Method for producing agricultural tryptophan through fermentation and application

Abstract

The invention relates to the technical field of biological engineering, and discloses a method for producing agricultural tryptophan through fermentation and application, and the method comprises the following steps: after thalli are accumulated, adding L-serine in a pulse manner, switching to double-carbon-source material supplementation of glucose and glycerol, and starting energy metabolism uncoupling control at the same time; according to the energy metabolism decoupling control, a decoupling agent is fed, a real-time respiration quotient is used as a feedback signal, and a closed-loop system is used for automatically adjusting the flow acceleration rate, so that the respiration quotient is maintained in a target range. According to the invention, intracellular energy charge is reduced through uncoupling so as to relieve glycolysis inhibition and strengthen carbon flux, double carbon sources are utilized to maintain redox balance, and pulse feeding is matched to eliminate terminal synthesis bottleneck. According to the method, the metabolic competition problem of cell growth and product synthesis is effectively solved, the conversion rate and fermentation yield of tryptophan are effectively improved, and the obtained product is suitable for preparing an agricultural fertilizer synergist or a feed additive.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a method and application for the fermentation production of agricultural tryptophan. Background Technology

[0002] L-Tryptophan is an important precursor in plant growth and development, and is commonly used in agricultural production as a fertilizer enhancer, plant growth regulator, and feed additive. Currently, deep liquid fermentation using microorganisms is the main industrial route for producing L-Tryptophan, with commonly used strains including Escherichia coli and Corynebacterium glutamicum.

[0003] The microbial fermentation synthesis of tryptophan involves a long biosynthetic pathway and a complex metabolic network, involving the supply of key precursors such as erythrose-4-phosphate and phosphoenolpyruvate, as well as the participation of coenzymes such as ATP and NAD(P)H. Existing fermentation processes often face a competitive conflict between cell growth and product synthesis for carbon metabolic flux. If cell growth is prioritized during fermentation, a large amount of carbon will be used to synthesize cellular structural substances, resulting in insufficient carbon flux to the tryptophan synthesis pathway. Conversely, simply restricting growth leads to a decrease in cell metabolic activity, making it impossible to maintain long-term acid production capacity. Furthermore, due to competition between glycolysis and aromatic amino acid synthesis for intermediate metabolites, the supply of key precursors often becomes the rate-limiting step.

[0004] On the other hand, tryptophan synthesis is highly sensitive to intracellular redox conditions. In fermentation systems using a single carbon source, the production and consumption of intracellular reducing coenzymes are difficult to dynamically balance, easily leading to overflow metabolism, resulting in the accumulation of byproducts such as acetic acid, inhibiting cell growth, and increasing the difficulty of subsequent separation and purification. Simultaneously, the final step in L-tryptophan synthesis depends on the supply of L-serine. However, under conventional fermentation conditions, intracellular L-serine synthesis is subject to strict metabolic regulation, and its concentration is often insufficient to saturate the active site of tryptophan synthase, causing the accumulation of the intermediate metabolite indole. Indole is not only toxic to cells but also feedback-inhibits the activity of upstream key enzymes, further limiting the increase in tryptophan production. Existing techniques mostly focus on genetically modifying strains, lacking effective methods to systematically address these metabolic bottlenecks through dynamic correlation control of fermentation process parameters. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method and application for the fermentation production of agricultural tryptophan, which solves the problems in existing microbial fermentation processes for L-tryptophan production, such as competition between cell growth and product synthesis for carbon metabolic flux, insufficient supply of key precursors in the tryptophan synthesis pathway, and imbalance of redox coenzymes, which make it difficult to improve substrate conversion rate and final yield.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for producing agricultural tryptophan by fermentation and its application.

[0007] In a first aspect, the present invention provides a method for producing agricultural tryptophan by fermentation, employing the following technical solution: A method for producing agricultural tryptophan by fermentation includes the following steps: Tryptophan-producing strains were inoculated into fermentation medium and fed-batch culture was carried out to accumulate cell biomass. After accumulating cell biomass, L-serine, as a co-inducer, was added to the fermentation system in a pulse, and the feeding strategy was switched from a single carbon source to a dual carbon source mixed feeding of glucose and glycerol. At the same time, energy metabolism uncoupling control was initiated. The energy metabolism uncoupling control includes adding an uncoupling agent to the fermentation broth and using the respiratory quotient during fermentation as a feedback signal. The flow rate of the uncoupling agent is automatically adjusted through a closed-loop control system to maintain the real-time respiratory quotient within the target range, thereby enhancing the upstream carbon metabolism flux and driving tryptophan synthesis.

[0008] By adopting the above technical solution, this invention utilizes a combination of chemical and physical regulation methods to achieve the redistribution and optimization of microbial metabolic flux. Its specific mechanism of action is as follows: Mechanism of energy metabolism uncoupling that enhances upstream glycolysis flux: The added uncoupling agent, acting as a proton carrier, embeds itself in the phospholipid bilayer of the microbial cell membrane, disrupting the transmembrane proton gradient and membrane potential, leading to a decrease in the proton kinetic potential. This process causes the energy generated by the electron transport chain to be dissipated as heat instead of being coupled with ATP synthase to generate ATP, directly resulting in a decrease in intracellular ATP concentration and an increase in ADP concentration. The low intracellular energy charge state relieves the allosteric inhibition of key rate-limiting enzymes in the glycolysis pathway, especially phosphofructokinase. According to metabolic regulatory mechanisms, in order to maintain basic survival energy, the cell is forced to increase the rate of glycolysis, thereby increasing the production rate of tryptophan precursors such as phosphoenolpyruvate and erythrose-4-phosphate, solving the problem of precursor supply limitation.

[0009] Steady-state maintenance mechanism of respiratory quotient feedback closed-loop control: Uncoupling agents exhibit a narrow dose window between promoting metabolic flux and producing cytotoxicity. The respiratory quotient (RQ), as a macroscopic parameter characterizing the degree of substrate oxidation and the type of anabolism, can reflect the metabolic state of cells in real time. When the RQ is controlled at a specific target value, it indicates that the proportion of substrate carbon source flowing towards complete oxidation and incomplete oxidation (overflow metabolism / product synthesis) is in a predetermined dynamic equilibrium. By using a PID closed-loop control system to link the flow rate of the uncoupling agent to the RQ value, metabolic fluctuations caused by changes in cell concentration or uncoupling agent degradation can be precisely offset, maintaining a high-flux metabolic homeostasis without inducing cell lysis.

[0010] Kinetic pull mechanism of synergistic inducer pulse addition: The key step in L-tryptophan biosynthesis involves the condensation of indole and L-serine to L-tryptophan, catalyzed by tryptophan synthase. While increased upstream flux provides a sufficient carbon skeleton, insufficient supply of the co-substrate L-serine leads to the accumulation of the intermediate indole, which can then be toxic to cells and feedback-inhibit the upstream enzyme system. This invention utilizes reaction kinetics principles to increase the substrate saturation in the enzymatic reaction system through pulsed addition of high concentrations of L-serine. According to the law of mass action, the high substrate concentration directly drives the forward reaction rate, rapidly converting the upstream carbon metabolic flux into the target product and eliminating the metabolic bottleneck.

[0011] Redox equilibrium mechanism with synergistic effect of dual carbon sources: Under intense uncoupling metabolic conditions, a single glucose carbon source can easily lead to an intracellular NADH / NAD ratio increase. + Imbalance exists. Glycerol and glucose enter the central carbon metabolism at different points, and glycerol has a higher reducing power than glucose. The mixed feeding strategy of glucose and glycerol regulates the composition of the intracellular reducing coenzyme pool through the glycerol metabolic pathway, providing a suitable coenzyme environment for the energy-intensive and highly reducing tryptophan synthesis pathway, and maintaining the physiological functions and redox balance of cells under high-load production conditions.

