization of composite inorganic salts and trace elements, thereby optimizing the nutritional balance between bacterial growth and product synthesis;

[0023] 2. The present invention adopts a staged control strategy during the fermentation process. In the early stage, a low rotation speed is maintained to avoid shear force damage to the bacteria. In the middle stage, the dissolved oxygen level and residual sugar concentration are controlled to maintain the growth and metabolic needs of the strain, ensuring high activity of the strain and efficient synthesis of ectoine.

[0024] 3. The present invention achieves 5L to 5m through a systematic amplification strategy 3 The fermentation tank has high efficiency conversion. The fermentation content of 5L tank has stably reached more than 120g / L, and the highest can reach 134.29g / L, which is the highest content reported so far. After pilot scale-up verification, the fermentation content of 50L tank has reached 86.5g / L, the fermentation content of 500L tank has reached 93.05g / L, and the content of 5m 3 The fermentation content in the tank reaches 90.13g / L; the output is stable, providing a reliable process basis for industrial production;

[0025] 4. The present invention reduces the generation of by-products through optimized culture medium and fermentation control, reduces the difficulty of subsequent extraction and purification, and has a positive effect on ensuring product purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 OD during the fermentation process of Example 1 of the present invention 600 , residual sugar and ectoine concentration change curve;

[0027] Figure 2 OD during the fermentation process of Example 4 of the present invention 600 , residual sugar and ectoine concentration change curve. DETAILED DESCRIPTION

[0028] The technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0029] The present invention optimizes the ectoine fermentation process from three aspects: culture medium optimization, segmented control and amplification process.

[0030] The fermentation medium of the present invention includes a base medium and trace elements, specifically as follows:

[0031] Table 1 Composition of base medium in the fermentation medium of the present invention

[0032]

[0033] Table 2 Trace element composition in the fermentation medium of the present invention

[0034]

[0035] The supplementation of trace elements can significantly enhance the metabolic activity of bacteria, which has a positive effect on increasing the yield and conversion rate of ectoine.

[0036] The fermentation process is as follows:

[0037] The ectoine synthesis strain is streaked onto a solid LB culture medium and cultured at 32-40° C. for 16-24 hours; the cultured strain is picked and inoculated into a liquid LB culture medium and cultured at 32-40° C. for 12-20 hours to obtain a first-level seed liquid; the first-level seed liquid is inoculated into a seed tank culture medium with an inoculation amount of 2-10%, and fermentation is carried out at 32-40° C. for 6-10 hours to obtain a second-level seed liquid; the second-level seed liquid is inoculated into a fermentation culture medium of the present invention with an inoculation amount of 5-20%, ampicillin, streptomycin and an inducer IPTG are added, and fermentation is carried out at 32-40° C. for 36-60 hours.

[0038] During the fermentation process, the carbon source is fed with glucose at a concentration of 500-800 g / L. When the base sugar is consumed to 2 g / L, the sugar is fed to maintain the residual sugar concentration at 0.5-10 g / L. Preferably, the residual sugar concentration is maintained at about 2 g / L to avoid substrate inhibition and enable efficient product synthesis. When fermentation is performed in a fermenter, when OD 600 When the reaction temperature reaches 6 to 15, 0.1 mmol of IPTG (isopropyl-β-D-thiogalactopyranoside) is added to start the synthesis of ectoine.

[0039] The fermentation process is controlled in stages as follows:

[0040] Initial conditions at 0h: temperature 32-40℃, speed set at 30-200rpm according to the volume of the fermenter, ventilation volume 0.8-1.2VVM, pH 7.0±0.2, automatically controlled by ammonia water; in the initial stage (0-12h), the dissolved oxygen is controlled to be ≥40%, and the speed is maintained at a low level (30-200rpm) to avoid excessive shear force damaging the bacteria; in the middle stage (after 12h), the dissolved oxygen drops below 40%, and the ventilation volume is gradually increased to 3-4VVM, and then the stirring speed is linked to maintain the dissolved oxygen at 18-23%; in the later stage, the speed is first reduced, and then the ventilation volume is reduced to reduce energy consumption.

