A dynamic control method and system for an l-valine fermentation process

Abstract

The present application relates to the technical field of L-valine fermentation, and particularly relates to a dynamic regulation method for an L-valine fermentation process, which comprises the following steps: inoculating an L-valine production strain into a fermentation tank, and starting a first fermentation stage with the goal of rapid proliferation of the bacterial cells; continuously obtaining the real-time NADH / NAD + ratio of the cells in the fermentation system by means of an online monitoring system; when the NADH / NAD + ratio is found to have a continuously rising trend and reaches or exceeds a first preset threshold T1, the system determines that the metabolic flow of the cells has been switched from growth domination to product synthesis domination, and a process switching is triggered by an automatic control system; the system enters a second fermentation stage, in which the core goal is to maximize L-valine synthesis, and the process parameters are simultaneously adjusted; the present application can intelligently capture the conversion window of the optimal production stage, and cooperatively optimize multiple process parameters, thereby significantly improving the yield, production intensity and conversion rate while shortening the fermentation cycle, which fully proves the advancement and practicality of the method in the intelligent manufacturing of L-valine.

Description

Technical Field

[0001] This invention relates to the field of L-valine fermentation technology, specifically to a method and system for dynamic control of the L-valine fermentation process. Background Technology

[0002] L-valine, an important branched-chain amino acid, is widely used in the feed, food, and pharmaceutical industries. Currently, microbial fermentation remains the mainstream method for producing L-valine. To improve yield and production efficiency, metabolic engineering techniques are commonly used to construct high-performance strains. Common strategies include blocking competitive metabolic pathways, enhancing the expression levels of key enzymes, and optimizing intracellular cofactor balance. Simultaneously, optimizing the fermentation process itself, especially the rational design of multi-stage fermentation regulation strategies, is equally crucial for unleashing the potential of engineered strains.

[0003] In existing technologies, multi-stage fermentation control largely relies on fixed process switching time points or setting switching thresholds based on biomass indicators (such as OD600). For example, when fermentation reaches a specific number of hours, or when OD600 rises to between 70 and 100, process parameters are switched manually or via program commands, such as reducing dissolved oxygen to induce the cells to transition from the growth phase to the product synthesis phase. However, this approach has significant shortcomings: due to factors such as differences in inoculum size, batch fluctuations in culture media, and deviations in reactor oxygen transfer characteristics, fixed time points or fixed biomass thresholds often fail to accurately match the actual metabolic state transition points within the cells. If the switch is too early, the cell mass has not accumulated sufficiently, limiting the later synthesis capacity; if the switch is too late, a large amount of carbon source is used for cell amplification rather than target product accumulation, dragging down both conversion rate and economic benefits. Although recent studies have attempted to introduce more refined intracellular regulation approaches, there is still a lack of effective schemes that directly use core physiological signals for dynamic process control. Therefore, a new strategy that can sense the "rhythm" of intracellular metabolism and achieve precise regulation is urgently needed. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing L-valine fermentation processes, which rely on fixed parameters for stage transitions and cannot adapt to real-time cell states. This invention provides a dynamic regulation method and system for the L-valine fermentation process. This method aims to achieve more precise control of metabolic pathways, thereby maximizing the L-valine production performance of existing engineered strains without further genetic modification.

[0005] This invention is achieved through the following technical solution: A method for dynamic regulation of L-valine fermentation involves online monitoring of real-time physiological state signals of cells during fermentation, and dynamic decision-making and switching of process parameters based on these signals. The physiological state signals include at least one characteristic indicator that characterizes intracellular reducing power levels and metabolic flow inversion. This characteristic indicator is the real-time ratio of the oxidized to reduced state concentrations of intracellular nicotinamide adenine dinucleotide (NADH / NAD). + Real-time ratio (hereinafter also referred to as "RNR").

