A process for the fermentative production of phenylalanine

Abstract

The application belongs to the technical field of microbial fermentation, and discloses a process for fermentatively producing phenylalanine, wherein a strain is inoculated into a seed culture medium for culture, seed liquid is obtained through acclimatization and optimization of the culture medium and culture conditions, inoculation into a fermenter for fermentation, and sugar is fed during the fermentation process to obtain high-yield acid fermentation liquor. Through multiple batches of sugar feeding into the fermentation liquor during the fermentation process, the application reduces the accumulation of metabolic by-products during the fermentation process, and improves the yield of phenylalanine.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a process for fermenting and producing phenylalanine. Background Technology

[0002] L-phenylalanine is an important intermediate in pharmaceuticals and food chemicals. It participates in the synthesis of important neurotransmitters and hormones in animals and has wide applications in the pharmaceutical and food industries.

[0003] As a carrier for anticancer drugs, phenylalanine can deliver these drugs to tumor sites, inhibiting tumor growth while significantly reducing drug toxicity. Furthermore, phenylalanine is also a major raw material for the synthesis of the low-calorie sweetener aspartame. In recent years, with the increasing market demand for oncology drugs and the sweetener aspartame, the supply of phenylalanine has also been growing year by year, placing higher demands on its industrial production. Previously, phenylalanine production methods were mainly divided into chemical synthesis, enzymatic methods, and microbial fermentation methods. In recent years, due to the high production costs, limited material sources, and serious environmental pollution associated with chemical synthesis and enzymatic methods, the main production method for phenylalanine has evolved to the relatively economical and environmentally friendly microbial fermentation method.

[0004] However, the technology for producing phenylalanine by fermentation in my country is not yet mature, and the acid production capacity and conversion rate are not high, resulting in high production costs. Therefore, there is an urgent need for new technological breakthroughs and production processes to improve the acid production capacity and conversion rate of phenylalanine while reducing production costs.

[0005] The fermentation strain for producing L-phenylalanine, Corynebacterium glutamicum CICC20909, was deposited by the applicant at the China Industrial Microbial Culture Collection Center (CICC) in September 2022. It can be obtained commercially through the website http: / / m.china-cicc.org / search / ?classtype=0&keyword=20909. Summary of the Invention

[0006] This invention provides a process for fermenting phenylalanine production. By domesticating fermentation strains, optimizing the culture medium, fermentation formula, and control parameters, the acid production rate of the strains is increased, thereby improving phenylalanine yield. This invention offers a high degree of controllability, low input costs, and precise control of the fermentation process, avoiding fermentation runaway and increased costs. It also reduces labor intensity, and the entire production process is simple and easy to operate.

[0007] The present invention is achieved through the following technical solution.

[0008] A process for producing phenylalanine by fermentation, characterized in that the process includes the following steps:

[0009] Step 1) Inoculate the domesticated seed liquid into a fermenter containing fermentation medium at a rate of 5-15% and ferment it. Control the temperature at 30-33℃, the tank pressure at 0.04-0.06Mpa, the pH at 6.5-7.0, the stirring speed at 200-400rpm, and the fermentation time at 24h to obtain fermentation liquid 1.

[0010] Step 2) Add glucose solution to fermentation broth 1 to control the residual sugar to 0.5%. The time mark for adding glucose solution is when the bottom sugar is below 0.3%. The fermentation time is 24-36 hours to obtain fermentation broth 2.

[0011] Furthermore,

[0012] The method for preparing the domestication seed solution is as follows:

[0013] The activated Corynebacterium glutamicum CICC 20909 was inoculated into the acclimatization medium, and the pH was controlled at 5.0, the pressure at 0.05 MPa, and the dissolved oxygen at 30%, and cultured for 12 h.

[0014] Continue to lower the pH to 4.0, keeping other conditions unchanged, and incubate for 12 hours;

[0015] Continue to lower the pH to 3.0, keeping other conditions unchanged, and incubate for 12 hours.

[0016] Preferably,

[0017] The fermentation medium components include: glucose 40 g / L, magnesium sulfate heptahydrate 3 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, ammonium sulfate 5 g / L, sodium citrate 1 g / L, monosodium glutamate 1 g / L, corn steep liquor 20 g / L, fumaric acid 0.5 g / L, and L-tyrosine 0.5 g / L.