[0012] Preferably, the uncoupling agent is selected from 2,4-dinitrophenol or carbonyl cyanide m-chlorophenylhydrazone. When 2,4-dinitrophenol is selected, its stock solution concentration is 20 mmol / L to 40 mmol / L, and the initial feed rate is 0.01 μmol / h per OD. 600 Up to 0.03 micromoles per hour per OD 600 When using carbonyl cyanide m-chlorophenylhydrazone, the stock solution concentration is 1 mmol / L to 2 mmol / L, and the initial feed rate is 0.001 μmol / h per OD. 600 Up to 0.003 micromoles per hour per OD 600 .

[0013] By adopting the above technical solution, specific concentration gradients and feeding strategies were set for different types of proton carriers, ensuring the precise initiation of the decoupling effect and avoiding irreversible cell damage caused by excessively high local concentrations.

[0014] Preferably, the target value of the respiratory quotient is in the range of 1.05 to 1.20; the closed-loop control system adopts a PID control algorithm to dynamically increase or decrease the flow rate of the uncoupling agent based on the deviation between the real-time respiratory quotient value and the target value.

[0015] By employing the above technical solution, the respiratory quotient is precisely controlled within the range of 1.05 to 1.20, placing the cells in a critical metabolic state transitioning from fully aerobic respiration to overflow metabolism. In this state, substrate consumption rate is maximized, and the carbon skeleton primarily flows towards product synthesis rather than cellular biomass accumulation, thus optimizing production efficiency.

[0016] Preferably, the pulsed addition of L-serine is used to adjust the optical density (OD) of the fermentation broth. 600 When the concentration reaches 80 to 100, the addition method is to add it all at once within 10 to 30 minutes, so that the instantaneous concentration of L-serine in the fermentation broth reaches 0.6 g / L to 1.5 g / L.

[0017] By adopting the above technical solution, pulse addition is performed when the bacterial biomass reaches a high level. On the one hand, it utilizes a high-density biocatalyst base, and on the other hand, the instantaneous high concentration of serine can enhance the catalytic efficiency of tryptophan synthase, thereby achieving high-intensity synthesis of the product.

[0018] Preferably, in the dual carbon source mixed feed, the molar ratio of glucose to glycerol is 1.5:1 to 2.5:1; and the total residual sugar concentration in the fermentation broth is controlled to be less than 3 grams per liter.

[0019] By adopting the above technical solution, the molar ratio is close to the stoichiometric ratio of carbon skeleton and reducing power required for tryptophan synthesis; at the same time, the limited feeding strategy that controls the low residual sugar concentration, combined with the use of uncoupling agents, effectively prevents the generation of a large amount of byproducts such as acetic acid from overflow metabolism, and ensures that the carbon source mainly flows to the target product.

[0020] Preferably, the fed-batch culture adopts an exponential feeding strategy, with the specific growth rate controlled at 0.18 to 0.22 per hour; and while initiating energy metabolism uncoupling control, the fermentation temperature is reduced from 36 to 38 degrees Celsius to 33 to 35 degrees Celsius and maintained until the end of fermentation.

[0021] By adopting the above technical solutions, exponential feeding ensures the rapid growth of cells in the early stage; the cooling strategy in the middle and late stages of fermentation maintains the stability of key enzymes, reduces the thermal stress caused by the decoupling agent, and prolongs the duration of product synthesis.

[0022] Preferably, during the fed culture and subsequent fermentation process, the pH of the fermentation broth is controlled to be 6.9 to 7.1 by adding ammonia water, and the dissolved oxygen is maintained at above 30% by adjusting the stirring speed and aeration rate; the fermentation culture medium contains ammonium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium citrate and trace elements.

[0023] By adopting the above technical solutions, maintaining a high dissolved oxygen level is a necessary condition to ensure the normal progress of the oxidative phosphorylation process and the effectiveness of the uncoupling agent. A stable pH environment ensures the activity of the metabolic enzyme system.

[0024] Preferably, the preparation method of the uncoupling agent includes: accurately weighing the uncoupling agent powder, dissolving it in dimethyl sulfoxide as a co-solvent to prepare a concentrated solution, then diluting it with sterile water to a fixed volume, wherein the final volume content of dimethyl sulfoxide is not higher than 20% (v / v), and then filtering it through a filter membrane for sterilization.

[0025] By adopting the above technical solution and strictly controlling the content of the co-solvent dimethyl sulfoxide, the hydrophobic uncoupling agent is fully dissolved, while the non-specific damage to the cell membrane structure by high concentrations of organic solvents is avoided, thus ensuring the stability of the process and the reproducibility of experimental results.

[0026] Preferably, after fermentation, an alkaline solution is added to the fermentation broth to adjust the pH and the mixture is heated to 50 to 70 degrees Celsius. The mixture is stirred until the produced tryptophan crystals are completely dissolved. Then, solid-liquid separation is performed to remove the bacterial cells, and the resulting clear liquid is used to prepare agricultural tryptophan products.

[0027] By adopting the above technical solution, and taking advantage of the physical characteristic that tryptophan is prone to crystallization in fermentation broth, the method of separating the bacterial cells after dissolving by heating and adjusting the alkali effectively reduces the loss of product entrainment in the bacterial residue and improves the yield.

[0028] Secondly, this invention provides an application of fermentation production of agricultural tryptophan, specifically the application of tryptophan produced by the fermentation production method described in this invention in the preparation of agricultural fertilizer enhancers, plant growth regulators, and feed additives.

[0029] By adopting the above technical solution, the tryptophan fermentation broth or crude product produced by the method of this invention has high production efficiency and low cost. This product not only contains L-tryptophan but is also rich in organic acids and trace elements produced during metabolism. When used in the preparation of agricultural fertilizer synergists or plant growth regulators, it can promote crop root growth and improve crop stress resistance; when used as a feed additive, it can effectively balance the amino acid composition of feed and improve the problem of limited animal growth caused by tryptophan deficiency.

[0030] This invention provides a method for producing agricultural tryptophan through fermentation and its application. It has the following beneficial effects: 1. This invention effectively solves the problem of competition for carbon sources between cell growth and product synthesis during fermentation by introducing an energy metabolism uncoupling control strategy based on respiratory quotient as a feedback signal. By maintaining a specific respiratory quotient level through closed-loop control, the cells are placed in a low-energy-charge metabolic state, relieving the inhibition of key enzymes in the glycolysis pathway and forcing carbon metabolism flux to flow to synthetic precursors such as phosphoenolpyruvate. This metabolic remodeling improves the conversion rate of carbon sources to tryptophan, thereby increasing the fermentation yield of the final product.

[0031] 2. This invention utilizes a pulsed feeding strategy with L-serine to eliminate the rate-limiting bottleneck at the end of tryptophan biosynthesis. High-concentration pulsed addition of a synergistic inducer during the stable cell growth phase increases the substrate saturation of the tryptophan synthase reaction system, accelerating the conversion of precursor indole to tryptophan based on kinetic principles. This not only avoids the cytotoxicity and feedback inhibition caused by the accumulation of intermediate metabolite indole, but also improves the product synthesis rate and production intensity in the later stages of fermentation.