[0041] During the process of process amplification, the amplification is carried out according to the principle of matching the volume oxygen transfer coefficient and the stirring shear force.

[0042] Example 1

[0043] The strain used in this example is an engineered strain of Escherichia coli MG1655 that expresses the ectABC gene cluster, knocks out the crr gene, and overexpresses the lysC gene. The construction method is described in the Chinese invention patent "CN118028204A Ectoin synthetic strain, its construction method, and application."

[0044] In this embodiment, the strain is fermented and cultured in the following steps:

[0045] S1. Plate culture

[0046] The strain was inoculated into LB solid medium by streaking, cultured at 37°C for 16-20 h;

[0047] S2, first-level seed

[0048] Pick the cultured bacteria, inoculate into a 50 mL shake flask containing liquid LB medium, place it in a shaker at 37°C and culture at 200 rpm for 12-16 hours as the primary seed liquid;

[0049] S3, secondary seed

[0050] The seed tank culture medium is: glucose 25g / L, yeast extract powder 5g / L, peptone 4g / L, potassium dihydrogen phosphate 2g / L, magnesium sulfate 1g / L, ferrous sulfate 0.01g / L, manganese sulfate 0.01g / L, and ampicillin with a final concentration of 100mg / L and streptomycin 50mg / L are added at the beginning of fermentation.

[0051] The activated first-level seed liquid is inoculated into the seed tank with an inoculation volume of 10% (v / v), an aeration volume of 1VVM, a stirring speed of 300-600rpm, and the dissolved oxygen is controlled at 20%. The fermentation culture is carried out at 37°C, and the pH is controlled at 7.0±0.2 with ammonia water throughout the process, and the culture is carried out for 6-8h.

[0052] S4, 5L tank fermentation

[0053] The fermentation medium is: 20 g / L glucose, 9 g / L ammonium sulfate, 4 g / L ammonium dihydrogen phosphate, 3 g / L citric acid, 4 g / L potassium sulfate, 2 g / L magnesium sulfate, 0.6 g / L ferrous sulfate, 0.3 g / L manganese sulfate, 3.2 mg / L vitamin B1, 3 mg / L vitamin B6, 2 mg / L NiCl2, 2 mg / L CuSO4, 2 mg / L CoCl2, 0.04 mg / L, and 0.04 mg / L NaMoO4. At the beginning of fermentation, ampicillin at a final concentration of 100 mg / L and streptomycin at 50 mg / L were added.

[0054] The cultured secondary seed liquid was inoculated into the seed tank with an inoculation volume of 10% (v / v), a temperature of 37°C, and ammonia water was used to control the pH at 7.0±0.2 throughout the process. When the sugar consumption in the fermentation tank dropped below 2g / L, 600g / L of glucose was added to maintain the glucose concentration in the fermentation tank at around 2g / L. When OD600 was ≥7, 0.1mmol / L of IPTG was added. In the early stage of fermentation (0-12h), the dissolved oxygen was controlled to be ≥40%, and the speed was 200-300rpm. In the middle stage (after 12h), the dissolved oxygen dropped to below 40%. The ventilation volume was gradually increased to 4VVM, and the stirring was set to 300-800rpm to correlate the dissolved oxygen, controlling the dissolved oxygen to be ≥20%. The tank pressure was 0.05MPa. In the later stage, the metabolism slowed down, and the speed was reduced first, and then the ventilation volume was reduced to reduce energy consumption.

[0055] During the fermentation process, OD 600 , residual sugar and ectoine concentration changes as shown in Figure 1 As shown, OD 600 It shows a trend of first increasing and then decreasing; the residual sugar shows a trend of first decreasing, and then fluctuates repeatedly between 0 and 5g / L with the supplementation; the tetrahydropyrimidine shows a trend of continuous increase, and the growth rate tends to be slow in the later stage of fermentation.

