A method for regulating N-acetylglucosamine fermentation based on oxygen uptake rate

Abstract

This invention provides a fermentation method for N-acetylglucosamine based on oxygen uptake rate regulation, comprising the following steps: S1: Activating and seeding an engineered bacterium that produces N-acetylglucosamine through induction with isopropyl-β-D-thiogalactoside to obtain a seed culture; S2: Inoculating the seed culture obtained in step S1 into a fermenter containing fermentation medium for fermentation, monitoring the oxygen uptake rate (OUR) of the fermentation broth during fermentation, and adding ammonium sulfate when the OUR rises; S3: Adding glucose solution during fermentation and continuing cultivation until the fermentation endpoint. This method, by monitoring the oxygen uptake rate in real time during fermentation and using its characteristic rise as the trigger signal for adding ammonium sulfate, achieves precise intervention at the metabolic inflection point of the bacteria, effectively inhibiting the abnormal rise of OUR caused by metabolic imbalance, prolonging the efficient synthesis period of the product, and thus significantly improving the yield and conversion rate of N-acetylglucosamine.

Description

Technical Field

[0001] This invention belongs to the field of fermentation engineering technology, and particularly relates to a fermentation method for N-acetylglucosamine based on oxygen uptake rate regulation. Background Technology

[0002] N-acetylglucosamine (GlcNAc) is an important building block of many polysaccharides in living organisms and has wide applications in medicine, food, cosmetics, and other fields. Microbial fermentation is the main method for producing N-acetylglucosamine.

[0003] In the biosynthesis pathway of N-acetylglucosamine, glutamine serves as the amino group donor, and its synthesis requires the consumption of ammonium ions. Therefore, ensuring a sufficient nitrogen source supply during fermentation is one of the key strategies for increasing N-acetylglucosamine yield. Existing technologies typically employ nitrogen supplementation to enhance yield. For example, patent document CN104988196A discloses a method for adding urea during fermentation; patent document CN117821546A discloses a method for supplementing with a composite nitrogen source comprising corn steep liquor powder, yeast powder, and ammonium sulfate; and patent document CN120310865A discloses a method for adding arginine and histidine to the feed. These methods have all improved the yield or conversion rate of N-acetylglucosamine to some extent.

[0004] However, in actual production, due to the complexity of the fermentation system, the metabolic state of the cells is constantly changing. Conventional nitrogen source supplementation strategies, such as constant-rate feeding or feeding based on parameters such as residual sugar concentration, often fail to accurately match the metabolic needs of the cells at different physiological stages, especially in the middle and late stages of N-acetylglucosamine synthesis, where product synthesis efficiency often encounters bottlenecks.

[0005] On the other hand, oxygen uptake rate (OUR) is a key physiological parameter reflecting the respiratory and metabolic activity of microorganisms, and can indicate the physiological state of the cells in real time and accurately. There are existing reports on the use of OUR for fermentation process regulation. For example, patent document CN121249824A discloses a method for improving the production efficiency of Bacillus subtilis lipopeptide sodium using OUR feedback control. This method addresses the problem of OUR decline during fermentation by continuously adding threonine and ammonium sulfate, aiming to maintain OUR at a high and stable level to promote product synthesis. Summary of the Invention

[0006] This invention proposes a fermentation method for N-acetylglucosamine based on oxygen uptake rate regulation. By monitoring the oxygen uptake rate in real time during fermentation and using its characteristic rise as a trigger signal for ammonium sulfate supplementation, precise intervention at the metabolic inflection point of the cells is achieved. This effectively inhibits the abnormal rise of OUR caused by metabolic imbalance, stabilizes the respiratory metabolic activity of the cells, prolongs the efficient synthesis period of the product, and thus significantly improves the yield and conversion rate of N-acetylglucosamine.

[0007] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0008] A fermentation method for N-acetylglucosamine based on oxygen uptake rate regulation includes the following steps:

[0009] S1: The engineered bacteria that produce N-acetylglucosamine by inducing expression through isopropyl-β-D-thiogalactoside (IPTG) are activated and cultured to obtain seed culture;

[0010] S2: Inoculate the seed culture obtained in step S1 into a fermenter containing fermentation medium for fermentation culture. Monitor the oxygen uptake rate (OUR) of the fermentation broth during fermentation. When the OUR rises, add ammonium sulfate to the fermentation broth.

[0011] S3: Add glucose solution during fermentation and continue culturing until the fermentation endpoint to obtain a fermentation broth containing N-acetylglucosamine.

[0012] This invention identifies a characteristic rise in OUR (oxygen uptake) during the mid-to-late stages of N-acetylglucosamine fermentation by real-time monitoring of oxygen uptake during the fermentation process. This rise is then used as a key point for fermentation regulation, providing a scientific basis for precise intervention. In microbial fermentation, changes in oxygen uptake directly reflect the dynamic evolution of the overall metabolic activity of the microorganisms. This invention, through continuous tracking of the OUR curve, reveals a characteristic rise in OUR at a specific stage of N-acetylglucosamine fermentation (approximately 12-16 hours). Further analysis by the inventors reveals that this rise is not a positive signal of increased metabolic activity, but rather indicates a shift in the internal metabolic balance of the microorganisms—at this point, the product synthesis rate slows significantly, indicating a mismatch between carbon and nitrogen source supply and the microbial synthesis requirements. While existing technologies have reported the use of OUR for fermentation regulation, their focus is usually on maintaining a high OUR level to promote growth and product synthesis, failing to recognize the metabolic bottleneck revealed by the OUR rise phenomenon, and even less so as to use it as a starting point for regulation. This invention is the first to identify an increase in OUR as a metabolic turning point that requires intervention, shifting the regulatory strategy from "passive maintenance" to "active intervention," which has clear physiological significance.

