A method for increasing the production level of tryptophan based on controlling the specific oxygen consumption rate

Abstract

The application discloses a method for improving tryptophan production level based on control of specific oxygen consumption rate, and belongs to the technical field of amino acid fermentation. In order to improve the yield and conversion rate of tryptophan and reduce the synthesis of by-products, the application provides a method for improving tryptophan production level based on control of specific oxygen consumption rate. In the process of producing tryptophan by fermentation of Escherichia coli, when the amount of oxygen consumed by unit dry weight of bacteria per unit time is slowly decreased from the peak to a target value in the early stage of the fermentation process, the amount of oxygen consumed by unit dry weight of bacteria per unit time is controlled in stages by supplementing a compound nitrogen material. The method can balance the yield and conversion rate, achieve the goal of improving the yield and conversion rate, and is suitable for large-scale industrial production.

Description

Technical Field

[0001] This invention belongs to the field of amino acid fermentation technology, specifically relating to a method for improving tryptophan production based on controlling the specific oxygen consumption rate. Background Technology

[0002] L-Tryptophan, also known as α-aminoindolepropionic acid, has the molecular formula: C1 11 H 12 N2O2 is a precursor to 5-hydroxytryptamine, an important neurotransmitter in the human body, and is also one of the essential amino acids. It has good nutritional and medicinal value and is widely used in the feed, pharmaceutical and health care industries as a major amino acid fermentation product.

[0003] L-Tryptophan production primarily utilizes genetically engineered *E. coli* or *Corynebacterium glutamicum* strains as production strains, achieving industrial-scale manufacturing through microbial fermentation. Microbial fermentation offers advantages such as low raw material costs, simple process control, and reliable product quality. In recent years, achieving low-cost, high-efficiency production of tryptophan has become crucial for major domestic amino acid manufacturers to drive continuous technological advancement. Therefore, to reduce costs and increase profits, higher demands are being placed not only on the nutritional composition of the culture medium but also on the rationality of fermentation process control.

[0004] The oxygen consumption capacity of microorganisms during aerobic fermentation reflects the intensity of respiratory metabolism. Especially in highly aerobic tryptophan fermentation, controlling the oxygen consumption rate significantly impacts the production of substrates, products, and byproducts in *E. coli*, ultimately affecting tryptophan concentration and conversion rate. Therefore, rationally controlling the oxygen consumption rate is crucial for achieving high yield and high conversion rate. Although some literature reports that controlling dissolved oxygen or oxygen uptake can improve tryptophan expression during fermentation, none have fully considered the impact of cell concentration on oxygen consumption per unit cell during fermentation; nor have there been reports of optimizing tryptophan fermentation and improving yield and conversion rate by continuously feeding to control the amount of oxygen consumed per unit cell dry weight per unit time. Summary of the Invention

[0005] To improve the yield and conversion rate of tryptophan and reduce the synthesis of byproducts, this invention provides a method for improving tryptophan production based on controlling the specific oxygen consumption rate. The method involves controlling the oxygen consumption per unit dry weight per unit time during the fermentation process of Escherichia coli. When the oxygen consumption per unit dry weight per unit time slowly decreases from its peak to 5.0 mmol / h / g in the early stage of fermentation, the amount of oxygen consumed per unit dry weight per unit time is controlled to be 2.5-4.0 mmol / h / g by supplementing with compound nitrogen feed.

[0006] Further specifying, the composition of the composite nitrogen material is 0.2 g / L amino acid powder, 0.3 g / L corn steep liquor powder, 0.2 g / L yeast powder, 0.03 g / L ammonium sulfate, 0.01 g / L defoamer, with the balance being water, and pH 8.0.

[0007] Furthermore, the fermentation process employs a stirring linkage to control dissolved oxygen levels to be no less than 20%.

[0008] Further specifying that during the fermentation process, when the glucose concentration in the fermentation broth is lower than 1.0 g / L after 2-4 hours of fermentation, a glucose solution with a concentration of 650 g / L is added to maintain the glucose concentration in the fermentation broth at 0.01-0.2 g / L during the fermentation process.

[0009] To further limit this, when the amount of oxygen consumed per unit dry weight per unit time is reduced to 5.0 mmol / h / g, the amount of oxygen consumed per unit dry weight per unit time is controlled in stages by supplementing with compound nitrogen material.