[0018] Preferably,

[0019] The concentration of the glucose solution is greater than 400 g / L.

[0020] Preferably,

[0021] The high-salt acclimatization culture medium comprises: 25 g / L glucose, 2 g / L magnesium sulfate heptahydrate, 0.6 g / L potassium dihydrogen phosphate, 0.6 g / L dipotassium hydrogen phosphate, 2 g / L ammonium sulfate, 0.5 g / L sodium citrate, 0.2 g / L monosodium glutamate, 8 g / L yeast extract, and 10-20 g / L sodium chloride.

[0022] Preferably,

[0023] The fermentation time in step 2) is 36 hours.

[0024] Preferably,

[0025] The high-salt acclimatization culture medium comprises: 25 g / L glucose, 2 g / L magnesium sulfate heptahydrate, 0.6 g / L potassium dihydrogen phosphate, 0.6 g / L dipotassium hydrogen phosphate, 2 g / L ammonium sulfate, 0.5 g / L sodium citrate, 0.2 g / L monosodium glutamate, 8 g / L yeast extract, and 20 g / L sodium chloride.

[0026] Preferably,

[0027] The concentration of the glucose solution is 500 g / L.

[0028] The technical solution of the present invention has the following outstanding advantages and uniqueness:

[0029] This invention improves the activity of the starting strain by acclimating it to acid and osmotic pressure, avoiding the decline in activity caused by high acidity and high osmotic pressure in the local microenvironment, extending the fermentation time, and increasing the fermentation yield of phenylalanine.

[0030] This invention utilizes a fed-batch sugar method to precisely control the residual sugar value during fermentation. Glucose intake is crucial for phenylalanine fermentation; excessively high sugar concentrations not only reduce yield but also increase production costs, while excessively low concentrations inhibit the bacterial strain's productivity, preventing it from reaching its maximum potential. This invention employs a fed-batch sugar method to reduce the accumulation of metabolic byproducts during fermentation, thereby further increasing the yield of phenylalanine while simultaneously reducing production costs. Attached Figure Description

[0031] Figure 1 Effects of strain domestication on L-phenylalanine fermentation content. Detailed Implementation

[0032] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The products and methods of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the products and methods described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention. To further understand this invention, the following detailed description is provided in conjunction with embodiments.

[0033] Example 1

[0034] strain domestication

[0035] 1. The starting strain Corynebacterium glutamicum CICC 20909 was plated on TSA plates with different salinities. The maximum salinity tolerance of Corynebacterium glutamicum CICC 20909 was measured to be 20 g / L sodium chloride, in order to improve its resistance to osmotic pressure.

[0036] 2. Activate Corynebacterium glutamicum CICC 20909 and then inoculate it into a high-salt acclimatization medium. The components of the high-salt acclimatization medium are: glucose 25 g / L, magnesium sulfate heptahydrate 2 g / L, potassium dihydrogen phosphate 0.6 g / L, dipotassium hydrogen phosphate 0.6 g / L, ammonium sulfate 2 g / L, sodium citrate 0.5 g / L, monosodium glutamate 0.2 g / L, yeast extract 8 g / L, and sodium chloride 20 g / L.

[0037] Acclimation process: Activated Corynebacterium glutamicum CICC 20909 was inoculated into the acclimation medium, and the pH was controlled at 5.0, the pressure at 0.05 MPa, and the dissolved oxygen at 30%. After 12 hours of cultivation, the growth was found to be good.

[0038] The pH was further lowered to 4.0, while other conditions remained unchanged. After culturing for 12 hours, the growth was observed to be good.

[0039] The pH was further lowered to 3.0, while other conditions remained unchanged. After culturing for 12 hours, the growth was observed to be good.

[0040] The pH was further lowered to 2.0, while other conditions remained unchanged. After culturing for 12 hours, it was observed that the proliferation of the strain slowed down and some of the strains died.

[0041] The domestication seed solution obtained at pH 3.0 was selected for subsequent experiments.

[0042] Example 2

[0043] A process for producing phenylalanine by fermentation, the specific operating steps of which are as follows:

[0044] The domesticated seed solution with a pH of 3.0 was selected and prepared according to Example 1.