[0013] Based on this discovery, this invention further investigated effective intervention methods during the OUR recovery phase and found that supplementing with ammonium sulfate during this phase effectively regulates the respiratory metabolic state of the cells, and this is not simply a matter of nitrogen supplementation. During fermentation, ammonia water, acting as a pH regulator, is continuously added, providing the cells with a basic source of ammonium ions. Therefore, under conventional understanding, the necessity of additional ammonium sulfate supplementation is not significant. However, this invention found that supplementing with ammonium sulfate at this specific OUR recovery point can produce a regulatory effect beyond simply supplementing the nitrogen source: appropriate amounts of ammonium sulfate can effectively inhibit abnormal OUR recovery, maintaining the cell's respiratory metabolism at a level favorable for product synthesis; conversely, excessive or improper supplementation of ammonium sulfate may lead to a continuous decrease in OUR, which is detrimental to product accumulation. This finding indicates that during the OUR recovery phase, ammonium sulfate acts more like a "metabolic regulator"—it helps the cells re-establish metabolic balance by affecting the activity of respiratory chain-related enzymes or regulating the expression of nitrogen metabolism-related genes, rather than simply supplementing ammonium ions that may not be lacking in the culture medium. This precise regulation based on the physiological needs of bacteria is fundamentally different from the strategy of continuously adding ammonium sulfate to maintain a constant ammonium ion concentration in existing technologies, both in terms of regulatory logic and mechanism of action.

[0014] This metabolic regulation is further manifested in the synergistic utilization of carbon and nitrogen sources. The biosynthesis of N-acetylglucosamine requires carbon sources to provide energy and a molecular skeleton, while nitrogen sources provide amino groups. When metabolic imbalance occurs during the recovery phase of the OUR (respiratory osmotic pressure), the efficiency of carbon source utilization often decreases, and some carbon flow may be diverted to maintenance energy consumption or byproduct generation pathways, leading to product synthesis being hindered. In this invention, the addition of ammonium sulfate at this point helps restore the normal respiratory and metabolic function of the cells, enabling the subsequently added glucose solution to be converted into the target product more efficiently. This "carbon and nitrogen source linkage regulation" mode, using OUR as a bridge, allows the fermentation system to maintain a relatively stable metabolic state for a longer period, effectively delaying the metabolic decline of the cells, thereby extending the product synthesis cycle and avoiding premature termination of fermentation due to metabolic imbalance.

[0015] In addition, unlike traditional feeding strategies that rely on fixed time points or offline detection data, this invention uses oxygen uptake rate, a real-time physiological indicator, as the basis for regulation. It can dynamically respond to individual differences in metabolic inflection points of different batches and different cell states, so that each batch of fermentation can obtain an intervention opportunity that matches its actual metabolic process.

[0016] Preferably, the ammonium sulfate supplementation method in step S2 is intermittent fed-batch. Intermittent fed-batch can dynamically match the metabolic fluctuations of the cells during the OUR recovery phase, avoiding metabolic inhibition caused by excessive supply. During the OUR recovery phase, the metabolic state of the cells is not constant but fluctuates. With continuous fed-batch, ammonium sulfate continuously enters the fermentation system at a constant rate, which may lead to excessive accumulation of ammonium ions during periods of lower metabolic demand. According to the basic principles of microbial metabolic regulation, excessive ammonium ions may affect the activity of key enzymes such as glutamine synthase through feedback inhibition mechanisms, or change intracellular pH homeostasis, thereby inhibiting the respiratory metabolism of the cells and causing a continuous decrease in OUR—this is precisely the phenomenon observed during the research and development of this invention. Therefore, this invention adopts an intermittent fed-batch method of ammonium sulfate, achieving "on-demand supply" and avoiding the metabolic burden caused by excessive accumulation of ammonium ions.

[0017] The intermittent addition is as follows: using the rise and fall of OUR (Oxygen Flow Rate) as a feedback signal, ammonium sulfate is added when OUR rises, stops when OUR falls, and resumes when OUR rises again. This invention uses the rise and fall of OUR as the start and stop signal for ammonium sulfate addition, essentially using the "physiological language" of the bacteria as the regulatory command: when the bacterial metabolism shows an imbalance signal (OUR rises), intervention is immediately initiated; when the bacterial metabolism tends to stabilize (OUR falls), intervention is paused to avoid over-treatment. This dynamic response mechanism allows the ammonium sulfate supply timeline to precisely match the timeline of bacterial metabolic fluctuations, achieving true "on-demand supply." It is worth noting that neither continuous feed nor timed intermittent feed can respond to real-time changes in cell metabolism: continuous feed may lead to excessive accumulation of ammonium ions during the metabolic plateau, affecting the activity of key enzymes such as glutamine synthase through feedback inhibition mechanisms, thereby inhibiting respiratory metabolism—this is precisely the phenomenon observed during the development of this invention where uniform feed resulted in a continuous decrease in OUR; timed intermittent feed may result in misalignment due to the fixed time interval not matching the actual metabolic rhythm of the cells, leading to "feeding when it should have been added and adding when it shouldn't have been added." This invention, however, uses feedback control with OUR rise and fall as start / stop signals, ensuring that each ammonium sulfate supply precisely falls at the moment when the cells exhibit metabolic fluctuations, and each pause corresponds to a period when cell metabolism tends to stabilize, thus maximizing the regulatory effect in dynamic equilibrium while minimizing potential metabolic interference. Furthermore, in actual production, even with the same strain and culture medium formulation, the fermentation process may still vary between different batches—OUR recovery may occur at 12 hours or be delayed to 16 hours; the magnitude and duration of the recovery may also vary from batch to batch. Traditional feeding strategies that rely on fixed time points cannot adapt to such differences. However, this invention uses OUR rise and fall as a start / stop signal for feedback control, thereby achieving "batch adaptability" and significantly improving the robustness and reproducibility of the process.