[0010] Further specifying, the phased control refers to controlling the amount of oxygen consumed per unit dry weight per unit time to be 3.5-4.0 mmol / h / g during fermentation 8-20h; controlling the amount of oxygen consumed per unit dry weight per unit time to be 3.0-3.5 mmol / h / g during fermentation 20-32h; and controlling the amount of oxygen consumed per unit dry weight per unit time to be 2.5-3.0 mmol / h / g from fermentation 32h to the end of fermentation.

[0011] Further specifying, the process prior to fermentation also includes slant culture, primary seed culture, and secondary seed culture of Escherichia coli;

[0012] The slant culture medium consisted of 10 g / L peptone, 5 g / L yeast extract, 10 g / L beef extract, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 15 g / L agar powder, and 0.05 g / L tetracycline, with the remainder being water.

[0013] The seed culture medium consisted of 10 g / L glucose, 4 g / L yeast extract, 0.5 g / L citric acid, 1 g / L ammonium sulfate, 1 g / L potassium dihydrogen phosphate, 1 mg / L vitamin B1, 0.35 mg / L biotin, 1.5 g / L magnesium sulfate heptahydrate, 2.8 mg / L ferrous sulfate heptahydrate, 2 mL / L trace element mixed solution, and the remainder was water.

[0014] The culture medium used for fermentation consisted of 30 g / L glucose, 10 g / L yeast extract, 2.5 g / L citric acid, 4 g / L ammonium sulfate, 5 g / L potassium dihydrogen phosphate, 2.8 g / L magnesium sulfate heptahydrate, 90 mg / L ferrous sulfate heptahydrate, 5 mg / L vitamin B1, 2 mg / L biotin, and 4 mL / L of a mixed solution of trace elements.

[0015] Further specifying, the primary seed culture involves inoculating the bacterial cells after slant culture into a seed culture medium and culturing them at 37°C, 220 rpm in a shake flask for 10-12 hours.

[0016] Further specifying, the secondary seed culture involves inoculating the primary seed solution into the seed culture medium at an inoculation rate of 1%, and culturing it for 10-12 hours at 36-37°C, an aeration ratio of 0.5 VVM, 200 rpm, a tank pressure of 0.03-0.05 MPa, a pH of 7.0-7.2, and dissolved oxygen ≥30%.

[0017] Further specifying, the fermentation involves inoculating the secondary seed culture at a rate of 10% into the fermentation medium and culturing it at 36-37°C, with an aeration ratio of 1.0 VVM, dissolved oxygen ≥20%, tank pressure of 0.03-0.05 MPa, and pH of 6.8-7.0 for 36-40 hours.

[0018] The specific oxygen consumption rate mentioned in this invention refers to the amount of oxygen consumed per unit dry weight of bacteria per unit time.

[0019] The beneficial effects of this invention are:

[0020] This invention regulates bacterial metabolic activity by controlling the feeding rate, thereby controlling the specific oxygen consumption rate. Compared with existing technologies, this invention has the following advantages:

[0021] 1. Compared to conventional feeding methods that rely on dissolved oxygen and pH to supplement sugar and ammonia, this invention achieves continuous feeding by adjusting the specific oxygen consumption rate during fermentation. This involves maintaining the cell's metabolic activity by continuously supplementing effective carbon and nitrogen nutrients during the tryptophan synthesis stage. Maintaining a suitable specific oxygen consumption rate ensures stable product synthesis, sustained high-speed acid production, and stable cell metabolism. With automated control technology, this strategy can be directly scaled up to industrial production scale.

[0022] 2. This invention's continuous feeding and staged control of specific oxygen consumption rate can improve tryptophan conversion and reduce by-products, thus benefiting product quality control. Specifically, based on actual industrial production needs, it allows for control of the specific oxygen consumption rate at different stages to manage the conversion of carbon atoms between products, cells, and maintenance processes. Maintaining a specific oxygen consumption rate of 3.5-4.0 mmol / h / g promotes cell growth and maintains metabolic activity, but results in more by-product synthesis. Maintaining a specific oxygen consumption rate above 2.5-3.0 mmol / h / g promotes tryptophan synthesis, but substrate conversion to CO2 release leads to a decrease in conversion rate, and subsequent cell aging and death can cause premature discharge. Maintaining a specific oxygen consumption rate of 3.0-3.5 mmol / h / g achieves a balance between acid production and conversion rate, thus improving both. Attached Figure Description

[0023] Figure 1 The curves showing the change in specific oxygen consumption rate during the fermentation process of each embodiment are shown.

