Process of using biological fermentation to produce l-histidine

Abstract

A process of using biological fermentation to produce L-histidine, comprising the following steps: introducing an Escherichia coli seed solution into a fermentation tank containing a fermentation culture medium, the culture temperature ranging from 35°C to 37°C; adding a glucose solution containing adenosine monophosphate in a fed-batch manner to maintain the content of reducing sugar at 46 g / L, and stopping the fed-batch addition of the glucose solution 4 h before the end of fermentation; and adding ammonia water in the fed-batch manner to maintain the pH value in the fermentation tank at 6.9 to 7.1, adding a defoamer in the fed-batch manner to perform defoaming, the fermentation time being 40 h to 50 h, and obtaining an L-histidine fermentation broth. The process has high fermentation efficiency and low production costs, and is suitable for industrial production.

Description

A process for producing L-histidine by biological fermentation TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological fermentation, and particularly relates to a process for producing L-histidine by biological fermentation. BACKGROUND

[0002] L-histidine (L-Histidine) is a basic amino acid with an imidazole nucleus in the molecule, and its chemical name is alpha-amino beta-imidazolyl propionic acid. L-histidine is a semi-essential amino acid for the human body, and is widely used in the pharmaceutical, food and feed industries, and also used in the production of tobacco products and cosmetics. The production of L-histidine is mainly based on protein hydrolysis, but this method has problems such as complicated operation steps, serious environmental pollution and high production cost. In recent years, microbial fermentation method has been successfully applied to the industrial production of many amino acids, and has the advantages of short fermentation period, low cost, mild reaction conditions, easy process control and high yield, and is also the preferred method for industrial production of L-histidine. The production of L-histidine by microbial fermentation has broad prospects, and its yield has a lot of room for improvement.

[0003] The existing technology for biological fermentation of histidine mainly includes screening of strains, optimization of culture process and addition of stimulating factors.

[0004] Document 1: Biotechnology, 2008, Zhao Jianyun et al., found that the addition of calcium gluconate in the fermentation medium can increase the flux of HMP pathway, which is beneficial to the synthesis of histidine in Corynebacterium glutamicum CLW0911.

[0005] Document 2: Microbial Technology Communication, 2009, Zhu Wenze et al., provides a method for improving the yield of L-histidine fermentation process, according to the principle of adjusting the distribution of cell metabolic flow, the method of adding citric acid or its salt in the culture medium is used to effectively improve the yield of L-histidine fermentation in Corynebacterium glutamicum TL1105.

[0006] Document 3: CN112481325A, the acid production mechanism of different strains and the tolerance to stimulating factors are quite different, but it has no reference significance. Although the existing technology has made a lot of research on the acid production mechanism of glutamic acid, for example, adding appropriate amount of sodium malate in the fermentation medium can improve the amount of histidine produced by Corynebacterium glutamicum, but sodium malate has no obvious stimulating effect on Serratia marcescens ATCC31026; and the ability of Serratia marcescens to produce histidine is significantly improved by adding calcium acetate and malonic acid.

[0007] The pathway of energy and material metabolism of prokaryotic cells is a very complex network, and it is still not clear how the changes in genes in the metabolic pathway will affect the energy and material flow. Due to the great difference in metabolic pathways of different strains, it is necessary to test different fermentation strains through experimental means, and how to improve the fermentation performance of a specific strain is a technical problem to be solved. SUMMARY

[0008] The application provides a process for producing L-histidine by biological fermentation. The process has high fermentation efficiency and low production cost, and is suitable for large-scale industrial production.

[0009] Aromatic or heterocyclic amino acids including histidine, tryptophan, valine and phenylalanine are all related to the HMP pathway, and erythrose-4-phosphate in the pathway is a key raw material for synthesizing aromatic and heterocyclic amino acids (phenylalanine, tyrosine, tryptophan and histidine). The tryptophan production capacity of Escherichia coli TR2023D-1 is greatly increased compared with the original strain, indicating that the metabolic flow of the HMP pathway is increased, and the synthesis capacity of other amino acids related to the HMP pathway, including histidine, is also likely to be up-regulated, and the strain belongs to a phenylalanine and tyrosine deficient strain, and there is no need to regulate the synthesis of phenylalanine and tyrosine.

[0010] Based on the above theory, the histidine production performance of Escherichia coli TR2023D-1 for producing tryptophan is studied and optimized, and the strain is preserved in the China General Microbiological Culture Collection Center, with a preservation number of CGMCC No. 27549 (CN117778225A, already preserved).

[0011] The application is realized by the following technical solutions.