[0045] Fermentation medium components: glucose 40 g / L, magnesium sulfate heptahydrate 3 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, ammonium sulfate 5 g / L, sodium citrate 1 g / L, monosodium glutamate 1 g / L, corn steep liquor 20 g / L, fumaric acid 0.5 g / L, L-tyrosine 0.5 g / L.

[0046] The domesticated seed liquid was inoculated into a fermenter containing fermentation medium at a rate of 15% for fermentation. The temperature was controlled at 32℃, the pressure at the fermenter was 0.05 MPa, the pH was 6.5, the air flow rate was 2.5 m3 / h, the stirring speed was 400 rpm, and the fermentation time was 24 h to obtain fermentation liquid 1.

[0047] Glucose was added to fermentation broth 1. The time for adding sugar was marked when the base sugar was below 0.3% (w / v). The concentration of the added sugar was 500 g / L. The residual sugar was controlled at 0.5% (w / v) during fermentation. The fermentation time was 24-36 h to obtain fermentation broth 2.

[0048] Example 3

[0049] A process for producing phenylalanine by fermentation, the specific operating steps of which are as follows:

[0050] The domesticated seed solution with a pH of 3.0 was selected and prepared according to Example 1.

[0051] Fermentation medium components: glucose 40 g / L, magnesium sulfate heptahydrate 3 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, ammonium sulfate 5 g / L, sodium citrate 1 g / L, monosodium glutamate 1 g / L, corn steep liquor 20 g / L, fumaric acid 0.5 g / L, L-tyrosine 0.5 g / L.

[0052] The domesticated seed liquid was inoculated into a fermenter containing fermentation medium at a rate of 10% for fermentation. The temperature was controlled at 33℃, the pressure at the fermenter was 0.05 MPa, the pH was 6.7, the air flow rate was 2.2 m3 / h, the stirring speed was 300 rpm, and the fermentation time was 24 h to obtain fermentation liquid 1.

[0053] Glucose was added to fermentation broth 1. The time for adding sugar was marked when the base sugar was below 0.3%. The concentration of added sugar was 600 g / L. The residual sugar was controlled at 0.5% during fermentation. The fermentation time was 36 h, resulting in fermentation broth 2.

[0054] Example 4

[0055] The effect of strain domestication on the efficiency of phenylalanine production by Corynebacterium glutamicum CICC 20909.

[0056] Preparation of seed culture for control group 1:

[0057] Corynebacterium glutamicum CICC 20909 was activated and then inoculated into seed culture medium.

[0058] The culture medium consisted of: 25 g / L glucose, 2 g / L magnesium sulfate heptahydrate, 0.6 g / L potassium dihydrogen phosphate, 0.6 g / L dipotassium hydrogen phosphate, 2 g / L ammonium sulfate, 0.5 g / L sodium citrate, 0.2 g / L monosodium glutamate, and 8 g / L yeast extract. The culture process involved inoculating activated Corynebacterium glutamicum CICC 20909 into the culture medium, maintaining a pH of 5.0, a pressure of 0.05 MPa, and dissolved oxygen at 30%, and culturing for 36 hours to obtain the seed culture.

[0059] Preparation of seed culture in control group 2:

[0060] Corynebacterium glutamicum CICC 20909 was activated and then inoculated into a high-salt acclimatization medium.

[0061] The culture medium consisted of: 25 g / L glucose, 2 g / L magnesium sulfate heptahydrate, 0.6 g / L potassium dihydrogen phosphate, 0.6 g / L dipotassium hydrogen phosphate, 2 g / L ammonium sulfate, 0.5 g / L sodium citrate, 0.2 g / L monosodium glutamate, 8 g / L yeast extract, and 20 g / L sodium chloride. The culture process involved inoculating activated Corynebacterium glutamicum CICC 20909 into the culture medium, maintaining a pH of 5.0, a pressure of 0.05 MPa, and dissolved oxygen at 30%, and culturing for 36 hours to obtain the seed culture.

