Method for producing L-tryptophan through microbial fermentation

Abstract

The invention discloses a method for producing L-tryptophan through microbial fermentation, and belongs to the technical field of microbial fermentation. Escherichia coli KV-2 is used as an original strain, and the final target product L-tryptophan is obtained in a fed-batch fermentation mode. In addition, by controlling the culture temperature and the dissolved oxygen level in stages, the growth metabolism and anabolism rates of the thalli are better balanced and regulated; the highest fermentation content of the L-tryptophan finally obtained by using the technical scheme is about 63.4 g / L, which is higher than the fermentation level reported by the existing tryptophan. On one hand, the yield of the L-tryptophan produced by a microbial fermentation method can be improved, and on the other hand, guidance is provided for industrial large-scale production of the L-tryptophan.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial fermentation, in particular to a method for producing L-tryptophan by microbial fermentation. BACKGROUND

[0002] L-tryptophan belongs to aromatic amino acids, is one of the eight essential amino acids, animals and humans cannot synthesize themselves, can only be supplemented by food intake, and L-tryptophan is an important component in the growth and development, metabolism of animals and humans, so it has wide application in the fields of feed, food, medicine, agriculture and forestry. According to data statistics, at present, the annual output of L-tryptophan is more than 50,000 tons, but it is still in short supply.

[0003] In the early stage, the production method of L-tryptophan mainly includes protein hydrolysis and chemical synthesis, but due to the problems of high pollution and high cost, it is gradually replaced by microbial fermentation. In recent years, with the continuous development of synthetic biology, biotechnology and other biological sciences, microbial fermentation has become the main way of L-tryptophan production.

[0004] Escherichia coli is the main microorganism used for microbial fermentation production of L-tryptophan at present, with the continuous development of strain modification technology, the source problem of high-efficiency production of L-tryptophan has been well solved; along with it, the stable performance of excellent strains puts forward higher requirements for reasonable medium composition and precise fermentation control technology. SUMMARY

[0005] The purpose of the present application is to provide a method for producing L-tryptophan by microbial fermentation, in order to solve the problems existing in the prior art. According to the characteristics of the metabolic pathway of intracellular synthesis of L-tryptophan in Escherichia coli and the theory that temperature significantly affects enzyme activity, in the fermentation process, the culture temperature is set to 37℃ in the early stage, which is beneficial to the rapid growth of the bacteria, and the culture temperature is set to 34℃ after 24h, which is more conducive to the efficient promotion of tryptophan synthesis by enhancing the expression or activity of related enzymes in the tryptophan synthesis pathway. In addition, the composition of the fermentation medium is screened, and a certain amount of phenylalanine and tyrosine mixture is added during the fermentation process. By selecting the above optimized control conditions, the titer and yield of tryptophan are significantly improved, and the above optimized culture conditions are more conducive to industrialization, and have good industrial application prospect.

[0006] To achieve the above purpose, the present application provides the following scheme:

[0007] The application provides a method for producing L-tryptophan by microbial fermentation, which comprises inoculating seed liquid of L-tryptophan-producing Escherichia coli into a fermentation medium, and fermenting for 24-48 hours under the condition that the pH is 6-8, and feeding the medium during the fermentation process, and controlling the fermentation temperature in stages, wherein the fermentation temperature is controlled to be 35-39 DEG C in the 0-24 hour stage, and the fermentation temperature is controlled to be 33-35 DEG C in the 24-48 hour stage, and the dissolved oxygen and OUR are controlled in stages, wherein the OUR is controlled to be 0-400 mmol / L / h and the dissolved oxygen is controlled to be 20-80% in the 0-20 hour stage, and the OUR is controlled to be 50-170 mmol / L / h and the dissolved oxygen is controlled to be 10-40% in the 20-48 hour stage.

[0008] The L-tryptophan-producing Escherichia coli is Escherichia coli KV-2, and the preservation number is CGMCC NO.34654.

[0009] Preferably, the feeding medium comprises 600-700 g / L glucose, 1-3 g / L magnesium sulfate heptahydrate, 0.1-0.9 g / L phenylalanine and 0.1-0.9 g / L tyrosine.