[0018] Preferably, the ammonium sulfate in step S2 is added in solution form, with a concentration of 70-110 g / L and a flow rate of 20-40 g / h. This concentration range balances supply efficiency and metabolic burden. If the ammonium sulfate concentration is too low (below 70 g / L), a large amount of liquid needs to be added to achieve an effective dosage adjustment, which may dilute the fermentation system and increase the risk of contamination. If the concentration is too high (above 110 g / L), the excessively high local instantaneous osmotic pressure can easily cause stress damage to the cells, and solubility limitations may affect the stability of the feed rate. This feed rate effectively inhibits the rise in OUR (Oxidation Tolerance) without triggering feedback inhibition due to excessively rapid nitrogen supply causing an instantaneous excessive accumulation of ammonium ions. If the rate is too low, the adjustment effect will be insignificant; if the rate is too high, it may lead to a continuous decrease in OUR.

[0019] Preferably, the concentration of the ammonium sulfate solution is 90 g / L, and the flow rate is 30 g / h. At this concentration, ammonium sulfate is stable and does not easily crystallize during the feeding process, ensuring the continuity and accuracy of the feeding. Simultaneously, the amount of ammonium ions carried per unit volume is moderate, avoiding the dilution effect on the fermentation system caused by excessive replenishment to achieve the desired dosage. Moreover, at this rate, the amount of ammonium sulfate supplied each time is just sufficient to stabilize the respiratory metabolism of the cells, without accumulating during the metabolically stable period due to excessive supply in a single operation.

[0020] Preferably, step S2 further includes: when the OD value in the fermentation broth reaches 32, adding the inducing agent IPTG and reducing the tank temperature from 34℃±0.5℃ to 33℃±0.5℃. When the OD value reaches 32, the cells have entered the late logarithmic growth phase, and the biomass accumulation is sufficient. Initiating induction at this time can ensure a sufficient number of enzyme-producing cells and avoid the drawbacks of inhibiting cell growth due to premature induction and prolonging the fermentation cycle due to late induction. The 1℃ temperature reduction moderately slows down the growth and metabolic rate of the cells, diverting more carbon sources and energy to the N-acetylglucosamine synthesis pathway. At the same time, the mild cooling stress helps maintain the metabolic activity of the cells and avoids physiological damage caused by drastic cooling.

[0021] Preferably, during fermentation, the pH of the fermentation broth is controlled at 6.9±0.1 by adding ammonia. This pH condition can maintain the normal growth and metabolic activity of the cells. The addition of ammonia not only adjusts the pH but also continuously replenishes ammonium ions, providing a basic nitrogen source for the cells. This allows the subsequent addition of ammonium sulfate during the OUR recovery phase to focus on "metabolic regulation" rather than simply supplementing the nitrogen source.

[0022] Preferably, the control method for adding glucose solution in step S3 is as follows: Addition begins when the residual glucose concentration in the fermentation broth decreases to 0.5 g / 100 mL. Before induction, the residual glucose concentration is controlled at 0.3-0.5 g / 100 mL. After induction, the glucose addition rate is adjusted to control the dissolved oxygen (DO) at 37.5% ± 2.5%, and the glucose concentration is controlled at 0-0.05 g / 100 mL. Controlling the residual sugar at 0.3-0.5 g / 100 mL before induction ensures sufficient carbon source supply during the cell growth stage. After induction, adjusting the sugar addition rate controls the DO at 37.5% ± 2.5%, while simultaneously reducing the residual sugar to 0-0.05 g / 100 mL, forming a closed-loop control system of "DO feedback guiding sugar addition". This differentiated control strategy before and after induction not only satisfies the different carbon source requirements at different physiological stages, but also avoids metabolic fluctuations caused by excessive or insufficient carbon sources through the dual constraints of DO and residual sugars, providing a stable carbon source supply environment for the efficient synthesis of products after supplementing ammonium sulfate during the OUR recovery stage.

[0023] Preferably, the fermentation endpoint in step S3 is when the product growth is less than 5 g / L every 4 hours. This invention uses a product growth of less than 5 g / L every 4 hours as the endpoint for discharge, changing the traditional practice of a fixed fermentation cycle. This avoids resource waste and increased costs caused by excessively long fermentation times, and ensures that each batch is harvested at the moment of highest product synthesis efficiency, achieving precise control over the timing of fermentation termination.

[0024] Preferably, the seed culture in step S1 includes mother bottle seed culture and seed tank seed culture, wherein the mother bottle seed culture is carried out until OD. 610 Transplant when the seed reaches 3-4, and cultivate the seeds in the seed tank to OD. 600 Transplanting occurs when the culture reaches 14-15 minutes. These two criteria ensure that the seed culture entering the next stage of cultivation is in the late logarithmic growth phase, with high cell activity and vigorous metabolism. This provides a consistent and vigorous seed culture for subsequent OUR monitoring and precise ammonium sulfate control in the fermenter, guaranteeing the stability and reproducibility of the entire fermentation process from the source.

[0025] In summary, this N-acetylglucosamine fermentation method based on oxygen uptake rate regulation achieves precise intervention at the metabolic inflection point of the cells by monitoring the oxygen uptake rate in real time during fermentation and using its characteristic rebound as a trigger signal for ammonium sulfate supplementation. This effectively inhibits the abnormal rebound of OUR caused by metabolic imbalance, stabilizes the respiratory metabolic activity of the cells, prolongs the efficient synthesis period of the product, and thus significantly improves the yield and conversion rate of N-acetylglucosamine. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is a monitoring curve of OUR in an embodiment of the present invention. Detailed Implementation

[0028] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0029] A fermentation method for N-acetylglucosamine based on oxygen uptake rate regulation includes the following steps:

[0030] S1: The engineered bacteria that produce N-acetylglucosamine by inducing expression through isopropyl-β-D-thiogalactoside (IPTG) are activated and cultured to obtain seed culture;

[0031] S2: Inoculate the seed culture obtained in step S1 into a fermenter containing fermentation medium for fermentation culture. Monitor the oxygen uptake rate (OUR) of the fermentation broth during fermentation. When the OUR rises, add ammonium sulfate to the fermentation broth.