[0012] The application provides a process for producing L-histidine by biological fermentation, which includes the following steps:

[0013] The seed liquid of Escherichia coli is inoculated into a fermentation tank containing a fermentation medium, the culture temperature is 35-37°C, the content of reducing sugar is maintained at 4-6 g / L by adding a glucose solution containing adenosine monophosphate, and the glucose solution is stopped flowing 4 hours before the end of fermentation; the pH in the fermentation tank is maintained at 6.9-7.1 by adding ammonia water, and a defoaming agent is added to defoam; the fermentation time is 40-50 h, and the L-histidine fermentation liquor can be obtained.

[0014] The fermentation medium components are: glucose 50-80 g / L, corn syrup 5-20 g / L, ammonium sulfate 5-10 g / L, potassium dihydrogen phosphate 4-6 g / L, citric acid 1-3 g / L, magnesium sulfate heptahydrate 1-2 g / L, sodium malate 0.2-0.5 g / L, ferrous sulfate heptahydrate 50-200 mg / L, indole propionic acid 5-20 mg / L, thiamine 0.4-0.6 mg / L, and biotin 0.1-0.3 mg / L.

[0015] As any preferred embodiment, the fermentation medium components are: glucose 60 g / L, corn syrup 10 g / L, ammonium sulfate 8 g / L, potassium dihydrogen phosphate 5 g / L, citric acid 2 g / L, magnesium sulfate heptahydrate 1.5 g / L, sodium malate 0.4 g / L, ferrous sulfate heptahydrate 100 mg / L, indole propionic acid 10 mg / L, thiamine 0.5 mg / L, and biotin 0.2 mg / L; the pH is controlled at 7.0.

[0016] As any preferred embodiment, the fermentation medium components are: glucose 60 g / L, corn syrup 10 g / L, ammonium sulfate 8 g / L, potassium dihydrogen phosphate 5 g / L, citric acid 2 g / L, magnesium sulfate heptahydrate 1.5 g / L, sodium malate 0.3 g / L, ferrous sulfate heptahydrate 100 mg / L, indole propionic acid 15 mg / L, thiamine 0.5 mg / L, and biotin 0.2 mg / L; the pH is controlled at 7.0.

[0017] As any preferred embodiment, the glucose solution components are: glucose 500 g / L + adenosine monophosphate 15 g / L.

[0018] As any preferred embodiment, the glucose solution components are: glucose 600 g / L + adenosine monophosphate 20 g / L.

[0019] As any preferred embodiment, the Escherichia coli is Escherichia coli TR2023D-1.

[0020] As any preferred embodiment, the inoculation amount of the Escherichia coli seed liquid is 5-10%.

[0021] As any preferred embodiment, the fermentation time is 48 h.

[0022] As any preferred embodiment, the reducing sugar content is 5 g / L.

[0023] As any preferred embodiment, the fermentation temperature is 36℃.

[0024] The beneficial effects obtained by the present application mainly include, but are not limited to, the following aspects:

[0025] Metabolic network is a very complex multi-enzyme synchronous catalytic reaction system, only changing one step of the rate-limiting reaction, the influence on the synthesis of product is often not great, so to optimize a metabolic network, the main nodes that have influence on the product must be adjusted at the same time, so that the metabolic flux is redistributed to benefit the accumulation of the target product. In this application, the performance of E. coli TR2023D-1 in producing histidine is greatly improved by optimizing the fermentation medium and stimulating factors, and the synthesis efficiency of tryptophan in the HMP pathway is weakened accordingly. Malate can inhibit the activity of pyruvate kinase, reduce the accumulation of pyruvate, and cause the accumulation of phosphoenolpyruvate, which is an inhibitor of phosphofructokinase, the rate-limiting enzyme of the EMP pathway. Therefore, the accumulation of phosphoenolpyruvate reduces the activity of phosphofructokinase, thereby reducing the flux of the EMP pathway and relieving the metabolic overflow between the EMP and TCA pathways. At the same time, the total glucose flux does not change, so more glucose enters the HMP synthesis pathway, and the yield of L-histidine is correspondingly improved. Anthranilate synthase is a key enzyme in the synthesis of tryptophan, and indolepropionic acid, as an allosteric inhibitor of anthranilate synthase, can increase the metabolic flow in the direction of histidine synthesis by weakening the activity of the enzyme and reducing the metabolic flux of tryptophan in the HMP pathway. Adenosine monophosphate (AMP) is a direct precursor of ATP synthesis, which can improve the synthesis efficiency of histidine by synthesizing ATP or activating key enzymes in the synthesis of histidine. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1: Effect of indolepropionic acid on the fermentation of E. coli TR2023D-1 to produce histidine.