[0032] 3. This invention employs a dual-carbon-source synergistic feeding process using glucose and glycerol, optimizing the intracellular redox coenzyme balance. Given the high reducing power of glycerol, its mixed metabolism with glucose can provide a suitable NADH / NAD ratio for high-intensity uncoupling metabolism. + The ratio satisfies the high reducing power requirement of tryptophan synthesis; this regulatory method maintains the physiological stability of cells in the non-growth-coupled phase, reduces the accumulation of overflow metabolic byproducts such as acetic acid, and helps simplify subsequent separation and purification steps. Detailed Implementation

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the preparation examples, comparative examples, and test examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Preparation Examples 1-4: Preparation Example 1: Preparation of Uncoupling Agent Stock Solution This preparation example provides a method for preparing a 20 mM 2,4-dinitrophenol stock solution, comprising the following steps: In a sterile laminar flow hood, accurately weigh 0.0921 g of 2,4-dinitrophenol powder and place it in a sterile 100 mL stoppered conical flask. Add 5.0 mL of dimethyl sulfoxide (DMSO) and vortex at room temperature until the 2,4-dinitrophenol powder is completely dissolved, forming a clear, orange-yellow concentrate. While stirring, slowly dilute the concentrate with sterile deionized water and transfer it to a 25 mL sterile volumetric flask. Continue to dilute to the mark with sterile deionized water and mix thoroughly. The final 2,4-dinitrophenol stock solution has a concentration of 20 mM, with a DMSO content of 20% (v / v). Filter the stock solution sterile using a 0.22 μm pore size polyethersulfone membrane, dispense it into sterile brown screw-top bottles, and store at 4°C in the dark for later use.

[0035] Preparation Example 2: Preparation of Uncoupling Agent Stock Solution This preparation example provides a method for preparing a 1 mM stock solution of carbonyl cyanide m-chlorophenylhydrazone, comprising the following steps: In a sterile laminar flow hood, accurately weigh 0.0051 g of carbonyl cyanide m-chlorophenylhydrazone powder and place it in a sterile 50 mL stoppered conical flask. Add 5.0 mL of dimethyl sulfoxide and vortex at room temperature until the carbonyl cyanide m-chlorophenylhydrazone powder is completely dissolved, forming a clear yellow concentrate. Under stirring, slowly dilute the concentrate with sterile deionized water and transfer it to a 25 mL sterile volumetric flask. Continue to dilute to the mark with sterile deionized water and mix thoroughly. The final carbonyl cyanide m-chlorophenylhydrazone stock solution has a concentration of 1 mM, with a dimethyl sulfoxide content of 20% (v / v). Filter the stock solution sterile using a 0.22 μm pore size polyethersulfone (PES) membrane, dispense it into sterile brown screw-top bottles, and store at 4°C protected from light.

[0036] Preparation Example 3: Preparation of Synergistic Inducer Solution This preparation example provides a method for preparing an L-serine solution with a concentration of 150 g / L, including the following steps: Accurately weigh 150.0 g of L-serine powder and add it to a clean beaker containing approximately 800 mL of deionized water. Heat and stir the suspension to 60-70°C until the L-serine is completely dissolved, forming a clear solution. After cooling the solution to room temperature, transfer it to a 1 L volumetric flask, dilute to the mark with deionized water, and mix thoroughly. Dispense the prepared solution into multiple autoclaved glass refill bottles and sterilize them at 115°C for 15 minutes using moist heat. After sterilization, allow them to cool naturally to room temperature for later use.

[0037] Preparation Example 4: Preparation of Feed Carbon Source Solution The preparation method of the feed carbon source solution provided in this example, namely a glucose solution with a concentration of 650 g / L and a glycerol solution with a concentration of 650 g / L, includes the following steps: Preparation of glucose solution: Accurately weigh 650.0 g of D-glucose monohydrate and add it to a clean beaker containing approximately 500 mL of deionized water. Stir until completely dissolved. Transfer the dissolved solution to a 1 L volumetric flask, dilute to the mark with deionized water, and mix thoroughly. Pour the prepared solution into an autoclaved glass feed bottle and sterilize at 115°C for 15 minutes. After cooling, it is ready for use.

[0038] Preparation of glycerol solution: Measure 474.5 mL (or accurately weigh 650.0 g) of glycerol with a purity of not less than 99.5% using a clean graduated cylinder. Add this to a clean beaker containing approximately 400 mL of deionized water and stir until completely mixed. Transfer the mixture to a 1 L volumetric flask, dilute to the mark with deionized water, and mix thoroughly. Transfer the prepared solution to an autoclaved glass feed bottle and sterilize at 121°C for 20 minutes. After cooling, it is ready for use.

[0039] Examples 1-5: Example 1: This embodiment provides a method for producing agricultural tryptophan by fermentation and its application, specifically including the following steps: First, the culture media were prepared. The seed culture medium consisted of 10.0g yeast extract, 20.0g peptone, 5.0g sodium chloride, and 20.0g glucose, dissolved in deionized water and adjusted to pH 7.0. It was then sterilized at 121°C for 20 minutes using moist heat. The initial fermentation medium consisted of 3.0g ammonium sulfate, 3.0g potassium dihydrogen phosphate, 3.0g dipotassium hydrogen phosphate, and 1.5g sodium citrate, with the addition of 1.5mL / L trace element solution. These components were dissolved in 2.5L deionized water and placed in a 5L fermenter, then sterilized at 121°C for 20 minutes using moist heat. Separately, 200g / L magnesium sulfate heptahydrate and 500g / L glucose solutions were prepared and sterilized separately at 121°C for 20 minutes.

[0040] Then, seed activation and inoculation were performed. The preserved production strain was inoculated into a 250 mL shake flask containing 50 mL of seed culture medium and cultured in a constant temperature shaker at 37 °C and 220 rpm for 12 hours to obtain the seed culture. Before fermentation began, sterilized magnesium sulfate heptahydrate solution and glucose solution were aseptically added to the fermenter to bring the initial concentrations in the tank to 1.2 g / L and 25 g / L, respectively. Subsequently, the seed culture was inoculated into the fermenter at an inoculation rate of 8% (v / v), and sterile water was added to bring the initial fermentation volume to 3.0 L.

[0041] Next is Phase 1, the cell proliferation and biomass accumulation phase (0-16 hours). Initial fermentation conditions were set at a temperature of 37°C and a pH maintained at 7.0 by automatic addition of 25% ammonia. Dissolved oxygen was maintained above 30% by adjusting the stirring speed (initially 400 rpm) and aeration rate (initially 1.0 vvm). When the initial glucose was consumed and dissolved oxygen rapidly recovered (approximately 8 hours), the peristaltic pump was started, and exponential feeding of the 650 g / L glucose solution prepared in Example 4 was initiated. The exponential feeding strategy was set with a specific growth rate μ of 0.20 h. -1 This stage lasts until the 16th hour of fermentation, at which point the optical density of the fermentation broth reaches 95.

[0042] The process then proceeded to Phase Two, the metabolic remodeling and product synthesis induction phase (16-48 hours). At hour 16 of fermentation, a 150 g / L L-serine solution prepared in Preparation Example 3 was pumped in a single injection over 15 minutes using a high-precision metering pump, bringing its instantaneous concentration in the fermentation broth to 0.7 g / L. Immediately thereafter, another high-precision metering pump was started to continuously pump in a 20 mM stock solution of 2,4-dinitrophenol prepared in Preparation Example 1, with an initial addition rate set at 0.02 μM / h / OD. 600 Simultaneously, single glucose feeding was stopped, and a dual-carbon-source synergistic feeding mode was switched. Two independent feed pumps were used to pump in the 650 g / L glucose solution and 650 g / L glycerol solution prepared in Example 4, respectively, controlling the molar ratio of the two carbon sources at 2:1. A constant-rate mode was used to maintain the total residual sugar concentration in the fermentation broth below 3 g / L. The respiratory quotient value measured by an online respiratory gas analyzer was used as a feedback signal, with a target value set at 1.10. A PID control algorithm was used to automatically adjust the pump rate of the 2,4-dinitrophenol stock solution to achieve dynamic stability of the respiratory quotient value. During this stage, the fermentation temperature was reduced from 37°C to 34°C and maintained.