[0032] S3: Add glucose solution during fermentation and continue culturing until the fermentation endpoint to obtain a fermentation broth containing N-acetylglucosamine.

[0033] This invention identifies a characteristic rise in OUR (oxygen uptake) during the mid-to-late stages of N-acetylglucosamine fermentation by real-time monitoring of oxygen uptake during the fermentation process. This rise is then used as a key point for fermentation regulation, providing a scientific basis for precise intervention. In microbial fermentation, changes in oxygen uptake directly reflect the dynamic evolution of the overall metabolic activity of the microorganisms. This invention, through continuous tracking of the OUR curve, reveals a characteristic rise in OUR at a specific stage of N-acetylglucosamine fermentation (approximately 12-16 hours). Further analysis by the inventors reveals that this rise is not a positive signal of increased metabolic activity, but rather indicates a shift in the internal metabolic balance of the microorganisms—at this point, the product synthesis rate slows significantly, indicating a mismatch between carbon and nitrogen source supply and the microbial synthesis requirements. While existing technologies have reported the use of OUR for fermentation regulation, their focus is usually on maintaining a high OUR level to promote growth and product synthesis, failing to recognize the metabolic bottleneck revealed by the OUR rise phenomenon, and even less so as to use it as a starting point for regulation. This invention is the first to identify an increase in OUR as a metabolic turning point that requires intervention, shifting the regulatory strategy from "passive maintenance" to "active intervention," which has clear physiological significance.

[0034] Based on this discovery, this invention further investigated effective intervention methods during the OUR recovery phase and found that supplementing with ammonium sulfate during this phase effectively regulates the respiratory metabolic state of the cells, and this is not simply a matter of nitrogen supplementation. During fermentation, ammonia water, acting as a pH regulator, is continuously added, providing the cells with a basic source of ammonium ions. Therefore, under conventional understanding, the necessity of additional ammonium sulfate supplementation is not significant. However, this invention found that supplementing with ammonium sulfate at this specific OUR recovery point can produce a regulatory effect beyond simply supplementing the nitrogen source: appropriate amounts of ammonium sulfate can effectively inhibit abnormal OUR recovery, maintaining the cell's respiratory metabolism at a level favorable for product synthesis; conversely, excessive or improper supplementation of ammonium sulfate may lead to a continuous decrease in OUR, which is detrimental to product accumulation. This finding indicates that during the OUR recovery phase, ammonium sulfate acts more like a "metabolic regulator"—it helps the cells re-establish metabolic balance by affecting the activity of respiratory chain-related enzymes or regulating the expression of nitrogen metabolism-related genes, rather than simply supplementing ammonium ions that may not be lacking in the culture medium. This precise regulation based on the physiological needs of bacteria is fundamentally different from the strategy of continuously adding ammonium sulfate to maintain a constant ammonium ion concentration in existing technologies, both in terms of regulatory logic and mechanism of action.

[0035] This metabolic regulation is further manifested in the synergistic utilization of carbon and nitrogen sources. The biosynthesis of N-acetylglucosamine requires carbon sources to provide energy and a molecular skeleton, while nitrogen sources provide amino groups. When metabolic imbalance occurs during the recovery phase of the OUR (respiratory osmotic pressure), the efficiency of carbon source utilization often decreases, and some carbon flow may be diverted to maintenance energy consumption or byproduct generation pathways, leading to product synthesis being hindered. In this invention, the addition of ammonium sulfate at this point helps restore the normal respiratory and metabolic function of the cells, enabling the subsequently added glucose solution to be converted into the target product more efficiently. This "carbon and nitrogen source linkage regulation" mode, using OUR as a bridge, allows the fermentation system to maintain a relatively stable metabolic state for a longer period, effectively delaying the metabolic decline of the cells, thereby extending the product synthesis cycle and avoiding premature termination of fermentation due to metabolic imbalance.

[0036] In addition, unlike traditional feeding strategies that rely on fixed time points or offline detection data, this invention uses oxygen uptake rate, a real-time physiological indicator, as the basis for regulation. It can dynamically respond to individual differences in metabolic inflection points of different batches and different cell states, so that each batch of fermentation can obtain an intervention opportunity that matches its actual metabolic process.

[0037] Furthermore, in step S2, ammonium sulfate is added intermittently. Intermittent addition dynamically matches the metabolic fluctuations of the cells during the OUR recovery phase, avoiding metabolic inhibition caused by excessive supply. During the OUR recovery phase, the metabolic state of the cells is not constant but fluctuates. With continuous addition, ammonium sulfate enters the fermentation system at a constant rate, which may lead to excessive accumulation of ammonium ions during periods of lower metabolic demand. According to the basic principles of microbial metabolic regulation, excessive ammonium ions may affect the activity of key enzymes such as glutamine synthase through feedback inhibition mechanisms, or alter intracellular pH homeostasis, thereby inhibiting the respiratory metabolism of the cells and causing a continuous decrease in OUR—a phenomenon observed during the development of this invention. Therefore, this invention uses intermittent addition of ammonium sulfate to achieve "on-demand supply," avoiding the metabolic burden caused by excessive accumulation of ammonium ions.

[0038] The intermittent feeding method involves using the rise and fall of OUR (Oxygen Flow Rate) as a feedback signal. Ammonium sulfate is fed in when OUR rises, stopped when OUR falls, and resumed when OUR rises again. This invention uses the rise and fall of OUR as the start and stop signal for ammonium sulfate feeding, essentially using the "physiological language" of the bacteria as the regulatory command: when an imbalance signal appears in bacterial metabolism (OUR rises), intervention is immediately initiated; when bacterial metabolism tends to stabilize (OUR falls), intervention is paused to avoid over-feeding. This dynamic response mechanism precisely matches the timing of ammonium sulfate supply with the timing of bacterial metabolic fluctuations, achieving true "on-demand supply."