[0024] Figure 2 The curves show the changes in the content of glutamic acid, a byproduct, during the fermentation process in each embodiment. Detailed Implementation

[0025] The present invention will be further described below with reference to specific embodiments and accompanying drawings, but it is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0026] The tryptophan-producing Escherichia coli used in this invention is disclosed in Chinese patent application No. 2016111887431, entitled "A Clear Fermentation Culture Medium and a Method for Increasing L-Tryptophan", with strain accession number CGMCCNO.11073.

[0027] The culture medium involved in this invention is as follows:

[0028] The slant culture medium consisted of 10 g / L peptone, 5 g / L yeast extract, 10 g / L beef extract, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 15 g / L agar powder, and 0.05 g / L tetracycline, with the remainder being water.

[0029] The seed culture medium consisted of 10 g / L glucose, 4 g / L yeast extract, 0.5 g / L citric acid, 1 g / L ammonium sulfate, 1 g / L potassium dihydrogen phosphate, 1 mg / L vitamin B1, 0.35 mg / L biotin, 1.5 g / L magnesium sulfate heptahydrate, 2.8 mg / L ferrous sulfate heptahydrate, 2 mL / L trace element mixed solution, and the remainder was water.

[0030] The fermentation medium consisted of 30 g / L glucose, 10 g / L yeast extract, 2.5 g / L citric acid, 4 g / L ammonium sulfate, 5 g / L potassium dihydrogen phosphate, 2.8 g / L magnesium sulfate heptahydrate, 90 mg / L ferrous sulfate heptahydrate, 5 mg / L vitamin B1, 2 mg / L biotin, and 4 mL / L of a mixed solution of trace elements.

[0031] The compound nitrogen feed consists of 0.2 g / L amino acid powder, 0.3 g / L corn steep liquor powder, 0.2 g / L yeast powder, 0.03 g / L ammonium sulfate, 0.01 g / L defoamer, and the balance being water, with a pH of 8.0.

[0032] Example 1:

[0033] This embodiment provides a method for improving tryptophan production based on controlling the specific oxygen consumption rate. The method includes four stages: slant culture, primary seed culture, secondary seed culture, and fermentation, as detailed below:

[0034] Slant culture: Escherichia coli taken from the preservation tube was inoculated onto sterile slant culture medium and cultured at 37°C for 2 days.

[0035] Primary seed culture: scrape 1cm 2 The Escherichia coli cells cultured on slant agar were inoculated into a 500 mL Erlenmeyer flask containing 100 mL of sterilized seed culture medium and sealed with 8 layers of gauze. Culture conditions: 37℃, 220 rpm, 10 h.

[0036] Secondary seed culture: 10L of seed culture medium was placed in a 15L seed tank and sterilized. The pressure was maintained at around 0.05MPa using sterile air. Then, 100mL of the cultured primary seed solution (1% inoculum) was inoculated into the seed tank using the pressure difference method for culture. The culture conditions were 36℃, aeration ratio of 0.5VVM, initial stirring speed of 200rpm, tank pressure of 0.03-0.05MPa, pH 7.0, and dissolved oxygen was controlled at no less than 30% throughout the process by adjusting the air volume and stirring speed. The culture cycle was 10h.

[0037] Fermentation culture: 20L of fermentation medium was added to a 50L fermenter. After sterilization and cooling, the secondary seed liquid was transferred into the fermenter for cultivation. The inoculum size was 10%. The cultivation conditions were: temperature 36℃, aeration ratio 1.0VVM, initial rotation speed 200rpm, dissolved oxygen controlled at no less than 20% throughout the process by adjusting the air volume and rotation speed, tank pressure 0.03-0.05MPa, pH controlled at 6.8 by adding compound nitrogen and ammonia water, and the cultivation cycle was 36h.

[0038] Feeding control strategy: During fermentation, when the glucose concentration in the fermentation broth drops below 1.0 g / L after 2-4 hours of fermentation, a 650 g / L glucose solution is added to maintain the glucose concentration at 0.01-0.2 g / L. When the oxygen consumption per unit cell dry weight per unit time slowly decreases from its peak to 5.0 mmol / h / g in the early stage of fermentation, compound nitrogen feed is added to control the oxygen consumption per unit cell dry weight per unit time to 3.5-4.0 mmol / h / g.

[0039] During the fermentation process, the components of CO2, O2, N2, and Ar in the fermentation tail gas were determined using the PASS2000 process mass spectrometer from Shanghai Shunyu Hengping Co., Ltd. The capacitance data were measured online using an online live cell analyzer from Hamilton Company. The specific oxygen consumption rate during the fermentation process was continuously calculated using the "Zhongzhi" fermentation process analysis software package developed by Shanghai Kuaipu Intelligent Co., Ltd.