[0006] As a further improvement to the above solution, the following steps are included: S1: Introduce L-valine-producing strains into the fermenter to start the first fermentation stage aimed at rapid cell proliferation; S2: Using an online monitoring system, continuously obtain real-time NADH / NAD levels in cells within the fermentation system. + ratio; S3: When the NADH / NAD is detected + When the ratio shows a continuous upward trend and reaches or exceeds the first preset threshold T1, the system determines that the cell metabolic flow has shifted from growth-dominated to product synthesis-dominated, and the automatic control system immediately triggers process switching. S4: The system enters the second fermentation stage, which focuses on maximizing L-valine synthesis. At least two of the following process parameters are adjusted simultaneously: a) reduce the stirring speed and / or aeration rate to lower the dissolved oxygen concentration; b) switch the feeding strategy from nutrient-rich feed to promote cell growth to restrictive feed to maintain metabolism; c) fine-tune the pH value to optimize the reactivity of key enzymes.

[0007] As a further improvement to the above scheme, step S5 is also included: continuously monitoring NADH / NAD. + Regarding the fluctuation of the ratio, when the ratio deviates from the second preset range R2, the process parameters of the second stage are fine-tuned to stabilize the ratio back within the second preset range R2. The second preset range R2 is determined through batch experiments, and the NADH / NAD ratio corresponding to the highest product synthesis rate is taken. + The range of ±15% of the ratio.

[0008] As a further improvement to the above scheme, the L-valine producing strain is an Escherichia coli strain that has been metabolically engineered. The modification methods include knocking out genes related to the pyruvate competitive pathway and / or introducing key enzyme encoding genes optimized by cofactor preference.

[0009] As a further improvement to the above scheme, the first fermentation stage in step S1 is the rapid growth period of the cells, and the specific fermentation conditions are as follows: a: Inoculate the activated L-valine-producing strain into a fermenter containing fermentation broth at an inoculation rate of 8% to 12%, with an initial glucose concentration of 15 g / L to 25 g / L; b: The fermentation temperature should be controlled between 36 ℃ and 38 ℃; c: The pH of the fermentation broth is controlled between 6.8 and 7.2 by automatically adding alkali solution; d: Maintain dissolved oxygen concentration between 25% and 40% by adjusting stirring speed and aeration rate; e: The feeding adopts the exponential feeding mode to control the glucose concentration in the fermentation broth to be maintained between 1.0 g / L and 3.0 g / L.

[0010] As a further improvement to the above scheme, the specific fermentation conditions for the second fermentation stage in step S4 are as follows: a: When the NADH / NAD+ ratio monitored online reaches the preset threshold T1 and remains so for more than 5 minutes, the control system automatically switches the process parameters. b: Reduce the dissolved oxygen concentration from 25% to 40% in the first fermentation stage to below 5%; c: Reduce the stirring speed from 700 r / min to 850 r / min to 300 r / min to 450 r / min; d: Reduce ventilation from 1.5 vvm to 2.0 vvm to 0.5 vvm to 1.0 vvm; e: Maintain the fermentation temperature at 36 ℃ to 38 ℃; f: Adjust the pH of the fermentation broth from 6.8 to 7.2 in the first stage to 6.5 to 6.9; g: The feeding mode was switched from exponential feeding mode to constant rate limiting feeding mode, and the glucose concentration in the fermentation broth was maintained at 0.5 g / L to 1.5 g / L.

[0011] A dynamic control system for L-valine fermentation, characterized in that it comprises: a bioreactor body; The online physiological signal monitoring unit is used to measure the NADH / NAD ratio in cells of the fermentation system in real time. + ratio; The central control unit, which has a pre-stored decision model and preset thresholds, is responsible for receiving monitoring data, making logical judgments and issuing control commands. The process parameter execution unit, based on the instructions issued by the central control unit, performs coordinated control of the reactor's stirring system, aeration system, feeding system, and pH adjustment system.

[0012] As a further improvement to the above scheme, the online physiological signal monitoring unit adopts a non-invasive online fluorescence sensor based on fluorescence lifetime or a specific wavelength, or an online analysis device that combines high-frequency automatic sampling with rapid quenching and enzyme labeling detection.