[0039] It is worth noting that neither continuous feed nor timed intermittent feed can respond to real-time changes in cell metabolism: continuous feed may lead to excessive accumulation of ammonium ions during the metabolic plateau, affecting the activity of key enzymes such as glutamine synthase through feedback inhibition mechanisms, thereby inhibiting respiratory metabolism—this is precisely the phenomenon observed during the development of this invention where uniform feed resulted in a continuous decrease in OUR; timed intermittent feed may result in misalignment due to the fixed time interval not matching the actual metabolic rhythm of the cells, leading to "feeding when it should have been added and adding when it shouldn't have been added." This invention, however, uses feedback control with OUR rise and fall as start / stop signals, ensuring that each ammonium sulfate supply precisely falls at the moment when the cells exhibit metabolic fluctuations, and each pause corresponds to a period when cell metabolism tends to stabilize, thus maximizing the regulatory effect in dynamic equilibrium while minimizing potential metabolic interference. Furthermore, in actual production, even with the same strain and culture medium formulation, the fermentation process may still vary between different batches—OUR recovery may occur at 12 hours or be delayed to 16 hours; the magnitude and duration of the recovery may also vary from batch to batch. Traditional feeding strategies that rely on fixed time points cannot adapt to such differences. However, this invention uses OUR rise and fall as a start / stop signal for feedback control, thereby achieving "batch adaptability" and significantly improving the robustness and reproducibility of the process.

[0040] Furthermore, in step S2, ammonium sulfate is added in solution form with a concentration of 70-110 g / L and a flow rate of 20-40 g / h. This concentration range balances supply efficiency and metabolic burden. If the ammonium sulfate concentration is too low (below 70 g / L), a large amount of liquid needs to be added to achieve an effective dosage adjustment, potentially diluting the fermentation system and increasing the risk of contamination. If the concentration is too high (above 110 g / L), excessively high local instantaneous osmotic pressure can easily cause stress damage to the cells, and solubility limitations may affect the stability of the feed rate. This feed rate effectively inhibits the rise in OUR without triggering feedback inhibition due to excessively rapid nitrogen supply causing an instantaneous accumulation of ammonium ions. A rate that is too low will have little regulatory effect, while a rate that is too high may lead to a continuous decrease in OUR.

[0041] Furthermore, the concentration of the ammonium sulfate solution was 90 g / L, and the flow rate was 30 g / h. At this concentration, ammonium sulfate is stable and does not easily crystallize during the feeding process, ensuring the continuity and accuracy of the feeding. Simultaneously, the amount of ammonium ions carried per unit volume is moderate, avoiding the dilution effect on the fermentation system caused by excessive replenishment to achieve the desired dosage. Moreover, at this rate, the amount of ammonium sulfate supplied each time is just sufficient to stabilize the respiratory metabolism of the cells, without accumulating during the metabolic plateau due to excessive supply in a single operation.

[0042] Furthermore, step S2 also includes: when the OD value in the fermentation broth reaches 32, adding the inducing agent IPTG and lowering the tank temperature from 34℃±0.5℃ to 33℃±0.5℃. When the OD value reaches 32, the cells have entered the late logarithmic growth phase, and biomass accumulation is sufficient. Initiating induction at this time ensures a sufficient number of enzyme-producing cells while avoiding the drawbacks of inhibited cell growth due to premature induction and prolonged fermentation cycle due to delayed induction. The 1℃ temperature reduction moderately slows down the cell growth and metabolic rate, diverting more carbon and energy to the N-acetylglucosamine synthesis pathway. At the same time, the mild cooling stress helps maintain the metabolic activity of the cells and avoids physiological damage caused by drastic cooling.

[0043] Furthermore, during the fermentation process, the pH of the fermentation broth was controlled at 6.9±0.1 by adding ammonia. This pH condition can maintain the normal growth and metabolic activity of the cells. The addition of ammonia not only adjusts the pH but also continuously replenishes ammonium ions, providing a basic nitrogen source for the cells. This allows the subsequent addition of ammonium sulfate during the OUR recovery phase to focus on "metabolic regulation" rather than simply supplementing the nitrogen source.

[0044] Furthermore, the control method for adding glucose solution in step S3 is as follows: Addition begins when the residual glucose concentration in the fermentation broth decreases to 0.5 g / 100 mL. Before induction, the residual glucose concentration is controlled at 0.3-0.5 g / 100 mL. After induction, the glucose addition rate is adjusted to control DO at 37.5% ± 2.5%, and the glucose concentration is controlled at 0-0.05 g / 100 mL. Controlling residual sugar at 0.3-0.5 g / 100 mL before induction ensures sufficient carbon source supply during the cell growth stage. After induction, adjusting the sugar addition rate controls DO at 37.5% ± 2.5%, while simultaneously reducing residual sugar to 0-0.05 g / 100 mL, forming a closed-loop control system of "DO feedback guiding sugar addition". This differentiated control strategy before and after induction not only satisfies the different carbon source requirements at different physiological stages, but also avoids metabolic fluctuations caused by excessive or insufficient carbon sources through the dual constraints of DO and residual sugars, providing a stable carbon source supply environment for the efficient synthesis of products after supplementing ammonium sulfate during the OUR recovery stage.

[0045] Furthermore, in step S3, the fermentation endpoint is reached when the product growth is less than 5 g / L every 4 hours. This invention uses a product growth of less than 5 g / L every 4 hours as the endpoint for discharge, changing the traditional practice of a fixed fermentation cycle. This avoids resource waste and increased costs caused by excessively long fermentation times, and ensures that each batch is harvested at the point of highest product synthesis efficiency, achieving precise control over the timing of fermentation termination.