[0027] Figure 2: Effect of adenosine monophosphate on the fermentation of E. coli TR2023D-1 to produce histidine. DETAILED DESCRIPTION

[0028] Those skilled in the art can improve the process parameters according to the content herein. In particular, it should be pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in this application. The products and methods of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the products and methods described herein without departing from the content, spirit and scope of the present application, to realize and apply the technology of the present application. In order to further understand the present application, the present application will be described in detail in conjunction with the examples below.

[0029] Example 1

[0030] Histidine fermentation process

[0031] The E. coli TR2023D-1 was activated, inoculated into LB liquid medium, and cultured at 37°C for 36 h to obtain a primary seed liquid;

[0032] The primary seed liquid was inoculated into a secondary seed medium at a 15% inoculation amount, and cultured at 37°C until the OD 600 value was about 8 to obtain a secondary seed liquid;

[0033] The secondary seed medium comprised sucrose 30 g / L, potassium dihydrogen phosphate 5 g / L, dipotassium hydrogen phosphate 5 g / L, yeast extract 5 g / L, magnesium sulfate heptahydrate 1 g / L, ferrous sulfate heptahydrate 50 mg / L, and biotin 0.1 mg / L, and the pH was controlled at 7.0.

[0034] The secondary seed liquid was inoculated into an automatic control fermentation tank containing a fermentation medium at an 8% inoculation amount, the culture temperature was 36°C, the dissolved oxygen content was 20%, the glucose solution (comprising glucose 500 g / L and adenosine monophosphate 15 g / L) was added to maintain the reducing sugar content at 5 g / L, and the glucose solution was stopped adding 4 h before the end of fermentation; the 25% ammonia water was added to maintain the pH in the fermentation tank at 7.0, and the defoaming agent was added to defoam; the fermentation time was 48 h to obtain the L-histidine fermentation liquid;

[0035] The fermentation medium comprised glucose 60 g / L, corn syrup 10 g / L, ammonium sulfate 8 g / L, potassium dihydrogen phosphate 5 g / L, citric acid 2 g / L, magnesium sulfate heptahydrate 1.5 g / L, sodium malate 0.4 g / L, ferrous sulfate heptahydrate 100 mg / L, indole propionic acid 10 mg / L, thiamine 0.5 mg / L, and biotin 0.2 mg / L, and the pH was controlled at 7.0.

[0036] Example 2

[0037] Histidine fermentation process

[0038] The E. coli TR2023D-1 was activated, inoculated into LB liquid medium, and cultured at 37°C for 36 h to obtain a primary seed liquid;

[0039] The primary seed liquid was inoculated into a secondary seed medium at a 15% inoculation amount, and cultured at 37°C until the OD 600 value was about 8 to obtain a secondary seed liquid;

[0040] The secondary seed medium comprised sucrose 30 g / L, potassium dihydrogen phosphate 5 g / L, dipotassium hydrogen phosphate 5 g / L, yeast extract 5 g / L, magnesium sulfate heptahydrate 1 g / L, ferrous sulfate heptahydrate 50 mg / L, and biotin 0.1 mg / L, and the pH was controlled at 7.0.

[0041] The secondary seed liquid was inoculated into the automatic control fermentation tank containing the fermentation medium at an inoculation amount of 8%, the culture temperature was 36°C, the dissolved oxygen content was 20%, the reducing sugar content was maintained at 5g / L by adding glucose solution (components: glucose 600g / L + adenosine monophosphate 20g / L), and the glucose solution was stopped adding 4h before the end of fermentation; the pH in the fermentation tank was maintained at 7.0 by adding 25% ammonia water, and the defoamer was added to defoam; the fermentation time was 50h, and the L-histidine fermentation liquor was obtained;

[0042] The fermentation medium components were: glucose 60g / L, corn syrup 10g / L, ammonium sulfate 8g / L, potassium dihydrogen phosphate 5g / L, citric acid 2g / L, magnesium sulfate heptahydrate 1.5g / L, sodium malate 0.3g / L, ferrous sulfate heptahydrate 100mg / L, indole propionic acid 15mg / L, thiamine 0.5mg / L, and biotin 0.2mg / L; the pH was controlled at 7.0.

[0043] Comparative Example 1

[0044] Histidine fermentation process

[0045] The Escherichia coli TR2023D-1 was activated and inoculated into LB liquid medium, and cultured at 37°C for 36h to obtain the primary seed liquid;

[0046] The primary seed liquid was inoculated into the secondary seed culture medium at an inoculation amount of 15%, and cultured at 37°C until the OD600 value was about 8 to obtain the secondary seed liquid;

[0047] The secondary seed culture medium components were: sucrose 30g / L, potassium dihydrogen phosphate 5g / L, dipotassium hydrogen phosphate 5g / L, yeast extract 5g / L, magnesium sulfate heptahydrate 1g / L, ferrous sulfate heptahydrate 50mg / L, and biotin 0.1mg / L; the pH was controlled at 7.0.