[0040] After the fermentation process described above was completed, the relevant indicators of the fermentation broth were measured and analyzed. The results showed that the fermentation potency was 52.3 g / L and the conversion rate was 18.3%.

[0041] Example 2:

[0042] This embodiment provides a method for improving tryptophan production based on controlling the specific oxygen consumption rate. The method includes four stages: slant culture, primary seed culture, secondary seed culture, and fermentation, as detailed below:

[0043] Slant culture: Escherichia coli taken from the preservation tube was inoculated onto sterile slant culture medium and cultured at 37°C for 2 days.

[0044] Primary seed culture: scrape 1cm 2 The Escherichia coli cells cultured on slant agar were inoculated into a 500 mL Erlenmeyer flask containing 100 mL of sterilized seed culture medium and sealed with 8 layers of gauze. Culture conditions: 37℃, 220 rpm, 12 h.

[0045] Secondary seed culture: 10L of seed culture medium was placed in a 15L seed tank and sterilized. The pressure was maintained at around 0.05MPa using sterile air. Then, 100mL of the cultured primary seed solution (1% inoculum) was inoculated into the seed tank using the pressure difference method for culture. The culture conditions were 37℃, aeration ratio of 0.5VVM, initial stirring speed of 200rpm, tank pressure of 0.03-0.05MPa, pH 7.2, and dissolved oxygen was controlled at no less than 30% throughout the process by adjusting the air volume and stirring speed. The culture cycle was 12h.

[0046] Fermentation culture: 20L of fermentation medium was added to a 50L fermenter. After sterilization and cooling, the secondary seed liquid was transferred into the fermenter for cultivation. The inoculum size was 10%. The cultivation conditions were: temperature 37℃, aeration ratio 1.0VVM, initial rotation speed 200rpm. Dissolved oxygen was controlled to be no less than 20% throughout the fermentation process by adjusting the air volume and rotation speed. The tank pressure was 0.03-0.05MPa. The pH was controlled at 7.0 by adding compound nitrogen and ammonia. The cultivation cycle was 40h.

[0047] Feeding control strategy: When the glucose concentration in the fermentation broth drops below 1.0 g / L after 2-4 hours of fermentation, a 650 g / L glucose solution is added to maintain the glucose concentration at 0.01-0.2 g / L during fermentation. When the oxygen consumption per unit dry weight per unit time slowly decreases from its peak to 6.0 mmol / h / g in the early stage of fermentation, compound nitrogen feed is added to control the oxygen consumption per unit dry weight per unit time to 3.0-3.5 mmol / h / g.

[0048] During the fermentation process, the components of CO2, O2, N2, and Ar in the fermentation tail gas were determined using the PASS2000 process mass spectrometer from Shanghai Shunyu Hengping Co., Ltd. The capacitance data were measured online using an online live cell analyzer from Hamilton Company. The specific oxygen consumption rate during the fermentation process was continuously calculated using the "Zhongzhi" fermentation process analysis software package developed by Shanghai Kuaipu Intelligent Co., Ltd.

[0049] After the fermentation process described above was completed, the relevant indicators of the fermentation broth were measured and analyzed. The results showed that the fermentation titer was 58.3 g / L and the conversion rate was 19.3%.

[0050] Example 3:

[0051] This embodiment provides a method for improving tryptophan production based on controlling the specific oxygen consumption rate. The method includes four stages: slant culture, primary seed culture, secondary seed culture, and fermentation, as detailed below:

[0052] Slant culture: Escherichia coli taken from the preservation tube was inoculated onto sterile slant culture medium and cultured at 37°C for 2 days.

[0053] Primary seed culture: scrape 1cm 2 The Escherichia coli cells cultured on slant agar were inoculated into a 500 mL Erlenmeyer flask containing 100 mL of sterilized seed culture medium and sealed with 8 layers of gauze. Culture conditions: 37℃, 220 rpm, 11 h.

[0054] Secondary seed culture: 10L of seed culture medium was placed in a 15L seed tank and sterilized. The pressure was maintained at around 0.05MPa using sterile air. Then, 100mL of the cultured primary seed solution (1% inoculum) was inoculated into the seed tank using the pressure difference method for culture. The culture conditions were 37℃, aeration ratio of 0.5VVM, initial stirring speed of 200rpm, tank pressure of 0.03-0.05MPa, pH 7.1, and dissolved oxygen was controlled at no less than 30% throughout the process by adjusting the air volume and stirring speed. The culture cycle was 11h.