[0043] Finally, there is Phase Three, the high-intensity production and maintenance phase (48-96 hours). The respiratory quotient feedback control strategy and dual-carbon source feeding strategy from Phase Two are maintained. The constant-rate feeding of the total carbon source is further finely controlled to maintain the total residual sugar concentration in the fermentation broth within the range of 0.5-1.5 g / L. The fermentation process continues until the end of 96 hours. During fermentation, samples are taken periodically for analysis of subsequent test cases.

[0044] Example 2: This embodiment provides a method and application for the fermentation production of agricultural tryptophan, the main difference from Example 1 being the use of the carbonyl cyanide m-chlorophenylhydrazone as an energy metabolism uncoupling agent. This embodiment specifically includes the following steps: First, the culture medium was prepared and sterilized, the seeds were activated and inoculated, and the first stage of cell amplification and biomass accumulation was carried out. All operations and parameters were exactly the same as in Example 1. By the 16th hour of fermentation, the optical density of the fermentation broth reached 96.

[0045] The process then proceeded to Phase Two, the metabolic remodeling and product synthesis induction phase (16-48 hours). At hour 16 of fermentation, a 150 g / L L-serine solution prepared in Preparation Example 3 was pumped in a single injection over 15 minutes using a high-precision metering pump, bringing its instantaneous concentration in the fermentation broth to 0.7 g / L. Immediately thereafter, another high-precision metering pump was started to continuously pump in a 1 mM carbonyl cyanide m-chlorophenylhydrazone stock solution prepared in Preparation Example 2. The initial addition rate was adjusted according to its activity and set at 0.001 μM / h / OD. 600 Simultaneously, the process switched to a dual-carbon-source synergistic feeding mode. Two independent feed pumps were used to inject the 650 g / L glucose solution and 650 g / L glycerol solution prepared in Example 4, respectively, maintaining a molar ratio of 2:1 for the two carbon sources. A constant-rate mode was employed to keep the total residual sugar concentration in the fermentation broth below 3 g / L. The respiratory quotient measured by an online respiratory gas analyzer was used as a feedback signal, with a target value set at 1.10. A PID control algorithm was used to automatically adjust the pump rate of the carbonyl cyanide m-chlorophenylhydrazone stock solution to achieve dynamic stability of the respiratory quotient. During this stage, the fermentation temperature was reduced from 37°C to 34°C and maintained.

[0046] Finally, there is Phase Three, the high-intensity production and maintenance phase (48-96 hours). The respiratory quotient feedback control strategy and dual-carbon source feeding strategy from Phase Two are maintained, with fine-tuning of the constant-rate feeding of the total carbon source to keep the total residual sugar concentration in the fermentation broth within the range of 0.5-1.5 g / L. The fermentation process continues until the end of 96 hours. During fermentation, samples are taken periodically for analysis of subsequent test cases.

[0047] Example 3: This embodiment provides a method and application for the fermentation production of agricultural tryptophan, the main difference from Example 1 being the adjustment of the molar addition ratio of the two carbon sources during the feeding stage. This embodiment specifically includes the following steps: First, the culture medium was prepared and sterilized, the seeds were activated and inoculated, and the first stage of cell amplification and biomass accumulation was carried out. All operations and parameters were exactly the same as in Example 1. By the 16th hour of fermentation, the optical density of the fermentation broth reached 95.

[0048] The process then proceeded to Phase Two, the metabolic remodeling and product synthesis induction phase (16-48 hours). At hour 16 of fermentation, a 150 g / L L-serine solution prepared in Preparation Example 3 was pumped in a single injection over 15 minutes using a high-precision metering pump, bringing its instantaneous concentration in the fermentation broth to 0.7 g / L. Immediately thereafter, another high-precision metering pump was started to continuously pump in a 20 mM stock solution of 2,4-dinitrophenol prepared in Preparation Example 1, with an initial addition rate set at 0.02 μM / h / OD. 600 Simultaneously, single glucose feeding was stopped, and a dual-carbon-source synergistic feeding mode was switched. Two independent feed pumps were used to pump in the 650 g / L glucose solution and 650 g / L glycerol solution prepared in Example 4, respectively, while maintaining a molar ratio of 1.5:1 for the two carbon sources. The total carbon source was fed at a constant rate to maintain the total residual sugar concentration in the fermentation broth below 3 g / L. The respiratory quotient measured by an online respiratory gas analyzer was used as a feedback signal, with a target value set at 1.10. A PID control algorithm was used to automatically adjust the pump rate of the 2,4-dinitrophenol stock solution to achieve dynamic stability of the respiratory quotient. During this stage, the fermentation temperature was reduced from 37°C to 34°C and maintained.

[0049] Finally, there is Phase Three, the high-intensity production and maintenance phase (48-96 hours). The respiratory quotient feedback control strategy and the dual-carbon source feeding strategy with a glucose / glycerol molar ratio of 1.5:1, employed in Phase Two, are maintained. The constant-rate feeding of the total carbon source is further finely controlled to maintain the total residual sugar concentration in the fermentation broth within the range of 0.5-1.5 g / L. The fermentation process continues until the end of 96 hours. During fermentation, samples are taken periodically for analysis of subsequent test cases.

[0050] Example 4: This embodiment provides a method and application for the fermentation production of agricultural tryptophan, the main difference from Embodiment 1 being the adjustment of the control target value of the respiratory quotient. This embodiment specifically includes the following steps: First, the culture medium was prepared and sterilized, the seeds were activated and inoculated, and the first stage of cell amplification and biomass accumulation was carried out. All operations and parameters were exactly the same as in Example 1. By the 16th hour of fermentation, the optical density of the fermentation broth reached 95.

[0051] The process then proceeded to Phase Two, the metabolic remodeling and product synthesis induction phase (16-48 hours). At hour 16 of fermentation, a 150 g / L L-serine solution prepared in Preparation Example 3 was pumped in a single injection over 15 minutes using a high-precision metering pump, bringing its instantaneous concentration in the fermentation broth to 0.7 g / L. Immediately thereafter, another high-precision metering pump was started to continuously pump in a 20 mM stock solution of 2,4-dinitrophenol prepared in Preparation Example 1, with an initial addition rate set at 0.02 μM / h / OD. 600Simultaneously, the system switched to a dual-carbon-source synergistic feeding mode, using two independent feed pumps to inject the 650 g / L glucose solution and 650 g / L glycerol solution prepared in Preparation Example 4, respectively. The molar ratio of the two carbon sources was controlled at 2:1, and a constant-rate mode was used to maintain the total residual sugar concentration in the fermentation broth below 3 g / L. The respiratory quotient value measured by an online respiratory gas analyzer was used as a feedback signal, but the target value was set at 1.20. The pumping speed of the 2,4-dinitrophenol stock solution was automatically adjusted through a PID control algorithm to achieve dynamic stability of the respiratory quotient value. During this stage, the fermentation temperature was reduced from 37°C to 34°C and maintained.