[0013] Compared with the prior art, the beneficial effects of the present invention are: This invention shifts the decision-making process for fermentation stage transitions from relying on external time or biomass indicators to relying on key internal physiological state indicators, including intracellular NADH / NAD ratios. + The real-time ratio of NADH / NAD is a core signal that sensitively reflects the intensity of cellular metabolism, particularly glycolysis and the state of reducing power supply. During L-valine fermentation, when the cell transitions from rapid proliferation and growth to a product synthesis-dominated phase, characteristic changes occur in intracellular metabolic flux and reducing power demand, which are reflected in the NADH / NAD ratio. + This is reflected in the value. This dynamic change is related to L. The demand for reducing power by key enzymes such as acetylhydroxy acid synthase in the valine synthesis pathway changes synchronously and precedes the inflection point of product accumulation rate, thus serving as an endogenous marker signal for the transition in fermentation stages; this can be achieved by directly monitoring the NADH / NAD ratio in cellular metabolism. + This invention can accurately capture the moment when cells enter the optimal production state and intervene accordingly, avoiding the drawbacks of the "one-size-fits-all" approach of the fixed parameter method, and making the process adaptable to the subtle differences in each batch of fermentation.

[0014] This invention is a universal process optimization scheme that can be combined with any high-performance L-valine engineered strain to further explore its production potential without altering the strain's genetic background. Based on experimental data, the method of this invention, compared to the traditional fixed OD value conversion method, can increase the production intensity of L-valine by 15%-25%, improve the sugar-acid conversion rate by 5%-15%, and potentially shorten the total fermentation time.

[0015] The dynamic control system for L-valine fermentation provided by this invention is a framework that links biochemical sensing, real-time data processing and process control. It is a key step toward a fully digital and intelligent fermentation plant and has broad application prospects. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments 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.

[0017] Figure 1This is a schematic diagram illustrating the principle and flow of the present invention; Figure 2 This is a comparison of the L-valine concentration over time curves between the dynamic RNR feedback control group and the traditional fixed OD value control group in this invention. Detailed Implementation

[0018] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings.

[0019] The following is in conjunction with the appendix Figure 1 The technical solution of the present invention will be further described below.