[0046] Furthermore, the seed culture in step S1 includes mother bottle seed culture and seed tank seed culture, with the mother bottle seed culture reaching OD.610 Transplant when the seed reaches 3-4, and culture the seeds in the seed tank until OD. 600 Transplanting occurs when the culture reaches 14-15 minutes. These two criteria ensure that the seed culture entering the next stage of cultivation is in the late logarithmic growth phase, with high cell activity and vigorous metabolism. This provides a consistent and vigorous seed culture for subsequent OUR monitoring and precise ammonium sulfate control in the fermenter, guaranteeing the stability and reproducibility of the entire fermentation process from the source.

[0047] In summary, this N-acetylglucosamine fermentation method based on oxygen uptake rate regulation achieves precise intervention at the metabolic inflection point of the cells by monitoring the oxygen uptake rate in real time during fermentation and using its characteristic rebound as a trigger signal for ammonium sulfate supplementation. This effectively inhibits the abnormal rebound of OUR caused by metabolic imbalance, stabilizes the respiratory metabolic activity of the cells, prolongs the efficient synthesis period of the product, and thus significantly improves the yield and conversion rate of N-acetylglucosamine.

[0048] To clearly demonstrate the technical effects and inventiveness of the present invention, the technical solution of the present invention is described below through Examples 1, 2, and Comparative Example 1. Examples 1 and 2 both fall within the scope of the present invention's technical concept, i.e., ammonium sulfate is added when an increase in OUR is detected; the difference lies in the addition method—Example 1 uses a uniform flow rate addition, while Example 2 uses an intermittent flow rate addition. Comparative Example 1 shows the initial process without adding ammonium sulfate, serving as a benchmark for effect comparison.

[0049] The engineered bacteria used in the embodiments and comparative examples of this invention to produce N-acetylglucosamine are the *E. coli* engineered bacteria specifically disclosed in patent document CN121294482A (title: A method for constructing a high-yield N-acetylglucosamine-producing *E. coli* engineered bacteria). Those skilled in the art can reproduce and prepare this strain based on the teachings of this patent document, thereby implementing this invention.

[0050] Unless otherwise specified, the culture methods used in the embodiments and comparative examples of this invention are as follows:

[0051] Mother bottle seed culture medium (1L Erlenmeyer flask, 150mL liquid volume), seed culture medium (50L seed tank, 30L liquid volume), fermentation culture medium (50L fermenter, 18.3L liquid volume).

[0052] Fermentation fed-batch culture medium:

[0053] 1. Glucose solution (600g / kg): 16.67kg glucose monohydrate, 7.83kg purified water, 57.75g potassium dihydrogen phosphate. Mix thoroughly and sterilize at 118℃ for 20 minutes. 2. Inducer solution (0.32mM): 1.68g IPTG inducer, 50mL purified water. Dissolve and sterilize at 118℃ for 20 minutes. 3. Ammonium sulfate solution (90g / L): 90g ammonium sulfate, diluted to 1L with purified water. Dissolve and sterilize. 4. Ammonia water: 27-28% concentration, used for pH adjustment. 5. Defoamer (50% concentration): used for foam control.

[0054] Comparative Example 1: Initial process (without adding ammonium sulfate)

[0055] I. Seeds in Mother Bottle

[0056] Inoculation volume: 0.22 mL / 150 mL (1 L Erlenmeyer flask)

[0057] Breathable medium: breathable membrane

[0058] Incubation temperature: 37℃±1℃

[0059] Shaker speed: 220 rpm

[0060] Incubation time: based on controlling OD.

[0061] Transplantation Standard: OD 610 3-4

[0062] II. Seeds in a 50L seed jar

[0063] Inoculation amount: 1% (300ml seed culture in a shake flask inoculated into 30L culture medium);

[0064] Incubation temperature: 34℃±0.5℃;

[0065] Tank pressure: 0.05 MPa ± 0.005 MPa

[0066] Airflow control: After inoculation, control the airflow to 30L / min.

[0067] Stirring speed control: After inoculation, the initial stirring speed should be controlled at 250 rpm, and the stirring speed should not exceed 500 rpm.

[0068] DO control: Calibration method for DO electrode: Before transplanting, adjust the fermenter temperature, pressure, air flow rate, and stirring speed to 31.5℃, 0.05Mpa, 30L / min, and 500rpm, respectively. After the DO detection value stabilizes, calibrate the DO value to 100%. During seed culture, when the DO drops to 50%, increase the stirring speed to control the DO at 30%±10%.

[0069] pH control: Before transplanting, the pH of the seed culture medium was adjusted to 6.9 with ammonia water. During fermentation, ammonia water was added by a peristaltic pump controlled by software to control the pH of the fermentation broth at 6.9±0.1.

[0070] Incubation time: based on OD (Occurrence Discharge).

[0071] Transplantation criteria: Normal sterility under microscopic examination, OD 600 :14-15.

[0072] III. 50L Fermentation Tank

[0073] Inoculation amount: 16.8% (3.7L seed culture transferred into 18.3L culture medium).

[0074] Tank temperature: After transplanting, control the tank temperature at 34℃±0.5℃; when the OD reaches 32, add an inducer and then lower the temperature to 33℃±0.5℃ until the seed is removed from the tank.

[0075] Tank pressure: After transplanting, control the tank pressure at 0.06 MPa ± 0.005 MPa.

[0076] Air flow control: After inoculation, the initial air flow was controlled at 25 L / min. After 4 hours, the air flow was increased to 50 L / min. After induction, the air flow was reduced to 30 L / min and kept constant.

[0077] Stirring speed control: After inoculation, the initial stirring speed is controlled at 250 rpm, and the maximum stirring speed shall not exceed 750 rpm. After induction, the stirring speed is maintained at its maximum value for 1 hour, and then the speed is reduced by 15 rpm every hour until it reaches 400 rpm, after which it is maintained.