[0048] The secondary seed liquid was inoculated into the automatic control fermentation tank containing the fermentation medium at an inoculation amount of 8%, the culture temperature was 36°C, the dissolved oxygen content was 20%, the reducing sugar content was maintained at 5g / L by adding glucose solution (components: glucose 500g / L), and the glucose solution was stopped adding 4h before the end of fermentation; the pH in the fermentation tank was maintained at 7.0 by adding 25% ammonia water, and the defoamer was added to defoam; the fermentation time was 48h, and the L-histidine fermentation liquor was obtained;

[0049] The fermentation medium components were: glucose 60g / L, corn syrup 10g / L, ammonium sulfate 8g / L, potassium dihydrogen phosphate 5g / L, citric acid 2g / L, magnesium sulfate heptahydrate 1.5g / L, ferrous sulfate heptahydrate 100mg / L, thiamine 0.5mg / L, and biotin 0.2mg / L; the pH was controlled at 7.0.

[0050] Comparative Example 2

[0051] Histidine fermentation process

[0052] E. coli TR2023D-1 was activated, inoculated into LB liquid medium, and cultured at 37°C for 36 h with shaking to obtain a primary seed solution;

[0053] The primary seed solution was inoculated into a secondary seed culture medium at a 15% inoculation amount, and cultured at 37°C until the OD 600 value was about 8 to obtain a secondary seed solution;

[0054] The secondary seed culture medium comprised sucrose 30 g / L, potassium dihydrogen phosphate 5 g / L, dipotassium hydrogen phosphate 5 g / L, yeast extract 5 g / L, magnesium sulfate heptahydrate 1 g / L, ferrous sulfate heptahydrate 50 mg / L, and biotin 0.1 mg / L, and the pH was controlled at 7.0.

[0055] The secondary seed solution was inoculated into an automatic control fermentation tank containing a fermentation culture medium at an 8% inoculation amount, the culture temperature was 36°C, the dissolved oxygen content was 20%, the glucose solution (comprising glucose 500 g / L) was added to maintain the reducing sugar content at 5 g / L, and the glucose solution was stopped 4 h before the end of fermentation; the 25% ammonia water was added to maintain the pH in the fermentation tank at 7.0, and the defoaming agent was added to eliminate foam; the fermentation time was 48 h to obtain the L-histidine fermentation liquor;

[0056] The fermentation culture medium comprised glucose 60 g / L, corn steep liquor 10 g / L, ammonium sulfate 8 g / L, potassium dihydrogen phosphate 5 g / L, citric acid 2 g / L, magnesium sulfate heptahydrate 1.5 g / L, sodium malate 0.4 g / L, ferrous sulfate heptahydrate 100 mg / L, thiamine 0.5 mg / L, and biotin 0.2 mg / L, and the pH was controlled at 7.0.

[0057] Test 3

[0058] By optimizing the fermentation metabolic flow, the content changes of histidine and tryptophan in the fermentation liquors obtained in different examples were compared. Table 1 below shows the details.

[0059] Table 1

[0060] As shown in Table 1 above, on the basis of Comparative Example 1, the addition of sodium malate in Comparative Example 2 can improve the carbon source metabolic flow in the HMP pathway, thereby being beneficial to the synthesis of histidine and tryptophan; by weakening the tryptophan synthesis pathway and strengthening the histidine synthesis pathway, the production of tryptophan is greatly reduced in Examples 1-2, and accordingly, the production of histidine is obviously improved.

[0061] Test 4

[0062] 1. On the basis of Comparative Example 2, the effect of the amount of indolepropionic acid added to the culture medium on the production of histidine by Escherichia coli TR2023D-1 was studied. The amount of indolepropionic acid added was set at 0, 5, 10, 20, 40 and 80, a total of six concentration gradients, in units of mg / L. As shown in Figure 1, a low concentration of indolepropionic acid added can significantly improve the yield of histidine, and when the amount of indolepropionic acid added increases to 20 mg / L, the yield of histidine decreases. The possible reason is that the enzymes related to tryptophan synthesis are not weakened too much, otherwise it will lead to the decrease of the activity of Escherichia coli TR2023D-1, thereby causing the decline of the acid production capacity.