[0055] Fermentation culture: 20L of fermentation medium was added to a 50L fermenter. After sterilization and cooling, the secondary seed liquid was transferred into the fermenter for cultivation. The inoculum size was 10%. The cultivation conditions were: temperature 37℃, aeration ratio 1.0VVM, initial rotation speed 200rpm. Dissolved oxygen was controlled to be no less than 20% throughout the fermentation process by adjusting the air volume and rotation speed. The tank pressure was 0.03-0.05MPa. The pH was controlled at 6.9 by adding compound nitrogen and ammonia. The cultivation cycle was 38h.

[0056] Feeding control strategy: When the glucose concentration in the fermentation broth drops below 1.0 g / L after 2-4 hours of fermentation, a 650 g / L glucose solution is added to maintain the glucose concentration at 0.01-0.2 g / L during fermentation. When the oxygen consumption per unit dry weight per unit time slowly decreases from its peak to 5.0 mmol / h / g in the early stage of fermentation, compound nitrogen feed is added to control the oxygen consumption per unit dry weight per unit time to 2.5-3.0 mmol / h / g.

[0057] During the fermentation process, the components of CO2, O2, N2, and Ar in the fermentation tail gas were determined using the PASS2000 process mass spectrometer from Shanghai Shunyu Hengping Co., Ltd. The capacitance data were measured online using an online live cell analyzer from Hamilton Company. The specific oxygen consumption rate during the fermentation process was continuously calculated using the "Zhongzhi" fermentation process analysis software package developed by Shanghai Kuaipu Intelligent Co., Ltd.

[0058] After the fermentation process described above was completed, the relevant indicators of the fermentation broth were measured and analyzed. The results showed that the fermentation potency was 48.2 g / L and the conversion rate was 16.8%.

[0059] Example 4:

[0060] This embodiment provides a method for improving tryptophan production based on controlling the specific oxygen consumption rate. The method includes four stages: slant culture, primary seed culture, secondary seed culture, and fermentation, as detailed below:

[0061] Slant culture: Escherichia coli taken from the preservation tube was inoculated onto sterile slant culture medium and cultured at 37°C for 2 days.

[0062] Primary seed culture: scrape 1cm 2 The Escherichia coli cells cultured on slant agar were inoculated into a 500 mL Erlenmeyer flask containing 100 mL of sterilized seed culture medium and sealed with 8 layers of gauze. Culture conditions: 37℃, 220 rpm, 11 h.

[0063] Secondary seed culture: 10L of seed culture medium was placed in a 15L seed tank and sterilized. The pressure was maintained at around 0.05MPa using sterile air. Then, 100mL of the cultured primary seed solution (1% inoculum) was inoculated into the seed tank using the pressure difference method for culture. The culture conditions were 37℃, aeration ratio of 0.5VVM, initial stirring speed of 200rpm, tank pressure of 0.03-0.05MPa, pH 7.1, and dissolved oxygen was controlled at no less than 30% throughout the process by adjusting the air volume and stirring speed. The culture cycle was 11h.

[0064] Fermentation culture: 20L of fermentation medium was added to a 50L fermenter. After sterilization and cooling, the secondary seed liquid was transferred into the fermenter for cultivation. The inoculum size was 10%. The cultivation conditions were: temperature 37℃, aeration ratio 1.0VVM, initial rotation speed 200rpm. Dissolved oxygen was controlled to be no less than 20% throughout the fermentation process by adjusting the air volume and rotation speed. The tank pressure was 0.03-0.05MPa. The pH was controlled at 6.9 by adding compound nitrogen and ammonia. The cultivation cycle was 38h.

[0065] Feeding control strategy: When the glucose concentration in the fermentation broth drops below 1.0 g / L after 2-4 hours of fermentation, a 650 g / L glucose solution is added to maintain the glucose concentration at 0.01-0.2 g / L during fermentation. When the oxygen consumption per unit dry weight per unit time gradually decreases from its peak to 5.0 mmol / h / g in the early stages of fermentation, the oxygen consumption per unit dry weight per unit time is controlled in stages by adding compound nitrogen feed: From 8-28 hours of fermentation, the oxygen consumption per unit dry weight per unit time is controlled at 3.5-4.0 mmol / h / g; from 28-38 hours of fermentation, the oxygen consumption per unit dry weight per unit time is controlled at 3.0-3.5 mmol / h / g.