[0056] Example 2

[0057] The plate culture, primary seed, secondary seed formula and process are the same as those in Example 1.

[0058] 50L tank fermentation: the formula is the same as that of Example 1. The cultured secondary seed liquid is accessed into the fermentation tank with an inoculum size of 10% (v / v), a temperature of 37°C, and a pH of 7.0 ± 0.2 is controlled throughout the entire process with ammonia water. When the sugar consumption in the fermentation tank is below 2g / L, 600g / L of glucose is added, and the glucose concentration in the fermentation tank is maintained at about 2g / L. When OD600 is ≥8, IPTG 0.1mmol / L is added. In the initial stage of fermentation (0-12h), the dissolved oxygen is controlled to be ≥40%, with a rotation speed of 150-200rpm. In the middle stage (after 12h), the dissolved oxygen is reduced to below 40%. The ventilation volume is gradually increased to 3.5VVM, and the stirring setting is 200-600rpm associated with the dissolved oxygen to control the dissolved oxygen to be ≥20%. The tank pressure is 0.05MPa, and the metabolism in the later stage slows down. The rotation speed is first reduced, and then the ventilation volume is reduced to reduce energy consumption.

[0059] Example 3

[0060] The plate culture, primary seed, secondary seed formula and process are the same as those in Example 1.

[0061] 500L tank fermentation: formula is the same as Example 1. The cultured secondary seed liquid is accessed into the fermentor, the inoculum size is 10% (v / v), the temperature is 37 ° C, and the pH is controlled to 7.0 ± 0.2 with ammonia throughout the process. When the sugar consumption in the fermentor is below 2g / L, 600g / L of glucose is added, and the glucose concentration in the fermentor is kept at about 2g / L. When OD600 is ≥9, IPTG 0.1mmol / L is added. In the initial stage of fermentation (0-12h), the dissolved oxygen is controlled to be ≥40%, the speed is 100-150rpm, and the dissolved oxygen is reduced to below 40% in the middle stage (after 12h). The ventilation volume is gradually increased to 3.5VVM, and the stirring setting is 150-350rpm associated with the dissolved oxygen to control the dissolved oxygen ≥20%. The tank pressure is 0.05-0.08MPa. The metabolism in the later stage slows down, and the speed is first reduced, and then the ventilation volume is reduced to reduce energy consumption.

[0062] Example 4

[0063] The plate culture, primary seed, secondary seed formula and process are the same as those in Example 1.

[0064] 5m 3 Tank fermentation: The formula is the same as in Example 1. The cultured secondary seed liquid is accessed into the fermentation tank with an inoculum size of 15% (v / v), a temperature of 37°C, and a pH of 7.0±0.2 is controlled throughout the process with ammonia water. When the sugar consumption in the fermentation tank is below 2g / L, 600g / L of glucose is added to maintain the glucose concentration in the fermentation tank at about 2g / L. When OD600 is ≥9, IPTG 0.1mmol / L is added. In the initial stage of fermentation (0-12h), the dissolved oxygen is controlled to be ≥40%, the rotation speed is 30-60rpm, and the dissolved oxygen is reduced to below 40% in the middle stage (after 12h). The ventilation volume is gradually increased to 3VVM, and the stirring setting is 60-180rpm associated with the dissolved oxygen to control the dissolved oxygen to be ≥20%. The tank pressure is 0.05-0.08MPa. The metabolism slows down in the later stage, and the rotation speed is first reduced, and then the ventilation volume is reduced to reduce energy consumption.

[0065] During the fermentation process, OD 600 , residual sugar and ectoine concentration changes as shown in Figure 2 As shown, OD 600 It shows a trend of first increasing and then decreasing; the residual sugar shows a trend of first decreasing, and then fluctuates repeatedly between 0 and 5g / L with the supplementation; the tetrahydropyrimidine shows a trend of continuous increase, and the growth rate tends to be slow in the later stage of fermentation.