[0052] Finally, there is Phase Three, the high-intensity production and maintenance phase (48-96 hours). The respiratory quotient feedback control strategy (target value 1.20) and dual carbon source feeding strategy from Phase Two are maintained. The constant-rate feeding of the total carbon source is further finely controlled to maintain the total residual sugar concentration in the fermentation broth within the range of 0.5-1.5 g / L. The fermentation process continues until the end of 96 hours. During fermentation, samples are taken periodically for analysis of subsequent test cases.

[0053] Example 5: This embodiment provides a method and application for the fermentation production of agricultural tryptophan, the main difference from Example 1 being the amount of L-serine synergistic inducer added. This embodiment specifically includes the following steps: First, the culture medium was prepared and sterilized, the seeds were activated and inoculated, and the first stage of cell amplification and biomass accumulation was carried out. All operations and parameters were exactly the same as in Example 1. By the 16th hour of fermentation, the optical density of the fermentation broth reached 95.

[0054] The process then proceeded to Phase Two, the metabolic remodeling and product synthesis induction phase (16-48 hours). At hour 16 of fermentation, a 150 g / L L-serine solution prepared in Preparation Example 3 was pumped in a single injection over 15 minutes using a high-precision metering pump, adjusting the injection volume to achieve an instantaneous concentration of 1.5 g / L in the fermentation broth. Immediately thereafter, another high-precision metering pump was started to continuously pump in a 20 mM stock solution of 2,4-dinitrophenol prepared in Preparation Example 1, with an initial addition rate set at 0.02 μM / h / OD. 600 Simultaneously, the system switched to a dual-carbon-source synergistic feeding mode, using two independent feed pumps to inject the 650 g / L glucose solution and 650 g / L glycerol solution prepared in Preparation Example 4, respectively. The molar ratio of the two carbon sources was controlled at 2:1, and a constant-rate mode was used to maintain the total residual sugar concentration in the fermentation broth below 3 g / L. The respiratory quotient value measured by an online respiratory gas analyzer was used as a feedback signal, with a target value set at 1.10. The pumping speed of the 2,4-dinitrophenol stock solution was automatically adjusted using a PID control algorithm to achieve dynamic stability of the respiratory quotient value. During this stage, the fermentation temperature was reduced from 37°C to 34°C and maintained.

[0055] Finally, there is Phase Three, the high-intensity production and maintenance phase (48-96 hours). The respiratory quotient feedback control strategy and dual-carbon source feeding strategy from Phase Two are maintained. The constant-rate feeding of the total carbon source is further finely controlled to maintain the total residual sugar concentration in the fermentation broth within the range of 0.5-1.5 g / L. The fermentation process continues until the end of 96 hours. During fermentation, samples are taken periodically for analysis of subsequent test cases.

[0056] Comparative Examples 1-6: Comparative Example 1: Compared with Example 1, the difference is that only glucose is used as the sole carbon source for feeding throughout the process, and no uncoupling agents or exogenous synergistic inducers are added; all other aspects are the same.

[0057] Comparative Example 2: Compared with Example 1, the difference is that only glucose is used as the sole carbon source for feeding throughout the process, and L-serine solution is not pulsed and added at the 16th hour of fermentation; all other aspects are the same.

[0058] Comparative Example 3: Compared with Example 1, the difference is that only glucose is used as the sole carbon source for feeding throughout the process, and no 2,4-dinitrophenol uncoupling agent stock solution is added; all other aspects are the same.

[0059] Comparative Example 4: Compared with Example 1, the difference is that the 2,4-dinitrophenol uncoupling agent stock solution is not added, nor is the L-serine solution pulsedly added; all other aspects are the same.

[0060] Comparative Example 5: Compared with Example 1, the difference is that the addition of 2,4-dinitrophenol uncoupling agent is not controlled by respiratory quotient feedback closed loop, but is added at a constant rate of 0.285 mL / h after 16 hours of fermentation. All other aspects are the same.

[0061] Comparative Example 6: Compared with Example 1, the difference is that the target value of the respiratory quotient feedback control is set to 0.95, while all other aspects are the same.

[0062] Test Examples 1-5: Test Example 1: Verification of Energy Metabolic State and Uncoupling Effect This test case aims to verify, by detecting changes in intracellular adenosine nucleotide energy charge levels and macroscopic metabolic rates, whether the uncoupling agent and control strategy used in the example successfully established a non-physiological energy metabolism state and triggered the expected metabolic flux redistribution.

[0063] Experimental steps: When the fermentation process reached the metabolic remodeling and product synthesis induction stage, and the control parameters stabilized, samples were taken at 30, 36 and 42 hours of fermentation.

[0064] Quickly aspirate 2 mL of fermentation broth and immediately inject it into a centrifuge tube containing 0.5 mL of 3M perchloric acid solution pre-cooled to -20°C. Vigorously vortex to quench enzyme activity and extract intracellular metabolites. Then centrifuge at 4°C and 12,000 rpm for 5 minutes. Take the supernatant, add pre-cooled 1M potassium carbonate solution to adjust the pH to 6.5-7.0, centrifuge again to remove the precipitate, filter the supernatant through a 0.22 μm filter membrane, and store at -80°C for later analysis.

[0065] Nucleotide analysis was performed using high performance liquid chromatography (HPLC) with a C18 reversed-phase column. Mobile phase A consisted of 0.06 M dipotassium hydrogen phosphate and 0.04 M potassium dihydrogen phosphate buffer, while mobile phase B consisted of pure methanol. A gradient elution program was used at a flow rate of 1.0 mL / min and a detection wavelength of 254 nm. The concentrations of ATP, ADP, and AMP in the sample were quantified using the external standard method.

[0066] Based on online process data and sampling analysis data, a time window of 30 to 42 hours was selected to calculate the average specific oxygen consumption rate and specific glucose consumption rate within this interval. The specific oxygen consumption rate was obtained by dividing the oxygen uptake rate measured by the exhaust gas analyzer by the cell dry weight, and the specific glucose consumption rate was obtained by dividing the glucose consumption rate calculated from the feed weighing data by the cell dry weight.

[0067] The energy charge value of adenosine nucleotide is calculated using the formula AEC=([ATP]+0.5[ADP]) / ([ATP]+[ADP]+[AMP]).

[0068] Experimental data: Table 1: Comparison of energy metabolism parameters in different experimental groups during the production stage

[0069] in conclusion: According to the data in Table 1, in Comparative Example 1, under conventional fermentation conditions without the addition of uncoupling agents, the intracellular AEC remained at a high level of 0.88, indicating that the cells were in a physiological state with sufficient energy. The specific glucose consumption rate was only 0.34 g / g / h, and the specific oxygen consumption rate was 4.89 mmol / g / h. The metabolic flux was limited by the energy requirements for cell growth and maintenance, exhibiting typical characteristics of an economical model.

[0070] In Examples 1 and 2, after introducing uncoupling agents and implementing respiratory quotient feedback control, the AEC values ​​decreased to the range of 0.61-0.63. This value is lower than normal physiological levels, confirming that 2,4-dinitrophenol and the carbonyl cyanide m-chlorophenylhydrazone effectively disrupted the transmembrane proton kinetic potential, leading to reduced oxidative phosphorylation efficiency and inhibited ATP synthesis. As a compensatory response, the cell was forced to upregulate glycolysis flux to maintain the minimum energy level required for survival. The specific glucose consumption rate in Example 1 increased to 0.89 g / g / h, approximately 2.6 times that of Comparative Example 1; simultaneously, the specific oxygen consumption rate surged to 11.42 mmol / g / h, indicating that the electron transport chain was operating at high speed in an attempt to restore the proton gradient. This high-consumption, low-energy-charge state confirms the effectiveness of the energy metabolism uncoupling engine mechanism, namely, by artificially creating an energy deficit, forcibly driving the uptake and catabolism of upstream carbon sources.