[0010] Preferably, the method for controlling the fermentation temperature in stages is that the fermentation temperature is controlled to be 37 DEG C in the 0-24 hour stage, and the fermentation temperature is controlled to be 34 DEG C in the 24-48 hour stage.

[0011] Preferably, the method for controlling the dissolved oxygen and OUR in stages is that the OUR is controlled to be 0-200 mmol / L / h and the dissolved oxygen is controlled to be 40-60% in the 0-20 hour stage, and the OUR is controlled to be 80-140 mmol / L / h and the dissolved oxygen is controlled to be 20-30% in the 20-48 hour stage.

[0012] Preferably, the fermentation is carried out for 48 hours under the condition that the pH is 6.5-7.2.

[0013] Preferably, the seed liquid of the L-tryptophan-producing Escherichia coli is obtained by culturing seed medium comprising 10-30 g / L proteose peptone, 40-60 g / L glucose, 8-12 g / L potassium dihydrogen phosphate, 20-30 g / L dipotassium hydrogen phosphate, 8-12 g / L sodium chloride and 6-8 g / L yeast powder.

[0014] Preferably, the components of the fermentation medium are: glucose 2-10 g / L, cane molasses 1-3 g / L, yeast powder 1-5 g / L, KH2PO4 1-3 g / L, (NH4)2HPO4 1-3 g / L, MgSO4·7H2O 0.5-2.5 g / L, betaine 0.1-0.9 g / L, vitamin B1 1-9 mg / L, biotin 0.1-0.3 mg / L, ferrous sulfate heptahydrate 50-90 mg / L, anhydrous copper sulfate 0.5-1.5 mg / L, polyether antifoam agent 0.1-0.2%.

[0015] Preferably, the tank pressure during the fermentation process is 0.04-0.06 MPa.

[0016] Preferably, the aeration rate during the fermentation process is 1.0-4.0 VVM.

[0017] Preferably, the stirring speed during the fermentation process is 100-800 rpm

[0018] The present application discloses the following technical effects:

[0019] The present application takes Escherichia coli KV-2 as the starting strain, and obtains the final target product L-tryptophan through fed-batch fermentation. In the fermentation process, the aromatic amino acid synthesis pathway downstream of the shikimic acid is intervened, a mixture of phenylalanine and tyrosine is supplemented, the synthesis pathway of phenylalanine and tyrosine is feedback inhibited, more shikimic acid flows to the synthesis of tryptophan; in addition, the culture temperature and the dissolved oxygen level are controlled in stages, so that the growth metabolism and the synthesis metabolism rate of the bacteria are better balanced and regulated; the highest fermentation content of L-tryptophan obtained by using the technical scheme of the present application is about 63.4 g / L, which is higher than the reported fermentation level of tryptophan. The present application can improve the yield of L-tryptophan produced by microbial fermentation on the one hand, and provide guidance for the industrialization and scale production of L-tryptophan on the other hand.

[0020] Preservation information: Escherichia coli KV-2, classified as Escherichia coli, preserved on May 23, 2025, preserved by China General Microbiological Culture Collection Center (CGMCC), located at No. 1, Beichen West Road, Chaoyang District, Beijing, with the preservation number of CGMCC NO. 34654. DETAILED DESCRIPTION

[0021] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.

[0022] 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. Additionally, for a range of values of, for example, concentrations, amounts, and other

[0023] Unless otherwise defined, 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 methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference is not an admission that it is prior art with respect to the present application.

[0024] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. 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 are illustrative only.

[0025] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", and the like are open-ended terms that are intended to mean including but not limited to.

[0026] The L-tryptophan-producing Escherichia coli used in the present application is Escherichia coli KV-2.

[0027] Example 1: Method for producing L-tryptophan by microbial fermentation

[0028] The seed liquid culture medium is composed of 20 g / L of proteose peptone, 50 g / L of glucose, 10 g / L of potassium dihydrogen phosphate, 25 g / L of dipotassium hydrogen phosphate, 10 g / L of sodium chloride, 7 g / L of yeast powder, and 1 L of water.