[0078] DO Control: DO Electrode Calibration Method: Before transfer, adjust the fermenter temperature, pressure, air flow rate, and stirring speed to 34℃, 0.06 MPa, 50 L / min, and 750 rpm, respectively. After the DO value stabilizes, calibrate the DO value to 100%. Before induction, control DO at 35% ± 5% by increasing the stirring speed and air flow rate; after induction, control DO at 37.5% ± 2.5% by controlling the flow rate of the glucose solution. When foaming causes large fluctuations in DO, control the median fluctuation value to 35%. Induction Control: After induction, turn off the stirring speed linkage. When glucose is depleted and DO rapidly recovers, start feeding, controlling the stirring speed as described above, and then control DO by adjusting the feeding rate.

[0079] pH control: Before transplanting, the pH of the fermentation medium was adjusted to 6.9 with ammonia. During the fermentation process, ammonia was added by a peristaltic pump controlled by software to control the pH of the fermentation broth at 6.9±0.1.

[0080] Intermediate feeding:

[0081] (1) Add glucose solution (60g / kg): When the residual glucose concentration in the fermentation broth drops to about 0.5g / 100mL, start adding glucose solution to control the residual glucose concentration in the fermentation broth at 0.3-0.5g / 100mL. After induction, adjust the glucose feeding rate to control DO at 37.5%±2.5% and glucose concentration at 0~0.05g / 100mL. When foaming and DO fluctuations are large, control the glucose concentration at 0.1±0.05g / 100mL, and then control it towards 0.1g / 100mL.

[0082] (2) Add ammonia water (concentration 27-28%): During the fermentation process, the peristaltic pump automatically adds ammonia water through software pH control.

[0083] (3) Add induction agent: When the OD value reaches 32 during fermentation, add all the induction agent.

[0084] (4) Add defoamer (concentration 50%): Start adding defoamer when foaming begins, and add 2 mL every hour regardless of the foam condition afterwards.

[0085] Fermentation cycle: Remove from the tank if the product growth is less than 5g / L every 4 hours.

[0086] Inactivation in the tank: Stop adding ammonia 1 hour in advance to allow the pH to drop to around 6.0, then heat to 55℃ and maintain for 30 minutes.

[0087] The changes in metabolic parameters during the fermentation process of Comparative Example 1 are shown in Table 1.

[0088] Table 1

[0089] Fermentation cycle h <![CDATA[OD 600 ]]> <![CDATA[OUR mol / (m 3 ∙h)]]> Product g / L Conductivity μs / cm Conversion rate % 0 3.01 5 11710 8 44.7 139 12.2 10180 12 53.6 97 37.4 7040 16 59.6 87 56.7 5570 20 68.6 147 68.1 5340 24 75.4 146 74.4 4710 28 77.1 147 89 4130 32 78.3 145 106.6 3990 36 75 119 124.4 4520 40 73.2 115 135.2 8340 44 69.2 97 136.8 12920 48 62.5 73 124.3 14820 37.0

[0090] As shown in Table 1, the OUR (or osmotic pressure) rebounded rapidly after 16 hours (from 87 to 147), while the conductivity also rebounded rapidly after 36 hours. The product content stopped increasing after 44 hours. At the end of fermentation, the N-acetylglucosamine content was 124.3 g / L, and the conversion rate was 37.0%.

[0091] Example 1: Ammonium sulfate is added at a constant rate.

[0092] The fermentation process was the same as in Comparative Example 1, except that the oxygen uptake (OUR) was monitored during fermentation. When the OUR was observed to rise after approximately 16 hours of fermentation, a uniform feed of ammonium sulfate solution was initiated. The feed method was as follows: a 90 g / L ammonium sulfate solution was fed at a rate of 30 g / h, for a total of 500 mL (equivalent to 45 g of solid ammonium sulfate), without any start-stop control based on OUR fluctuations.

[0093] The changes in metabolic parameters during the fermentation process of Example 1 are shown in Table 2.

[0094] Table 2

[0095] Fermentation cycle h <![CDATA[OD 600 ]]> <![CDATA[OUR mol / (m 3 ∙h)]]> Product g / L Conductivity μs / cm Conversion rate % 0 2.6 11 12050 8 45.8 139 12.7 10320 12 56.2 94 37.0 7270 16 64.1 92 55.6 5740 20 69.4 72 74.6 5540 24 64.4 53 88.4 5040 28 61.6 35 94.0 5150 32 61.6 34 110.5 5380 36 62.7 35 110.7 5370 40 62.9 44 123.3 5020 44 64.1 47 133.0 5010 48 66.9 62 142.4 4710 52 65.4 58 146.2 4560 56 64.2 73 149.1 4530 50.3

[0096] As shown in Table 2, after uniform addition of ammonium sulfate, the OUR (or osmolarity) continuously decreased from 92 at 16 h to 35 at 28 h without any rebound. However, the excessively low OUR level led to a decrease in the sugar consumption rate. At the end of fermentation, the N-acetylglucosamine content was 149.1 g / L, and the conversion rate was 50.3%. The conductivity did not show a significant rebound.

[0097] Example 2: Using intermittent flow-through of ammonium sulfate

[0098] The fermentation process was the same as in Comparative Example 1, except that oxygen uptake (OUR) was monitored during fermentation. When OUR began to rise after approximately 16 hours of fermentation, intermittent feeding of ammonium sulfate solution was initiated. The feeding method was as follows: using the rise and fall of OUR as a feedback signal, when OUR rose, 90 g / L of ammonium sulfate solution was fed at a rate of 30 g / h; when OUR fell, the feeding was stopped; and when OUR rose again, the feeding resumed. The total amount of ammonium sulfate fed was approximately 60 g (based on ammonium sulfate solids).

[0099] The changes in metabolic parameters during the fermentation process in Example 2 are shown in Table 3.