[0066] During the fermentation process, the components of CO2, O2, N2, and Ar in the fermentation tail gas were determined using the PASS2000 process mass spectrometer from Shanghai Shunyu Hengping Co., Ltd. The capacitance data were measured online using an online live cell analyzer from Hamilton Company. The specific oxygen consumption rate during the fermentation process was continuously calculated using the "Zhongzhi" fermentation process analysis software package developed by Shanghai Kuaipu Intelligent Co., Ltd.

[0067] After the fermentation process described above was completed, the relevant indicators of the fermentation broth were measured and analyzed. The results showed that the fermentation titer was 60.8 g / L and the conversion rate was 20.5%.

[0068] Example 5:

[0069] This embodiment provides a method for improving tryptophan production based on controlling the specific oxygen consumption rate. The method includes four stages: slant culture, primary seed culture, secondary seed culture, and fermentation, as detailed below:

[0070] Slant culture: Escherichia coli taken from the preservation tube was inoculated onto sterile slant culture medium and cultured at 37°C for 2 days.

[0071] Primary seed culture: scrape 1cm 2 The Escherichia coli cells cultured on slant agar were inoculated into a 500 mL Erlenmeyer flask containing 100 mL of sterilized seed culture medium and sealed with 8 layers of gauze. Culture conditions: 37℃, 220 rpm, 11 h.

[0072] Secondary seed culture: 10L of seed culture medium was placed in a 15L seed tank and sterilized. The pressure was maintained at around 0.05MPa using sterile air. Then, 100mL of the cultured primary seed solution (1% inoculum) was inoculated into the seed tank using the pressure difference method for culture. The culture conditions were 37℃, aeration ratio of 0.5VVM, initial stirring speed of 200rpm, tank pressure of 0.03-0.05MPa, pH 7.1, and dissolved oxygen was controlled at no less than 30% throughout the process by adjusting the air volume and stirring speed. The culture cycle was 11h.

[0073] Fermentation culture: 20L of fermentation medium was added to a 50L fermenter. After sterilization and cooling, the secondary seed liquid was transferred into the fermenter for cultivation. The inoculum size was 10%. The cultivation conditions were: temperature 37℃, aeration ratio 1.0VVM, initial rotation speed 200rpm. Dissolved oxygen was controlled to be no less than 20% throughout the fermentation process by adjusting the air volume and rotation speed. The tank pressure was 0.03-0.05MPa. The pH was controlled at 6.9 by adding compound nitrogen and ammonia. The cultivation cycle was 38h.

[0074] Feeding control strategy: When the glucose concentration in the fermentation broth drops below 1.0 g / L after 2-4 hours of fermentation, a 650 g / L glucose solution is added to maintain the glucose concentration at 0.01-0.2 g / L during fermentation. When the oxygen consumption per unit dry weight per unit time gradually decreases from its peak to 5.0 mmol / h / g in the early stages of fermentation, the oxygen consumption per unit dry weight per unit time is controlled in stages by adding compound nitrogen feed: 3.5-4.0 mmol / h / g per unit dry weight per unit time during fermentation 8-20 hours; 3.0-3.5 mmol / h / g per unit dry weight per unit time during fermentation 20-32 hours; and 2.5-3.0 mmol / h / g per unit dry weight per unit time from 32 hours until the end of fermentation.

[0075] During the fermentation process, the components of CO2, O2, N2, and Ar in the fermentation tail gas were determined using the PASS2000 process mass spectrometer from Shanghai Shunyu Hengping Co., Ltd. The capacitance data were measured online using an online live cell analyzer from Hamilton Company. The specific oxygen consumption rate during the fermentation process was continuously calculated using the "Zhongzhi" fermentation process analysis software package developed by Shanghai Kuaipu Intelligent Co., Ltd.

[0076] After the fermentation process described above was completed, the relevant indicators of the fermentation broth were measured and analyzed. The results showed that the fermentation titer was 62.8 g / L and the conversion rate was 21.5%.

[0077] Comparative example:

[0078] This comparative example provides an initial fermentation method for producing tryptophan, which includes four stages: slant culture, primary seed culture, secondary seed culture, and fermentation, as detailed below:

[0079] Slant culture: Escherichia coli taken from the preservation tube was inoculated onto sterile slant culture medium and cultured at 37°C for 2 days.

[0080] Primary seed culture: scrape 1cm 2 The Escherichia coli cells cultured on slant agar were inoculated into a 500 mL Erlenmeyer flask containing 100 mL of sterilized seed culture medium and sealed with 8 layers of gauze. Culture conditions: 37℃, 220 rpm, 11 h.