[0066] The table shows examples 1-4, which are 5L, 50L, 500L, 5m 3 Fermentation results in tanks.

[0067] Table 3 Fermentation results of Examples 1-4

[0068]

[0069] From the results, the present invention not only achieved a high yield of 134.29 g / L and a high conversion rate of 47.50% in a 5L experimental tank, but also achieved a high conversion rate from 5L small-scale fermentation to 5m 3 Large-scale fermentation process scale-up. During the scale-up process, factors such as the characteristics of the fermentation tank, the growth characteristics of the strain, and the metabolic needs were comprehensively considered. Parameters such as inoculation volume, ventilation volume, agitation speed, and dissolved oxygen control were optimized and adjusted to ensure that high and stable yields of ectoine can be obtained in large-scale fermentation production, exceeding the current overall industry level.

[0070] In summary, the present invention provides a process for producing ectoine by efficient fermentation, which can effectively solve the problems existing in the prior art and provide a new idea and method for the industrial production of ectoine.

[0071] The present invention is not limited to the above-mentioned optimal implementation mode. Anyone can derive other forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, any technical solution that is the same or similar to that of the present application falls within the scope of protection of the present invention.

Claims

1. A process for producing tetrahydropyrimidine by efficient fermentation, characterized in that: The following steps are involved: S1. Culture of ectoine The ectoine synthesis strain is cultured on a plate, cultured as a first-level seed, and cultured as a second-level seed to obtain a second-level seed solution; S2. Fermentation The S1 secondary seed solution was inoculated into a fermentation tank containing fermentation medium, and the fermentation dissolved oxygen and rotation speed were controlled in stages to achieve efficient fermentation and production of ectoine. The fermentation medium comprises a base medium and trace elements; the base medium comprises glucose, ammonium sulfate, ammonium dihydrogen phosphate, citric acid, potassium sulfate, magnesium sulfate, ferrous sulfate, manganese sulfate, ampicillin and streptomycin; the trace elements comprise vitamin B1, vitamin B6, NiCl2, CuSO4, CoCl2 and NaMoO4; In the initial fermentation period of 0 to 12 hours, the dissolved oxygen is controlled to be ≥40%, and the rotation speed is maintained at a low level of 30 to 200 rpm. In the middle fermentation period after 12 hours, when the dissolved oxygen drops below 40%, the ventilation volume is gradually increased to 3 to 4 VVM, and then the stirring speed is linked to maintain the dissolved oxygen at 18 to 23%.

2. The process for producing ectoine by efficient fermentation according to claim 1, characterized in that: In the S1, the plate culture is to inoculate the strain into LB solid culture medium by streaking, and culture it at 32-40°C for 16-24 hours; the first-level seed culture is to pick the bacteria cultured on the plate, inoculate it into liquid LB culture medium, and culture it at 32-40°C for 12-20 hours to obtain the first-level seed liquid; the second-level seed culture is to inoculate the first-level seed liquid into the seed tank culture medium with an inoculation amount of 2-10%, and ferment and culture it at 32-40°C for 6-10 hours to obtain the second-level seed liquid.

3. The process for producing ectoine by efficient fermentation according to claim 2, characterized in that: The seed tank culture medium comprises 25 g / L glucose, 5 g / L yeast extract powder, 4 g / L peptone, 2 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, 0.01 g / L ferrous sulfate, and 0.01 g / L manganese sulfate, and ampicillin with a final concentration of 100 mg / L and streptomycin with a final concentration of 50 mg / L are added at the beginning of fermentation.