[0100] Table 3

[0101] Fermentation cycle h <![CDATA[OD 600 ]]> <![CDATA[OUR mol / (m 3 ∙h)]]> Product g / L Conductivity μs / cm Conversion rate % 0 3.0 23 13980 8 51.2 136 15.3 11480 12 65.4 122 46.5 6910 16 79.8 121 64.9 5290 20 85.7 130 83.3 4990 24 92.2 102 96.3 4140 28 86.8 90 108.2 4070 32 83.2 78 119.0 3880 36 84.6 64 135.4 3600 40 78.6 68 146.0 3600 44 81 60 159.6 4400 48 71.1 50 166.9 3750 52 69.5 42 170.9 3740 56 69.9 42 181.1 3460 50.4

[0102] As shown in Table 3, the rise in OUR was effectively suppressed after intermittent addition of ammonium sulfate, but unlike in Example 1, it did not continue to decline to an excessively low level. Instead, it remained within the range suitable for product synthesis. At the end of fermentation, the N-acetylglucosamine content reached 181.1 g / L, the conversion rate was 50.4%, and the conductivity remained at a low level.

[0103] The fermentation results of Comparative Example 1, Example 1, and Example 2 are summarized in Table 4.

[0104] Table 4

[0105] Process conditions Fermentation cycle (h) Product content (g / L) Conversion rate (%) Comparative Example 1 (without additional entries) 48 124.3 37.0% Example 1 (Uniform flow addition) 56 149.1 50.3% Example 2 (Intermittent Flow Feed) 56 181.1 50.4%

[0106] As can be seen from Table 4:

[0107] 1. Compared with Comparative Example 1, the fermentation cycle of both Example 1 and Example 2 was extended by 8 hours and the conversion rate was increased by about 13 percentage points, indicating that adding ammonium sulfate when OUR rises can effectively improve fermentation performance.

[0108] 2. Compared with Example 1, Example 2 showed a 32.0 g / L increase in product content (21.5%) within the same fermentation cycle, while the conversion rate remained similar. This indicates that, with a similar total ammonium sulfate supplementation, the intermittent feeding method of this invention, which uses OUR rise and fall as feedback signals, can more effectively promote the synthesis of N-acetylglucosamine, and the product accumulation effect is far superior to that of the uniform feeding method.

[0109] In addition, to make the experimental process and results more obvious, the following is attached: Figure 1 -OUR monitoring curves. Among them, Comparative Example 1 is the red curve, Example 1 is the black curve, and Example 2 is the blue curve.

[0110] In summary, this invention, by monitoring oxygen uptake rate in real time and intermittently adding ammonium sulfate based on OUR recovery, can precisely regulate the respiratory and metabolic state of the cells, effectively solving the technical problem of low product synthesis efficiency in the later stages of N-acetylglucosamine fermentation, and achieving significantly better technical results than existing processes.

[0111] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A fermentation method for N-acetylglucosamine based on oxygen uptake rate regulation, characterized in that, Includes the following steps: S1: The engineered bacteria that produce N-acetylglucosamine by inducing expression through isopropyl-β-D-thiogalactoside (IPTG) are activated and cultured to obtain seed culture; S2: Inoculate the seed culture obtained in step S1 into a fermenter containing fermentation medium for fermentation culture. Monitor the oxygen uptake rate (OUR) of the fermentation broth during fermentation. When the OUR rises, add ammonium sulfate to the fermentation broth. S3: Add glucose solution during fermentation and continue culturing until the fermentation endpoint to obtain a fermentation broth containing N-acetylglucosamine.

2. The N-acetylglucosamine fermentation method based on oxygen uptake rate regulation according to claim 1, characterized in that, The method of adding ammonium sulfate in step S2 is intermittent flow addition.

3. The N-acetylglucosamine fermentation method based on oxygen uptake rate regulation according to claim 2, characterized in that, The intermittent feeding method is as follows: using the rise and fall of OUR as a feedback signal, ammonium sulfate is fed in when OUR rises, feeding stops when OUR falls, and feeding continues when OUR rises again.

4. The N-acetylglucosamine fermentation method based on oxygen uptake rate regulation according to any one of claims 1-3, characterized in that, In step S2, the ammonium sulfate is added in solution form, with a concentration of 70-110 g / L and a flow rate of 20-40 g / h.

5. The N-acetylglucosamine fermentation method based on oxygen uptake rate regulation according to claim 4, characterized in that, The concentration of the ammonium sulfate solution is 90 g / L, and the flow rate is 30 g / h.

6. The N-acetylglucosamine fermentation method based on oxygen uptake rate regulation according to claim 1, characterized in that, Step S2 also includes: when the OD value in the fermentation broth reaches 32, adding the inducing agent IPTG and reducing the tank temperature from 34℃±0.5℃ to 33℃±0.5℃.

7. The N-acetylglucosamine fermentation method based on oxygen uptake rate regulation according to claim 1, characterized in that, During the fermentation process, the pH of the fermentation broth was controlled at 6.9±0.1 by adding ammonia water.

8. The N-acetylglucosamine fermentation method based on oxygen uptake rate regulation according to claim 1, characterized in that, The method for controlling the addition of glucose solution in step S3 is as follows: when the residual glucose concentration in the fermentation broth decreases to 0.5 g / 100 mL, the addition is started. Before induction, the residual glucose concentration is controlled at 0.3-0.5 g / 100 mL. After induction, the glucose addition rate is adjusted to control the DO at 37.5%±2.5%, and the glucose concentration is controlled at 0-0.05 g / 100 mL.

9. The N-acetylglucosamine fermentation method based on oxygen uptake rate regulation according to claim 1, characterized in that, The fermentation endpoint described in step S3 is when the product growth is less than 5 g / L every 4 hours, at which point the product is discharged from the tank.

10. The N-acetylglucosamine fermentation method based on oxygen uptake rate regulation according to claim 1, characterized in that, The seed culture in step S1 includes mother bottle seed culture and seed tank seed culture, wherein the mother bottle seed culture is carried out to OD. 610 Transplant when the seed reaches 3-4, and cultivate the seeds in the seed tank to OD. 600 Transplant when the seedlings reach 14-15 cm in height.

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