[0062] The above seed culture was fermented according to the process in Example 2. The fermentation time below refers to the total fermentation time (the total time for preparing fermentation broths 1 and 2). Figure 1 As shown, in control group 1 without high salt (high osmotic pressure) and pH acclimation, the maximum yield of phenylalanine was 14.8 g / L, and the longest fermentation time was 54 h. Continued fermentation caused a decrease in strain activity or death, and the yield no longer increased. After high salt acclimation alone, the maximum yield of phenylalanine was 16.4 g / L, and the longest fermentation time was 54 h. Continued fermentation caused a decrease in strain activity, and the yield no longer increased significantly. The experimental group (Example 2) that underwent high salt and pH acclimation could extend the fermentation time to 60 h, and the maximum fermentation yield was 19.7 g / L, which was 33.1% and 20.1% higher than that of control groups 1 and 2, respectively.

[0063] In the later stages of fermentation, with the increase of fermentation products and byproducts, the pH and osmotic pressure of the microenvironment of the bacteria increase significantly, which can lead to a decrease in the activity of the strain or even death. The domesticated strain can tolerate high pH and osmotic pressure and still maintain good activity in the later stages of fermentation, and can maintain a high fermentation yield. However, the initial strain Corynebacterium glutamicum CICC 20909 has a significantly shortened effective fermentation time because it has not been domesticated. Further extending the fermentation time has no effect on the fermentation yield.

[0064] Example 5

[0065] The effect of controlling residual sugar value in fermentation broth on fermentation yield.

[0066] Referring to the process in Example 2, the residual sugar content is controlled to a certain proportion by adding glucose. See Table 1 for details:

[0067] Table 1

[0068]

[0069] This invention achieves precise control of residual sugar levels during fermentation through a fed-batch sugar method. Glucose intake is crucial for phenylalanine fermentation; excessively high sugar concentrations not only reduce yield but also increase production costs, while excessively low concentrations inhibit the bacterial strain's productivity, preventing it from reaching its maximum potential. This invention employs a fed-batch sugar method, controlling the residual sugar level at 0.5%, which reduces the accumulation of metabolic byproducts during fermentation, thereby further increasing the phenylalanine yield while simultaneously reducing production costs.

[0070] While the specific embodiments of the present invention have been described above in conjunction with examples, they are not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that any modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection of the present invention.

Claims

1. A process for producing phenylalanine by fermentation, characterized in that, The process includes the following steps: Step 1) Inoculate the domesticated seed liquid into a fermenter containing fermentation medium at a rate of 5-15% and ferment it. Control the temperature at 30-33℃, the tank pressure at 0.04-0.06Mpa, the pH at 6.5-7.0, the stirring speed at 200-400rpm, and the fermentation time at 24h to obtain fermentation liquid 1. Step 2) Add glucose solution to fermentation broth 1 to control the residual sugar to 0.5%. The time mark for adding glucose solution is when the bottom sugar is below 0.3%. The fermentation time is 24-36 hours to obtain fermentation broth 2. The method for preparing the domestication seed solution is as follows: The activated Corynebacterium glutamicum CICC 20909 was inoculated into a high-salt acclimatization medium, and cultured for 12 hours with pH 5.0, pressure 0.05 MPa and dissolved oxygen 30% controlled. Continue to lower the pH to 4.0, keeping other conditions unchanged, and incubate for 12 hours; Continue to lower the pH to 3.0, keep other conditions unchanged, and incubate for 12 hours; The high-salt acclimatization culture medium comprises: 25 g / L glucose, 2 g / L magnesium sulfate heptahydrate, 0.6 g / L potassium dihydrogen phosphate, 0.6 g / L dipotassium hydrogen phosphate, 2 g / L ammonium sulfate, 0.5 g / L sodium citrate, 0.2 g / L monosodium glutamate, 8 g / L yeast extract, and 20 g / L sodium chloride.

2. The process according to claim 1, characterized in that, The fermentation medium components include: glucose 40 g / L, magnesium sulfate heptahydrate 3 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, ammonium sulfate 5 g / L, sodium citrate 1 g / L, monosodium glutamate 1 g / L, corn steep liquor 20 g / L, fumaric acid 0.5 g / L, and L-tyrosine 0.5 g / L.

3. The process according to claim 1, characterized in that, The concentration of the glucose solution is greater than 400 g / L.

4. The process according to claim 1, characterized in that, The fermentation time in step 2) is 36 hours.

5. The process according to claim 3, characterized in that, The concentration of the glucose solution is 500 g / L.

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