4. The process for producing ectoine by efficient fermentation according to claim 3, characterized in that: The secondary seed culture is heavy, the inoculation amount of the primary seed liquid is 10% (v / v), the ventilation volume is 1VVM, the stirring speed is 300-600rpm, the dissolved oxygen is controlled at 20%, the fermentation culture is carried out at 37°C, the pH is controlled at 7.0±0.2 throughout the process with ammonia water, and the culture is carried out for 6-8h.

5. The process for producing ectoine by efficient fermentation according to claim 1, characterized in that: In the S2, the contents of the components in the fermentation medium are 20-50 g / L of glucose, 6-12 g / L of ammonium sulfate, 3-8 g / L of ammonium dihydrogen phosphate, 2-5 g / L of citric acid, 3-8 g / L of potassium sulfate, 2-5 g / L of magnesium sulfate, 0.2-1 g / L of ferrous sulfate, 0.2-0.8 g / L of manganese sulfate, 10 mg / L of vitamin B12, 10 mg / L of vitamin B6, 1-5 mg / L of NiCl2, 0.5-3 mg / L of CuSO4, 0.01-0.1 mg / L of CoCl2, and 0.01-0.1 mg / L of NaMoO4. When fermentation begins, ampicillin and streptomycin are added to a final concentration of 50-100 mg / L and 50-100 mg / L respectively.

6. The process for producing ectoine by efficient fermentation according to claim 5, characterized in that: The contents of the components in the fermentation medium are 20 g / L glucose, 9 g / L ammonium sulfate, 4 g / L ammonium dihydrogen phosphate, 3 g / L citric acid, 4 g / L potassium sulfate, 2 g / L magnesium sulfate, 0.6 g / L ferrous sulfate, 0.3 g / L manganese sulfate, 3.2 mg / L vitamin B1, 3 mg / L vitamin B6, 2 mg / L NiCl2, 2 mg / L CuSO4, 0.04 mg / L CoCl2, and 0.04 mg / L NaMoO4. Ampicillin at a final concentration of 100 mg / L and streptomycin at 50 mg / L are added at the start of fermentation.

7. The process for producing ectoine by efficient fermentation according to claim 1, characterized in that: In the S2, during the fermentation process, the inoculation amount of the secondary seed liquid is 2-10% (v / v), the fermentation temperature is 32-40°C, and the pH is controlled at 7.0±0.2 with ammonia water throughout the process. When the sugar consumption in the fermentation tank drops below 2 g / L, glucose is added to maintain the glucose concentration in the fermentation tank at 0-5 g / L. IPTG is added when OD600 is ≥7-9.

8. The process for producing ectoine by efficient fermentation according to claim 1, characterized in that: Said S2 also includes a process amplification step, during which the process amplification is carried out according to the matching principle of volume oxygen transfer coefficient and stirring shear force.

9. The process for producing ectoine by efficient fermentation according to claim 1, characterized in that: In a 5L fermentation tank, the stirring speed is 200-300rpm at the initial stage of fermentation. In the middle stage of fermentation, the ventilation volume is gradually increased to 4VVM, and the stirring is set at 300-800rpm in association with dissolved oxygen to control the dissolved oxygen ≥20%; in a 50L fermentation tank, the stirring speed is 150-200rpm at the initial stage of fermentation. In the middle stage of fermentation, the ventilation volume is gradually increased to 3.5VVM, and the stirring is set at 200-600rpm in association with dissolved oxygen to control the dissolved oxygen ≥20%; in a 500L fermentation tank, the stirring speed is 100-150rpm at the initial stage of fermentation. In the middle stage of fermentation, the ventilation volume is gradually increased to 3.5VVM, and the stirring is set at 150-350rpm in association with dissolved oxygen to control the dissolved oxygen ≥20%; in a 5m 3 In the fermentation tank, the stirring speed is 30-60 rpm at the initial stage of fermentation. In the middle stage of fermentation, the ventilation volume is gradually increased to 3VVM, and the stirring speed is set to 60-180 rpm in conjunction with the dissolved oxygen, and the dissolved oxygen is controlled to be ≥20%.

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