[0029] The fermentation culture medium is composed of 6 g / L of glucose, 2 g / L of cane molasses, 3 g / L of yeast powder, 2 g / L of KH2PO4, 2 g / L of (NH4)2HPO4, 1.5 g / L of MgSO4·7H2O, 0.5 g / L of betaine, 5 mg / L of vitamin B1, 0.2 mg / L of biotin, 70 mg / L of ferrous sulfate heptahydrate, 1 mg / L of anhydrous copper sulfate, 1.5 g / L of polyether antifoam agent, and 1 L of water.

[0030] The feed medium is composed of 650 g / L glucose, 2 g / L magnesium sulfate heptahydrate, 0.5 g / L phenylalanine, 0.5 g / L tyrosine, 1 L of water, and sterilized and cooled to mix uniformly.

[0031] The method for producing L-tryptophan by microbial fermentation comprises the following steps:

[0032] Seed liquid culture method: the seed liquid in the seed tank is cultured at a ventilation rate of 1.0-2.0vvm, a stirring speed of 300-600rpm, a tank temperature of 30-35℃, a pH of 6.5-7.2, and an OD growth of 10-15 for 6-10h.

[0033] The cultured seed liquid is inoculated into the fermentation medium at an inoculation amount of 15% and placed in the fermentation tank for fermentation for 48h. The tank pressure is 0.04-0.06MPa, the ventilation rate is 1.0-4.0vvm, the stirring speed is 100-800rpm, ammonia water is supplied to maintain the pH at 6.5-7.2 during the fermentation process, and the reducing sugar content is controlled to be not higher than 0.5%. When the fermentation is carried out for 6h, the glucose in the initial medium is consumed, at which time the feeding of the feed medium is started. The addition amount is gradually increased as the fermentation period progresses. At this time, the addition amount is 10g / h, and the maximum addition amount is about 35-40g / h at 12h. Then, the rate is maintained until the end of the fermentation. The dissolved oxygen level and the fermentation culture temperature are controlled in stages. The fermentation culture temperature is controlled at 37℃ for 0-24h, and at 34℃ for 24h-48h. The OUR is controlled at 0-200mmol / L / h for 0-20h, and the dissolved oxygen is controlled at 40-60%. The OUR is controlled at 80-140mmol / L / h for 20h-48h, and the dissolved oxygen is controlled at 20-30%. After the fermentation is completed, the L-tryptophan fermentation broth is obtained.

[0034] Example 2: Effect of temperature control during fermentation on L-tryptophan synthesis

[0035] On the basis of Example 1, the effect of optimized temperature control conditions on L-tryptophan synthesis is studied. The difference from Example 1 is that three groups of temperature control conditions are set during the fermentation process to screen the best temperature control conditions.

[0036] Group 1: the fermentation temperature is controlled at 37℃ throughout the fermentation process.

[0037] Group 2: the fermentation temperature is controlled at 37℃ for 0-24h, and at 34℃ for 24-48h.

[0038] Group 3: the fermentation temperature is controlled at 34℃ throughout the fermentation process.

[0039] Under the experimental conditions, the temperature control conditions during the culture process are optimized, and the experimental results are shown in Table 1.

[0040] Table 1 L-tryptophan production amount at different fermentation temperatures

[0041]

[0042] The results in Table 1 show that the stepwise control of culture temperature is more suitable for the fermentation culture of tryptophan. When the whole process is controlled at 34℃, the L-tryptophan content produced by fermentation is 48.7g / L, and the L-tryptophan production capacity is much lower than that of the whole process fermentation at 37℃; when the fermentation is controlled at 37℃ for 0-24h and at 34℃ for 24-48h, the L-tryptophan content produced by fermentation is as high as 54.4g / L, which is much higher than that of the other two groups. The highest OD 600 of the bacterial concentration (OD 600 ) of group 3 is 57.5, which is much lower than the highest OD 600 under the other two culture conditions, indicating that the condition of 37℃ is conducive to the growth of the strain, and a certain concentration of biomass is the basis for the synthesis of tryptophan; from the results of group 1 and group 2, when the bacterial concentration reaches a certain value, the bacterial cells begin to rapidly synthesize tryptophan product, and controlling the temperature at 34℃ at this time is more conducive to the accumulation of the product, the main reason being that the enzyme activity is higher under this temperature condition. It can be seen that the final fermentation temperature for the fermentation of L-tryptophan in the present application is to control the fermentation temperature at 37℃ for 0-24h and at 34℃ for 24-48h.