[0020] The present invention provides a dynamic control method for L-valine fermentation process, which does not switch fermentation stages based on preset fermentation time or optical density (OD value) of culture medium, but rather monitors the real-time physiological state signals of cells during fermentation online, and makes dynamic decisions and switches process parameters based on the feedback of physiological state signals. The specific steps are as follows: S1: An L-valine-producing strain is introduced into the fermenter to initiate the first fermentation stage, which aims at rapid cell proliferation. This stage maintains a relatively high dissolved oxygen level. The L-valine-producing strain is a metabolically engineered *E. coli* strain. The engineering methods include knocking out genes related to the pyruvate competitive pathway and / or introducing key enzyme-encoding genes optimized by cofactor preference. The first fermentation stage is the rapid cell growth period, and the specific fermentation conditions are as follows: a: The activated L-valine-producing strain is inoculated into the fermenter containing fermentation broth at an inoculum size of 8% to 12%, with an initial glucose concentration of 15 g / L to 25 g / L; b: The fermentation temperature is controlled at 36 ℃ to 38 ℃; c: The pH of the fermentation broth is controlled at 6.8 to 7.2 by automatic addition of alkali; d: The dissolved oxygen concentration is maintained at 25% to 40% by adjusting the stirring speed and aeration rate; e: An exponential feeding mode is used to control the glucose concentration in the fermentation broth to be maintained at 1.0 g / L to 3.0 g / L. S2: Using an online monitoring system, continuously obtain real-time NADH / NAD levels in cells within the fermentation system. + ratio; S3: When the NADH / NAD is detected + When the ratio shows a continuous upward trend and reaches or exceeds the first preset threshold T1, the system determines that the cell metabolic flow has shifted from growth-driven to product synthesis-driven, and the automatic control system immediately triggers a process switch. The first preset threshold T1 is determined by conducting multiple batches of fermentation experiments using the same strain and culture medium to adjust the NADH / NAD ratio. +The dynamic change curve of the ratio and L The correlation analysis was performed on the valine accumulation rate curve to determine this. S4: The system enters the second fermentation stage, with the core objective of maximizing L-valine synthesis. At least two of the following process parameters are adjusted simultaneously: a) reducing the stirring speed and / or aeration rate to lower the dissolved oxygen concentration; b) switching the feeding strategy from nutrient-rich feed to promote cell growth to a restrictive feed to maintain metabolism; c) fine-tuning the pH to optimize the reactivity of key enzymes. The specific fermentation conditions for the second fermentation stage are as follows: a: When online monitoring of NADH / NAD + When the ratio reaches the preset threshold T1 and remains so for more than 5 minutes, the control system automatically switches the process parameters; b: reduce the dissolved oxygen concentration from 25% to 40% in the first fermentation stage to below 5%; c: reduce the stirring speed from 700 r / min to 850 r / min to 300 r / min to 450 r / min; d: reduce the aeration rate from 1.5 vvm to 2.0 vvm to 0.5 vvm to 1.0 vvm; e: maintain the fermentation temperature at 36 ℃ to 38 ℃; f: adjust the pH of the fermentation broth from 6.8 to 7.2 in the first stage to 6.5 to 6.9; g: switch the feeding mode from exponential feeding mode to constant-rate limiting feeding mode, and control the glucose concentration in the fermentation broth to maintain at 0.5 g / L to 1.5 g / L. Step S5: Continuously monitor NADH / NAD + Regarding the fluctuation of the ratio, when the ratio deviates from the second preset range R2, the process parameters of the second stage are fine-tuned to stabilize the ratio back within the second preset range R2. The second preset range R2 is determined through batch experiments, and the NADH / NAD ratio corresponding to the highest product synthesis rate is taken. + The range of ±15% of the ratio.

[0021] A dynamic regulation system for L-valine fermentation, specifically comprising: Bioreactor: Standard 5 L or larger fermenter, equipped with basic units such as agitator, aeration, temperature, pH, dissolved oxygen (DO) probes, and automatic feed pump.

[0022] The online RNR monitoring module can be implemented in one of two ways: Option A (non-invasive): Install an online fluorescence sensor to monitor intracellular autofluorescence or exogenous probe fluorescence associated with NADH using specific excitation / emission wavelengths, and convert it into relative RNR values ​​using a calibration model.

[0023] Option B (Invasive): Integrate a high-frequency automated sampler (e.g., sampling every 2 minutes), immediately and rapidly quench the sample before it enters the microfluidic enzyme labeling unit to measure NAD. + Calculate the absolute value of RNR based on NADH concentration.

[0024] Central control unit (PLC or industrial computer): Receives data from all sensors, including the RNR monitoring module, DO probe, and pH meter. Internally runs decision-making software that pre-stores the threshold T1 and switching logic. Once the condition is met, it immediately sends instructions to each actuator.

[0025] Process parameter execution module: Receives instructions from the central control unit and precisely adjusts the speed of the stirring motor, the opening of the venting solenoid valve, the speed of the feed pump, and the acid-base pump to achieve coordinated switching and stable control of process parameters.

[0026] The dynamic regulation method based on real-time cellular physiological state (RNR ratio) feedback proposed in this invention can intelligently capture the optimal production stage transition window and synergistically optimize multiple process parameters. While shortening the fermentation cycle, it significantly improves yield, production intensity and conversion rate, fully demonstrating the advanced nature and practicality of this method in the intelligent manufacturing of L-valine.

[0027] The present application will now be described in detail through exemplary embodiments. The embodiments described below are merely some, not all, of the embodiments described herein. All other embodiments obtained by those skilled in the art based on the embodiments of the present application without inventive effort should fall within the scope of protection of the present application.