[0071] Example 4 set a respiratory quotient target of 1.20, resulting in a further decrease in AEC to 0.54 and a peak specific glucose consumption rate of 1.12 g / g / h. Although carbon metabolism flux was further improved, the lower energy charge level affected cell tolerance and long-term viability (the overall benefits need to be determined in conjunction with subsequent yield data).

[0072] Comparative Example 5 used a constant-rate, open-ring addition of 2,4-dinitrophenol. Although the AEC decreased and the rate of glucose consumption increased, its effect was weaker than that of Example 1. This indicates that a fixed addition rate cannot adapt to the dynamic changes in cell concentration and physiological state during fermentation, and thus fails to maintain a stable uncoupling strength. Comparative Example 6 set the respiratory quotient target to 0.95. The data was close to that of Comparative Example 1, indicating that at this set value, the amount of uncoupling agent added by the PID controller was low, and it failed to effectively trigger metabolic remodeling.

[0073] In summary, by controlling the addition of uncoupling agents through respiratory quotient feedback, cells can be precisely locked in a specific low-energy-charge state, thereby relieving allosteric inhibition of the glycolysis pathway (such as the inhibition of phosphofructokinase by ATP), increasing carbon metabolism flux, and providing sufficient precursor flux for the synthesis of downstream L-tryptophan.

[0074] Test Example 2: Validation of flux and precursor pool abundance at key metabolic nodes This test case verifies the effect of uncoupling agents on upstream metabolic flux and the facilitation effect of L-serine on downstream synthetic pathways by quantitatively analyzing the concentration of key intracellular precursors and the accumulation of intermediate metabolites in the fermentation broth.

[0075] Experimental steps: When the fermentation process enters the product synthesis stage, 5 mL of fermentation broth is extracted from the fermenter using a rapid sampler and quickly injected into a centrifuge tube containing 20 mL of 60% methanol aqueous solution (containing 0.1% formic acid) pre-cooled to -40°C. The mixture is then immediately vortexed for 15 seconds to quench metabolic activity.

[0076] The mixture was centrifuged at -20°C and 10,000g for 10 minutes. The supernatant was collected and dried under a nitrogen stream. The residue was reconstituted with 500 μL of acetonitrile / water (50:50, v / v), and an isotope-labeled internal standard was added. The mixture was then filtered through a 0.22 μm PTFE membrane for intracellular metabolite analysis.

[0077] Intracellular phosphoenolpyruvate and erythrose-4-phosphate concentrations were detected using an ultra-high performance liquid chromatography-tandem mass spectrometry system. Chromatographic separation was performed using a HILIC amide column (100 mm × 2.1 mm, 1.7 μm), with gradient elution of acetonitrile and ammonium acetate buffer as the mobile phase. Mass spectrometry was performed in multiple reaction monitoring mode with negative ion mode scanning.

[0078] The supernatant of the fermentation broth after centrifugation was filtered through a 0.22 μm aqueous filter membrane, and the extracellular indole concentration was determined using a high-performance liquid chromatograph equipped with a UV detector. A C18 column was used, the mobile phase was methanol-water (60:40), and the detection wavelength was 280 nm.

[0079] All concentration data were normalized based on the cell dry weight at the time of sampling to calculate the intracellular metabolite abundance per unit cell.

[0080] Experimental data: Table 2: Comparison of Intracellular Abundance of Key Metabolic Intermediates and Accumulation of Byproducts

[0081] in conclusion: According to the data in Table 2, the intracellular PEP concentrations in Comparative Examples 1 and 3 remained only between 0.76 and 0.81 μmol / g DCW, while the E4P concentrations were between 0.28 and 0.31 μmol / g DCW. This indicates that without the addition of an uncoupling agent, the cells are subject to strict metabolic regulation. The intermediates produced by upstream glycolysis are mainly used to meet growth requirements, and the supply of precursors to the aromatic amino acid synthesis pathway is limited. Even though Comparative Example 3 was supplemented with the downstream substrate serine, the metabolic flux distribution was not altered due to the shortage of upstream precursors.

[0082] Data from Examples 1, 2, and Comparative Example 2 show that after the introduction of the uncoupling agent, the intracellular PEP concentration jumped to the range of 3.29-3.65 μmol / g DCW, and the E4P concentration increased to 1.15-1.24 μmol / g DCW, approximately 3-4 times higher than the control group. This difference confirms the energy metabolism uncoupling engine mechanism: by reducing the energy charge, allosteric inhibition is relieved, forcing an increase in glycolysis flux, leading to a large accumulation of key precursors PEP and E4P intracellularly, providing sufficient carbon skeleton for tryptophan synthesis.

[0083] While Comparative Example 2 achieved an expansion of the precursor library, it also showed an accumulation of indole as high as 487.6 mg / L in its fermentation broth. This indicates that in the absence of exogenous L-serine supplementation, the large influx of carbon flux from upstream caused severe congestion at the tryptophan synthase node, preventing the timely conversion of intermediate products into the final product and leading to indole extracellular leakage. In contrast, Example 1 maintained a high level of precursor while achieving an extracellular indole concentration of only 18.5 mg / L. This demonstrates the pathway channeling mechanism of the downstream precursor pulse: the pulse addition of exogenous L-serine effectively matched the upstream pulled flux, eliminated the rate-limiting step, and smoothly converted the accumulated metabolic flux into the target product L-tryptophan, avoiding the toxic accumulation of intermediate metabolites and the waste of carbon.

[0084] The data for Comparative Example 5 fell between those of Example 1 and the control group, and were accompanied by higher indole accumulation, indicating that constant-rate addition of the uncoupling agent could not precisely maintain metabolic balance, resulting in insufficient flux at some times and flux overload and poor conversion at other times. Example 1 employed a respiratory quotient feedback and serine synergistic strategy to achieve dynamic matching of precursor supply and consumption.

[0085] Test Example 3: Verification of Redox Equilibrium This test case aims to determine the ratio of intracellular pyridine nucleotide coenzymes under different carbon source feeding strategies, and to verify whether the dual carbon source synergistic strategy effectively improves the intracellular redox state under high-intensity metabolic flux, especially the supply of reducing power required for biosynthesis.

[0086] Experimental steps: When fermentation reaches the 40th hour, 3 mL of fermentation broth is quickly extracted from the special sampling port of the fermenter and immediately injected into a centrifuge tube containing quenching solution pre-cooled to -40°C. The tube is then rapidly vortexed to terminate the metabolic reaction.

[0087] Centrifuge at 10000g for 5 minutes at -20℃ and discard the supernatant. Add 1mL of pre-chilled acidic extraction buffer to the bacterial pellet to extract the oxidized coenzyme, and add 1mL of pre-chilled alkaline extraction buffer to a parallel sample to extract the reduced coenzyme.

[0088] The sample was sonicated on an ice bath for 30 seconds, then heated in a 95°C water bath for 5 minutes to completely destroy the enzyme activity and release the coenzyme. After cooling, the supernatant was collected by centrifugation and the pH was adjusted to around 7.0.

[0089] Quantitative analysis was performed using ultra-high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS). A hydrophilic interaction column was selected, and the mobile phase consisted of ammonium acetate buffer and acetonitrile gradient elution. Mass spectrometry was performed in multiple reaction monitoring (MRM) mode, and the absolute concentrations of each coenzyme were calculated using the standard curve method.

[0090] The ratio of reduced to oxidized coenzyme was calculated, and the data were the average of three repeated measurements.