[0043] Example 3 Effect of different components of the feeding medium on the synthesis of L-tryptophan

[0044] On the basis of the optimization results of Example 2, the effect of further optimizing the components of the feeding medium on the synthesis of L-tryptophan was studied, and the difference from Example 2 is only that three groups of different feeding media were set:

[0045] Feeding medium 1: 650g / L glucose, 2g / L magnesium sulfate heptahydrate, 1L water, mixed uniformly after sterilization and cooling;

[0046] Feeding medium 2: 650g / L glucose, 2g / L magnesium sulfate heptahydrate, 0.5g / L phenylalanine, 0.5g / L tyrosine, 1L water, mixed uniformly after sterilization and cooling;

[0047] Feeding medium 3: 650g / L glucose, 2g / L magnesium sulfate heptahydrate, 2g / L phenylalanine, 2g / L tyrosine, 1L water, mixed uniformly after sterilization and cooling.

[0048] Feeding medium 4: 650g / L glucose, 2g / L magnesium sulfate heptahydrate, 0.5g / L phenylalanine, 1L water, mixed uniformly after sterilization and cooling;

[0049] Feed medium 5: 650 g / L glucose, 2 g / L magnesium sulfate heptahydrate, 0.5 g / L tyrosine, water 1 L, sterilized and cooled down to mix.

[0050] The effects of different feed medium components on the synthesis of L-tryptophan were optimized under the experimental conditions, and the experimental results are shown in Table 2.

[0051] Table 2: L-tryptophan production amount under different feed medium components

[0052]

[0053] The results in Table 2 show that when phenylalanine and tyrosine are added to the feed medium, the content of L-tryptophan produced by microbial fermentation is higher than that without the addition of phenylalanine and tyrosine. According to the intracellular metabolic pathway analysis of Escherichia coli, tryptophan, phenylalanine and tyrosine are synthesized from chorismic acid through different metabolic pathways. When phenylalanine and tyrosine are gradually added during the fermentation process, the related enzymes for synthesizing phenylalanine and tyrosine from chorismic acid are inhibited, and more chorismic acid flows into the pathway for synthesizing tryptophan. However, different effects are produced by adding different concentrations of phenylalanine and tyrosine, and high concentrations of phenylalanine and tyrosine can inhibit the growth of the bacteria, thereby affecting the synthesis of tryptophan. In addition, the effect of separately adding phenylalanine or tyrosine is not as good as that of adding a mixture of phenylalanine and tyrosine. Therefore, the feed medium 2 in this example is finally selected as the feed medium of the present application.

[0054] Example 4: Effect of OUR control on L-tryptophan synthesis during fermentation

[0055] Based on the optimization results of Example 3, the effect of OUR control conditions on the synthesis of L-tryptophan was further optimized. The difference from Example 3 is that two different OUR control schemes were set for comparison. Real-time detection was performed by a tail gas mass spectrometer, and OUR was controlled by adjusting air flow, speed and tank pressure in different stages during the fermentation process, in order to investigate the effects of different OUR control strategies on the microbial fermentation production of L-tryptophan. The two control schemes are as follows:

[0056] Scheme one: during 0-20h fermentation, OUR is controlled at 0-200mmol / L / h, dissolved oxygen is controlled at 40-60%, during 20h-48h fermentation, OUR is controlled at 80-140mmol / L / h, dissolved oxygen is controlled at 20-30%; during 0-20h fermentation, air flow, rotation speed, tank pressure set value are respectively 20-35L / min, 300-600rpm and 0.04-0.06MPa; during 20-48h fermentation, air flow, rotation speed, tank pressure set value are respectively 20-30L / min, 300-500rpm and 0.04-0.06MPa.

[0057] Scheme two: during 0-20h fermentation, OUR is controlled at 0-200mmol / L / h, during 20h-48h fermentation, OUR is controlled at 160-200mmol / L / h; dissolved oxygen is controlled at 40-60% during the whole fermentation process. During 0-20h fermentation, air flow, rotation speed, tank pressure set value are respectively 20-35L / min, 300-600rpm and 0.04-0.06MPa; during 20-48h fermentation, air flow, rotation speed, tank pressure set value are respectively 30-35L / min, 500-600rpm and 0.04-0.06MPa.