[0063] 2. The amount of indolepropionic acid added to the fermentation medium was selected as 10 mg / L, and the effect of the addition of adenosine monophosphate in the glucose nutrient solution on the production of histidine by Escherichia coli TR2023D-1 was further studied. The amount of adenosine monophosphate added was set at 0, 5, 10, 15, 20, 25 and 30, a total of seven concentration gradients, in units of g / L. As shown in Figure 2, adenosine monophosphate is positively correlated with the efficiency of Escherichia coli TR2023D-1 in producing histidine, and when the amount of adenosine monophosphate added reaches 15 g / L, the yield of histidine remains stable.

[0064] 3. The above studies show that when the amount of indolepropionic acid added to the fermentation medium is 10 mg / L and the concentration of adenosine monophosphate in the nutrient solution is 15 g / L, the amount of histidine produced by Escherichia coli TR2023D-1 is 49.8 g / L, while the yield is 20.6 g / L without the addition of indolepropionic acid and adenosine monophosphate (Comparative Example 2), the yield is 32.9 g / L with the addition of indolepropionic acid only (Figure 1), and the yield is 28.5 g / L with the addition of adenosine monophosphate only.

[0065] The present application also verified the effect of the above optimal amounts of indolepropionic acid and adenosine monophosphate on Serratia marcescens ATCC31026, and the results showed that it did not significantly improve the yield of histidine, which also verified the conclusion that different strains have different mechanisms of acid production and different tolerances to stimulators.

[0066] The above describes the specific embodiments of the present application in combination with the examples, but is not a limitation on the protection scope of the present application. Those skilled in the art should understand that these modifications or improvements made without departing from the spirit of the present application should be within the scope of protection of the present application.

Claims

1. A process for producing L-histidine by biological fermentation, comprising the following steps: fermenting Escherichia coli seed liquid in a fermenter containing a fermentation medium, the fermentation temperature being 35-37℃, the reducing sugar content being maintained at 4-6 g / L by feeding glucose solution containing adenosine monophosphate, and the glucose solution being stopped feeding 4 hours before the end of fermentation; the pH in the fermenter being maintained at 6.9-7.1 by feeding ammonia water, and defoaming by feeding defoaming agent; the fermentation time being 40-50 hours, thereby obtaining L-histidine fermentation liquor; the fermentation medium comprises 50-80 g / L of glucose, 5-20 g / L of corn steep liquor, 5-10 g / L of ammonium sulfate, 4-6 g / L of potassium dihydrogen phosphate, 1-3 g / L of citric acid, 1-2 g / L of magnesium sulfate heptahydrate, 0.2-0.5 g / L of sodium malate, 50-200 mg / L of ferrous sulfate heptahydrate, 5-20 mg / L of indole propionic acid, 0.4-0.6 mg / L of thiamine, and 0.1-0.3 mg / L of biotin; the Escherichia coli is Escherichia coli TR2023D-1, which is preserved in the China General Microbiological Culture Collection Center with the preservation number of CGMCC No. 27549.

2. The process of claim 1, wherein, the fermentation medium comprises 60 g / L of glucose, 10 g / L of corn steep liquor, 8 g / L of ammonium sulfate, 5 g / L of potassium dihydrogen phosphate, 2 g / L of citric acid, 1.5 g / L of magnesium sulfate heptahydrate, 0.4 g / L of sodium malate, 100 mg / L of ferrous sulfate heptahydrate, 10 mg / L of indole propionic acid, 0.5 mg / L of thiamine, and 0.2 mg / L of biotin; and the pH is controlled at 7.

0.

3. The process of claim 1, wherein, the fermentation medium comprises 60 g / L of glucose, 10 g / L of corn steep liquor, 8 g / L of ammonium sulfate, 5 g / L of potassium dihydrogen phosphate, 2 g / L of citric acid, 1.5 g / L of magnesium sulfate heptahydrate, 0.3 g / L of sodium malate, 100 mg / L of ferrous sulfate heptahydrate, 15 mg / L of indole propionic acid, 0.5 mg / L of thiamine, and 0.2 mg / L of biotin; and the pH is controlled at 7.

0.

4. The process of claim 1, wherein, the glucose solution comprises 500 g / L of glucose + 15 g / L of adenosine monophosphate.

5. The process of claim 1, wherein, the glucose solution comprises 600 g / L of glucose + 20 g / L of adenosine monophosphate.

6. The process of claim 1, wherein, the inoculation amount of the Escherichia coli seed liquid is 5-10%.

7. The process of claim 1, wherein, the fermentation time is 48 hours.

8. The process of claim 1, wherein, the reducing sugar content is 5 g / L.

9. The process of claim 1, wherein, the fermentation temperature is 36℃.

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