[0091] Experimental data: Table 3: Comparison of intracellular redox coenzyme ratios and reducing power status

[0092] in conclusion: Based on the data in Table 3, in Comparative Example 2, under the conditions of single glucose feeding and the addition of 2,4-dinitrophenol as an uncoupling agent, the NADH / NAD ratio... + The ratio increased to 0.54, while the NADPH / NADP ratio... + The ratio dropped sharply to 0.31. This indicates that under the energy dissipation pressure induced by 2,4-dinitrophenol, cells enhanced the glycolysis pathway to replenish ATP through substrate-level phosphorylation, leading to the accumulation of catabolistic reducing power; however, because the carbon flux mainly flows to glycolysis, the flux into the pentose phosphate pathway is relatively reduced, resulting in insufficient regeneration capacity of the key biosynthetic reducing power NADPH. NADPH / NADP + The low ratio severely limits the efficiency of key reduction steps such as shikimate dehydrogenase in the tryptophan synthesis pathway, resulting in a metabolic bottleneck.

[0093] In contrast, Example 1 employed a dual-carbon source feeding strategy using glucose and glycerol, and under the same 2,4-dinitrophenol uncoupling conditions, the NADPH / NADP ratio was [not specified]. + The ratio remained at a high level of 0.84, superior to Comparative Example 2. This is because the location of glycerol's entry into the metabolic network (glycerol-3-phosphate) bypasses key upstream regulatory points in glycolysis, and the reducing power generated by its metabolic process differs from that of glucose. The introduction of glycerol shares the metabolic load of ATP production, allowing more glucose-6-phosphate to be diverted into the pentose phosphate pathway, thereby enhancing the NADPH regeneration cycle.

[0094] Furthermore, the NADH / NAD in Example 1 +The ratio was 0.38, lower than that of Comparative Example 2, indicating that the dual-carbon source system had better electron transport efficiency through the respiratory chain, or that there was more active intracellular transhydrogenase activity, achieving the conversion of NADH to NADPH. The data trend of Example 3 was consistent with that of Example 1, but the NADPH / NADP ratio was lower. + The slightly lower ratio suggests that a 2:1 molar ratio is more optimized for maintaining the strong reducing environment required for anabolism.

[0095] In summary, the dual-carbon-source synergistic strategy successfully solved the reducing power deficit problem that occurs when a single carbon source is in the uncoupled state. By reconstructing the intracellular redox balance, it provides sufficient NADPH driving force for high-intensity L-tryptophan biosynthesis, verifying the effectiveness of the resource optimization mechanism.

[0096] Test Example 4: Determination of Basic Fermentation Performance Parameters This test case monitors the cell growth kinetics, substrate residue levels, and accumulation of major metabolic byproducts in each fermentation batch to assess the stability of the process and the macroscopic control effect of metabolic flow.

[0097] Experimental steps: Throughout the fermentation process, 10 mL of fermentation broth was aseptically extracted from the sampling port of the fermenter every 4 hours. One mL of the sample was diluted with deionized water to an appropriate ratio, and the optical density was measured at a wavelength of 600 nm using a spectrophotometer. The cell dry weight was then calculated based on a pre-established standard curve.

[0098] The remaining sample was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was filtered through a 0.22 μm filter membrane for extracellular metabolite analysis.

[0099] Residual sugar and organic acid concentrations were determined using a high-performance liquid chromatography (HPLC) system equipped with a differential refractive index detector and a UV detector. A Bio-Rad Aminex HPX-87H size exclusion column was used, and the column temperature was maintained at 50°C.

[0100] The mobile phase was 5 mL of H₂SO₄ solution, the flow rate was set at 0.6 mL / min, and isocratic elution was performed.

[0101] Based on retention time and peak area of ​​standards, glucose, glycerol and acetic acid in the fermentation broth were qualitatively and quantitatively analyzed, and the data were recorded as average or peak values ​​during the fermentation process.

[0102] Experimental data: Table 4: Comparison of basic performance indicators of the fermentation process

[0103] in conclusion: According to the data in Table 4, the endpoint cell dry weight of Comparative Example 1 reached 68.4 g / L, which was higher than all other experimental groups that added uncoupling agents. This indicates that under natural physiological conditions without human intervention, microorganisms use most of the carbon source and energy they take in for their own proliferation and biomass synthesis, and the metabolic flux shows obvious growth-coupled characteristics. At this time, the acetic acid accumulation was low, indicating that the carbon flux and the TCA cycle processing capacity were relatively matched.

[0104] The cell dry weights of Examples 1 to 4 and Comparative Examples 2 and 5 were generally distributed in the range of 38.6-45.1 g / L, which was about 35%-40% lower than that of Comparative Example 1. This biomass inhibition phenomenon confirms that the uncoupling agent successfully disrupted the chemiospatial gradient of energy production, reduced ATP production efficiency, and forced the cells to shift their metabolic focus from biomass accumulation to maintaining survival and substrate oxidation, thus achieving a forced switch from growth mode to production mode.

[0105] Regarding byproduct control, Comparative Example 2 showed an acetic acid accumulation as high as 8.42 g / L. This is because uncoupling increased glycolysis flux, producing excess pyruvate and acetyl-CoA. Since the reducing power generated by glucose metabolism is primarily NADH, this leads to a TCA cycle imbalance due to the NADH / NAD ratio. + Excessive ratios lead to obstruction and severe overflow metabolism. In contrast, Example 1 employs a dual carbon source synergistic strategy, controlling the acetic acid concentration at a low level of 1.12 g / L. The introduction of glycerol modulates the intracellular redox state, and the pulsed addition of L-serine effectively diverts carbon skeleton flow to the tryptophan synthesis pathway, avoiding the accumulation and overflow of upstream intermediate metabolites.

[0106] Example 4 set a respiratory quotient target of 1.20, which further enhanced flux pull but also led to an increase in acetic acid accumulation to 4.85 g / L, with the lowest biomass among all groups. This suggests that excessively strong uncoupling intensity can exceed the cell's self-regulation threshold for overflow metabolism, resulting in some carbon sources being wasted on byproduct synthesis.

[0107] The high average residual sugar concentration in Comparative Example 5, accompanied by an accumulation of 3.67 g / L acetic acid, indicates that a constant uncoupling agent addition rate cannot adapt to changes in the physiological state of the cells during the later stages of fermentation, leading to metabolic oscillations and decreased substrate utilization. In contrast, Example 1, based on closed-loop control using the respiratory quotient, maintained a lower residual sugar level, demonstrating the advantages of steady-state control in the process.

[0108] Test Example 5: Performance Evaluation of L-Tryptophan Production This test case comprehensively evaluates the actual impact of different process strategies on L-tryptophan synthesis capacity by quantitatively analyzing the concentration of the fermentation endpoint product, combining substrate consumption data, calculating core technical indicators such as yield and production intensity.

[0109] Experimental steps: The fermentation process lasted for 96 hours, and 50 mL of fermentation broth was collected from the fermenter.

[0110] Since high-concentration L-tryptophan has low solubility at room temperature, a large amount of crystal precipitate exists in the sample. It is necessary to add an equal volume of 2M NaOH solution to the sample, heat it to 60℃ and stir for 30 minutes to ensure that all crystals are completely dissolved. Then cool it to room temperature and make up to 5 times the original volume with deionized water.

[0111] The diluted sample was centrifuged at 12,000 rpm for 10 minutes to remove bacterial cells and insoluble impurities. The supernatant was filtered through a 0.22 μm nylon membrane and the filtrate was collected for testing.