[0058] The influence of different OUR control conditions on L-tryptophan synthesis is optimized under the experimental conditions, and the experimental results are shown in Table 3:

[0059] Table 3: L-tryptophan yield under different OUR control conditions

[0060]

[0061] The results in Table 3 show that: during the fermentation process, the difference between the OUR control level and the dissolved oxygen control level can significantly affect the level of tryptophan produced by the bacteria. High dissolved oxygen and OUR levels are controlled in the early stage to promote rapid growth of the bacteria, and the dissolved oxygen is reduced in the later stage to promote more carbon metabolic flow to HMP (pentose phosphate) and tryptophan synthesis pathway rather than TCA cycle, thereby promoting rapid accumulation of tryptophan. Therefore, scheme one is finally selected as the most suitable OUR control condition of the present application.

[0062] The above-described embodiments are only 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 of the present application as defined by the claims.

Claims

1. A method for producing L-tryptophan by microbial fermentation, characterized by, The method is inoculating the seed liquid of the L-tryptophan-producing Escherichia coli into a fermentation medium, fermenting for 24-48 h under the condition that the pH is 6-8, supplementing the medium during the fermentation process, and controlling the fermentation temperature in stages, the fermentation temperature is controlled at 35-39 DEG C in the 0-24 h stage, the fermentation temperature is controlled at 33-35 DEG C in the 24-48 h stage, the dissolved oxygen and OUR are controlled in stages, the OUR is controlled at 0-400 mmol / L / h and the dissolved oxygen is controlled at 20-80% in the 0-20 h stage, the OUR is controlled at 50-170 mmol / L / h and the dissolved oxygen is controlled at 10-40% in the 20-48 h stage. The L-tryptophan-producing Escherichia coli is Escherichia coli KV-2, and the preservation number is CGMCC NO.34654.

2. The method of claim 1, wherein, The supplement medium is composed of 600-700 g / L glucose, 1-3 g / L magnesium sulfate heptahydrate, 0.1-0.9 g / L phenylalanine and 0.1-0.9 g / L tyrosine.

3. The method of claim 1, wherein, The method for controlling the fermentation temperature in stages is controlling the fermentation temperature at 37 DEG C in the 0-24 h stage and controlling the fermentation temperature at 34 DEG C in the 24-48 h stage.

4. The method of claim 1, wherein, The method for controlling the dissolved oxygen and OUR in stages is controlling the OUR at 0-200 mmol / L / h and the dissolved oxygen at 40-60% in the 0-20 h stage, and controlling the OUR at 80-140 mmol / L / h and the dissolved oxygen at 20-30% in the 20-48 h stage.

5. The method of claim 1, wherein, The fermentation is carried out for 48 h under the condition that the fermentation pH is 6.5-7.

2.

6. The method of claim 1, wherein, The seed liquid of the L-tryptophan-producing Escherichia coli is obtained by culturing a seed medium composed of 10-30 g / L peptone, 40-60 g / L glucose, 8-12 g / L potassium dihydrogen phosphate, 20-30 g / L dipotassium hydrogen phosphate, 8-12 g / L sodium chloride and 6-8 g / L yeast powder.

7. The method of claim 1, wherein, The fermentation medium is composed of 2-10 g / L glucose, 1-3 g / L cane molasses, 1-5 g / L yeast powder, 1-3 g / L KH2PO4, 1-3 g / L (NH4)2HPO4, 0.5-2.5 g / L MgSO4·7H2O, 0.1-0.9 g / L betaine, 1-9 mg / L vitamin B1, 0.1-0.3 mg / L biotin, 50-90 mg / L ferrous sulfate heptahydrate, 0.5-1.5 mg / L anhydrous copper sulfate and 0.1-0.2% polyether antifoam agent.

8. The method of claim 1, wherein, The tank pressure in the fermentation process is 0.04-0.06 MPa.

9. The method of claim 1, wherein, The aeration rate in the fermentation process is 1.0-4.0 vmv.

10. The method of claim 1, wherein, The stirring speed in the fermentation process is 100-800 rpm.

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