[0081] Secondary seed culture: 10L of seed culture medium was placed in a 15L seed tank and sterilized. The pressure was maintained at around 0.05MPa using sterile air. Then, 100mL of the cultured primary seed solution (1% inoculum) was inoculated into the seed tank using the pressure difference method for culture. The culture conditions were 37℃, aeration ratio of 0.5VVM, initial stirring speed of 200rpm, tank pressure of 0.03-0.05MPa, pH 7.1, and dissolved oxygen was controlled at no less than 30% throughout the process by adjusting the air volume and stirring speed. The culture cycle was 11h.

[0082] Fermentation culture: 20L of fermentation medium was added to a 50L fermenter. After sterilization and cooling, the secondary seed liquid was transferred into the fermenter for cultivation. The inoculum size was 10%. The cultivation conditions were: temperature 37℃, aeration ratio 1.0VVM, initial rotation speed 200rpm, dissolved oxygen was controlled to be no less than 20% throughout the fermentation process by adjusting the air volume and rotation speed, tank pressure 0.03-0.05MPa, pH was controlled at 6.9 by ammonia water, and the cultivation cycle was 38h.

[0083] Feeding control strategy: Instead of using compound nitrogen feed to control the specific oxygen consumption rate during fermentation, a 650 g / L glucose solution was added after 2-4 hours of fermentation when the glucose concentration in the fermentation broth fell below 1.0 g / L, maintaining the glucose concentration at 0.01-0.2 g / L throughout the fermentation process. Ammonia was added automatically based on pH, controlling the pH to be no lower than 6.9 and no higher than 7.0.

[0084] During the fermentation process, the components of CO2, O2, N2, and Ar in the fermentation tail gas were determined using the PASS2000 process mass spectrometer from Shanghai Shunyu Hengping Co., Ltd. The capacitance data were measured online using an online live cell analyzer from Hamilton Company. The specific oxygen consumption rate during the fermentation process was continuously calculated using the "Zhongzhi" fermentation process analysis software package developed by Shanghai Kuaipu Intelligent Co., Ltd.

[0085] After the fermentation process described above was completed, the relevant indicators of the fermentation broth were measured and analyzed. The results showed that the fermentation potency was 43.5 g / L and the conversion rate was 15.9%.

[0086] Results Analysis: To investigate the effect of specific oxygen consumption rate on tryptophan fermentation, different specific oxygen consumption rates were controlled in Examples 1-3 (Example 1: 3.5-4.0 mmol / h / g; Example 2: 3.0-3.5 mmol / h / g; Example 3: 2.5-3.0 mmol / h / g). Combining the fermentation titers and conversion rates of Examples 1-3, it can be seen that there is an optimal control range for the specific oxygen consumption rate. Comparative analysis of the tryptophan concentration and conversion rate in Examples 1-3 shows that Example 3, maintaining a specific oxygen consumption rate of 2.5-3.0 mmol / h / g, had lower acid production rate and conversion rate. This indicates that the added glucose was mainly used for cell growth. Since the reducing power and energy generated by the TCA cycle ultimately use oxygen as the electron acceptor, under low specific oxygen consumption rate conditions, it is clear that intermediate metabolites from glucose metabolism and tryptophan synthesis precursors were drawn away for cell synthesis, which is detrimental to tryptophan synthesis. Meanwhile, the byproducts lactic acid, acetic acid, and glutamate did not accumulate significantly (see...). Figure 2 In Example 1, maintaining an oxygen consumption rate of 3.5-4.0 mmol / h / g resulted in a faster TCA cycle and a significantly improved acid production ratio, as evidenced by the corresponding fermentation titer and conversion rate. However, the faster TCA cycle also led to a significantly higher accumulation of the byproduct glutamate, indicating that the intermediate α-ketoglutarate in the TCA cycle was converted into glutamate. In Example 2, maintaining an oxygen consumption rate of 3.0-3.5 mmol / h / g yielded unexpected results, with significantly better acid production and conversion rates than Examples 1 and 3. This suggests that maintaining an appropriate oxygen consumption rate is crucial for intracellular metabolic balance during fermentation, indicating an optimal control ratio for cell growth, tryptophan synthesis, and TCA cycle balance. Furthermore, the glutamate content in Example 2 showed a decreasing trend compared to Example 1. Therefore, based on Examples 1-3 and considering the varying oxygen consumption rate requirements at different fermentation stages, Examples 4 and 5 were designed to control the oxygen consumption rate in stages, taking into account the fermentation results of Examples 1-3. Example 4 illustrates a two-stage control of specific oxygen consumption rate, while Example 5 illustrates a three-stage control. Fermentation results show that the staged control of specific oxygen consumption rate resulted in higher tryptophan concentration and conversion rate compared to Examples 1-3. Furthermore, the byproduct glutamate did not accumulate significantly; in fact, it improved the conversion rate. The changes in specific oxygen consumption rate during fermentation for each example are shown in the figure. Figure 1 The results for the content of the main byproduct glutamic acid are shown in [the table below]. Figure 2Example 6 served as the initial process control, without using compound nitrogen supplementation to control the specific oxygen consumption rate. It was clearly observed that after 28 hours, the specific oxygen consumption rate remained significantly lower than in Examples 1-5, at only 1.5-1.8 mmol / h / g. This indicates that the bacterial metabolism was relatively poor in the later stages, with more glucose being consumed as energy, resulting in a lower tryptophan synthesis rate. No significant accumulation of the byproduct glutamate was observed.