[0112] Quantitative detection was performed using high performance liquid chromatography (HPLC). The chromatographic column was an Agilent ZORBAX Eclipse Plus C18. The mobile phase consisted of 20 mM potassium dihydrogen phosphate buffer and pure acetonitrile in a volume ratio of 90:10. The flow rate was 1.0 mL / min and the column temperature was 30 °C.

[0113] The concentration of L-tryptophan in the original fermentation broth was calculated using a diode array detector at a wavelength of 280 nm and based on the external standard curve.

[0114] Based on the total sugar consumption data and fermentation time in Test Example 4, the technical indicators were calculated according to the following formula: Glucose yield = Total L-tryptophan production / Total carbon source consumption Production intensity = L-tryptophan endpoint concentration / fermentation time Experimental data: Table 5: Comparison of L-tryptophan fermentation endpoint performance indicators among experimental groups

[0115] in conclusion: According to the data in Table 5, the final L-tryptophan concentration in Comparative Example 1 was only 18.34 g / L, with a yield of 0.094 g / g. In the absence of external metabolic intervention, the cell metabolism is strictly limited by its own feedback regulation mechanism. The carbon source is mainly used for biomass synthesis, and the flux to secondary metabolites is limited. This represents the upper limit of the yield of this engineered strain under the natural growth coupling mode.

[0116] Example 1, through the introduction of a three-pronged synergistic control strategy of push, pull, and feed, achieved a leap in L-tryptophan yield to 58.42 g / L, a 218% increase compared to Comparative Example 1, with the yield increasing to 0.232 g / g. The data indicate that the energy uncoupling induced by 2,4-dinitrophenol successfully disrupted metabolic equilibrium, increasing glycolysis throughput; the pulsed addition of L-serine effectively channeled the rate-limiting step at the end of the synthetic pathway, efficiently converting the upstream carbon skeleton into the final product; and the dual carbon source feeding ensured the reducing force balance required for high-throughput synthesis. All three are indispensable, exhibiting a significant synergistic effect.

[0117] Although the yield of Comparative Example 2 increased to 32.55 g / L, the efficiency was only 0.136 g / g. Combined with the high concentration of indole accumulation observed in Test Example 2, this indicates that an upstream-driven process without downstream pathway guidance leads to a large amount of carbon source being retained as intermediate metabolites or wasted through overflow metabolism, failing to efficiently convert into the target product. The yields of Comparative Examples 3 and 4 were 24.12 g / L and 26.78 g / L, respectively, showing limited improvement. This confirms that without a strong carbon metabolic flow driven by an uncoupling agent, simply optimizing downstream reaction kinetics or cofactor supply cannot fundamentally change the order of magnitude of the yield.

[0118] Example 4 increased the respiratory quotient target value to 1.20. Although the total glucose consumption reached a maximum of 287.4 g / L, indicating relatively active carbon metabolism, the final yield and productivity were actually lower than in Example 1. This is consistent with the large accumulation of acetic acid observed in Test Example 4, indicating that excessive uncoupling strength led to excessive oxidation of the carbon source to CO2 or diversion to overflow metabolites, reducing atom economy. This conversely proves that a respiratory quotient of 1.10 in Example 1 is a better operating point for balancing carbon flux pull and conversion efficiency.

[0119] Example 5 increased the pulse concentration of L-serine compared to Example 1, resulting in a slight increase in yield to 59.76 g / L and a production rate of 0.236 g / g. This indicates that under the conditions of Example 1, there is still some room for improvement in the substrate saturation of tryptophan synthase, and increasing the transduction intensity could further tap the production potential. However, the increase is relatively gradual compared to the control, suggesting that the process has approached the physiological limits of this strain.

[0120] Comparative Example 5, using open-loop control of 2,4-dinitrophenol addition, yielded 41.93 g / L, lower than Example 1. The fluctuating uncoupling strength caused cells to be in a state of energy surplus or deficiency at certain times, making it impossible to maintain a steady-state, efficient production window as with closed-loop control of the respiratory quotient.

Claims

1. A method for producing agricultural tryptophan by fermentation, characterized in that, Includes the following steps: Tryptophan-producing strains were inoculated into fermentation medium and fed-batch culture was carried out to accumulate cell biomass. After accumulating cell biomass, L-serine, as a co-inducer, was added to the fermentation system in a pulse, and the feeding strategy was switched from a single carbon source to a dual carbon source mixed feeding of glucose and glycerol. At the same time, energy metabolism uncoupling control was initiated. The energy metabolism uncoupling control includes adding an uncoupling agent to the fermentation broth and using the respiratory quotient during fermentation as a feedback signal. The flow rate of the uncoupling agent is automatically adjusted through a closed-loop control system to maintain the real-time respiratory quotient within the target range, thereby enhancing the upstream carbon metabolism flux and driving tryptophan synthesis.

2. The method for producing agricultural tryptophan by fermentation according to claim 1, characterized in that, The uncoupling agent is selected from 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone.

3. The method for producing agricultural tryptophan by fermentation according to claim 1, characterized in that, The target value range for the respiratory quotient is 1.05-1.20; The closed-loop control system employs a PID control algorithm to dynamically increase or decrease the amount of uncoupling agent fed based on the deviation between the real-time respiratory quotient and the target value.

4. The method for producing agricultural tryptophan by fermentation according to claim 1, characterized in that, The pulsed addition of L-serine was performed on the optical density (OD) of the fermentation broth. 600 When the concentration reaches 80-100, the L-serine is added all at once within 10-30 minutes to make the instantaneous concentration of L-serine in the fermentation broth reach 0.6-1.5 g / L.

5. The method for producing agricultural tryptophan by fermentation according to claim 1, characterized in that, In the dual-carbon-source mixed feed, the molar ratio of glucose to glycerol is 1.5:1 to 2.5:1; and the total residual sugar concentration in the fermentation broth is controlled to be below 3 g / L.

6. The method for producing agricultural tryptophan by fermentation according to claim 1, characterized in that, The fed-batch culture employed an exponential feed strategy, with a specific growth rate controlled at 0.18-0.22 h⁻¹. -1 Furthermore, while initiating energy metabolism uncoupling control, the fermentation temperature was reduced from 36-38℃ to 33-35℃ and maintained until the end of fermentation.

7. The method for producing agricultural tryptophan by fermentation according to claim 1, characterized in that, During the fed culture and subsequent fermentation process, the pH of the fermentation broth was controlled to be 6.9-7.1 by adding ammonia water, and the dissolved oxygen was maintained above 30% by adjusting the stirring speed and aeration rate. The fermentation medium contains ammonium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium citrate, and trace elements.

8. The method for producing agricultural tryptophan by fermentation according to claim 1, characterized in that, The preparation method of the uncoupling agent includes: weighing the uncoupling agent powder, dissolving it in dimethyl sulfoxide as a co-solvent to prepare a concentrated solution, then diluting it with sterile water to a fixed volume, wherein the final volume content of dimethyl sulfoxide is not higher than 20% (v / v), and then filtering it through a filter membrane for sterilization.

9. The method for producing agricultural tryptophan by fermentation according to claim 1, characterized in that, After fermentation, alkali solution is added to the fermentation broth to adjust the pH and the mixture is heated to 50-70℃. The mixture is stirred until the produced tryptophan crystals are completely dissolved. Then, solid-liquid separation is performed to remove the bacterial cells. The resulting clear liquid is used to prepare agricultural tryptophan products.

10. An application of fermentation for the production of agricultural tryptophan, characterized in that, The application of tryptophan produced by the fermentation method for producing agricultural tryptophan according to any one of claims 1-9 in the preparation of agricultural fertilizer synergists, plant growth regulators and feed additives.

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