[0087] This invention is not limited to the five embodiments provided. Any improvement of components or enhancement of fermentation potency using similar process strategies as described in this patent is within the scope of protection of this patent.

[0088] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. A method for improving tryptophan production based on controlling the specific oxygen consumption rate, characterized in that, The method involves controlling the oxygen consumption per unit dry weight per unit time during the fermentation of *E. coli* to produce tryptophan in stages, when the oxygen consumption per unit dry weight per unit time drops to 5.0 mmol / h / g. The compound nitrogen feed consists of 0.2 g / L amino acid powder, 0.3 g / L corn steep liquor powder, 0.2 g / L yeast powder, 0.03 g / L ammonium sulfate, 0.01 g / L defoamer, and the remainder is water, with a pH of 8.

0. The staged control refers to controlling the oxygen consumption per unit dry weight per unit time to 3.5-4.0 mmol / h / g during fermentation 8-20 h; 3.0-3.5 mmol / h / g during fermentation 20-32 h; and 2.5-3.0 mmol / h / g from fermentation 32 h until the end of fermentation.

2. The method according to claim 1, characterized in that, The fermentation process employs a stirring mechanism to control dissolved oxygen levels to be no lower than 20%.

3. The method according to claim 1, characterized in that, During the fermentation process, when the glucose concentration in the fermentation broth is lower than 1.0 g / L after 2-4 hours of fermentation, a glucose solution with a concentration of 650 g / L is added to maintain the glucose concentration in the fermentation broth at 0.01-0.2 g / L.

4. The method according to claim 1, characterized in that, The process prior to fermentation also includes slant culture, primary seed culture, and secondary seed culture of Escherichia coli. The slant culture medium consisted of 10 g / L peptone, 5 g / L yeast extract, 10 g / L beef extract, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 15 g / L agar powder, and 0.05 g / L tetracycline, with the remainder being water. The seed culture medium consisted of 10 g / L glucose, 4 g / L yeast extract, 0.5 g / L citric acid, 1 g / L ammonium sulfate, 1 g / L potassium dihydrogen phosphate, 1 mg / L vitamin B1, 0.35 mg / L biotin, 1.5 g / L magnesium sulfate heptahydrate, 2.8 mg / L ferrous sulfate heptahydrate, 2 mL / L trace element mixed solution, and the remainder was water. The culture medium used for fermentation consisted of 30 g / L glucose, 10 g / L yeast extract, 2.5 g / L citric acid, 4 g / L ammonium sulfate, 5 g / L potassium dihydrogen phosphate, 2.8 g / L magnesium sulfate heptahydrate, 90 mg / L ferrous sulfate heptahydrate, 5 mg / L vitamin B1, 2 mg / L biotin, and 4 mL / L of a mixed solution of trace elements.

5. The method according to claim 4, characterized in that, The primary seed culture involves inoculating the bacterial cells from the slant culture into a seed culture medium and culturing them at 37°C and 220 rpm in a shake flask for 10-12 hours.

6. The method according to claim 4, characterized in that, The secondary seed culture involves inoculating the primary seed culture solution into the seed culture medium at an inoculation rate of 1%, and culturing it for 10-12 h at 36-37℃, an aeration ratio of 0.5 VVM, 200 rpm, a tank pressure of 0.03-0.05 MPa, a pH of 7.0-7.2, and dissolved oxygen ≥30%.

7. The method according to claim 4, characterized in that, The fermentation process involves inoculating the secondary seed culture at a rate of 10% into the fermentation medium and culturing it at 36-37°C, with an aeration ratio of 1.0 VVM, dissolved oxygen ≥20%, tank pressure of 0.03-0.05 MPa, and pH 6.8-7.0 for 36-40 h.

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