[0028] Example 1 A method for dynamic regulation of L-valine fermentation process, specifically including the following steps: 1. Prepare the bacterial strain and fermentation broth (1) Production strain a. Constructing the engineered strain VL-06 of L-valine-producing Escherichia coli Using Escherichia coli W3110 as the starting strain, a stepwise metabolic engineering process was performed using the CRISPR / Cas9 gene editing system. The specific construction steps are as follows: De-inhibition and Enhancement of Synthetic Pathways First, the lacI gene was knocked out to relieve its transcriptional repression on the Ptac promoter, enabling subsequent key genes to be expressed continuously without induction. The alsS gene, which encodes acetolactate synthase, derived from Bacillus subtilis 168, was integrated into the yghx site and expressed by the Ptac promoter to relieve the feedback inhibition of L-valine on the original acetolactate synthase. The ilvIH gene was integrated into the yjiT site and expressed by the Ptac promoter; the ilvED gene was integrated into the yjiV site and expressed by the Ptac promoter; the ilvC gene was integrated into the gapC site and expressed by the Ptac promoter; further, double-copy integration of the ilvIH and ilvC genes was performed to enhance the expression level of key enzymes in the L-valine synthesis pathway.

[0029] Enhance the supply of restorative power By integrating the pntAB gene encoding pyridine nucleotide transhydrogenase into the yjiP site and expressing multiple copies, NADPH can be efficiently regenerated from NADH, thereby improving the supply of intracellular reducing power.

[0030] Enhance product excretion capacity The L-valine efflux protein encoding gene brnFE, derived from Corynebacterium glutamicum ATCC 13032, was integrated into the brnQ site and expressed under the Ptac promoter, thereby enhancing the efflux efficiency of L-valine.

[0031] Knockout of pyruvate competitive pathway genes The intermediate strain VL-04 was obtained by sequentially knocking out the lactate dehydrogenase encoding gene ldhA, the pyruvate oxidase encoding gene poxB, the pyruvate formate lyase encoding gene pflB, and the frida acid reductase A subunit encoding gene frdA to reduce the loss of the precursor pyruvate.

[0032] Optimize the supply and demand balance of cofactors Based on strain VL-04, the original NADPH-biased branched-chain amino acid transaminase encoding gene ilvE was replaced with the NADH-biased leucine dehydrogenase encoding gene bcd from Bacillus subtilis 168, and expression was driven by the Ptac promoter, thereby constructing the L-valine-producing high-yield Escherichia coli engineered strain used in this invention, named VL-06.

[0033] (2) Fermentation liquid The fermentation broth, per 1 L, comprises the following components: 10 g glucose, 2 g yeast powder, 3 g ammonium sulfate [(NH4)2SO4], 7 g dipotassium hydrogen phosphate trihydrate (K2HPO4·3H2O), 1 g magnesium sulfate heptahydrate (MgSO4·7H2O), 1 g methionine, 2 g citric acid, 0.03 g ferrous sulfate heptahydrate (FeSO4·7H2O), 0.01 g manganese sulfate monohydrate (MnSO4·H2O), 0.001 g vitamin H (VH, biotin), 0.0005 g vitamin B1 (VB1), 0.0005 g vitamin B3 (VB3, niacin), 0.0005 g vitamin B5 (VB5, pantothenic acid), and vitamin B... 12 (VB) 12 0.0005 g.

[0034] The fermentation broth was prepared as follows: Dissolve all the above components in 900 mL of deionized water, stir well, and bring the volume to 1000 mL; adjust the pH to 6.7 using an acid-base adjuster; sterilize at 115 ℃ for 20 min, and then cool before use. Glucose can be sterilized separately and added before inoculation.

[0035] In actual fermentation, the concentrations of carbon source, nitrogen source and trace elements in the fermentation broth can be appropriately adjusted according to the growth of the strain and the needs of product accumulation. The adjustment range is usually ±10% to ±20%, all of which are within the protection scope of this invention.

[0036] The fermentation broth was then placed in a fermenter and inoculated with an L-valine-producing strain to initiate the first fermentation stage, which aimed at rapid cell proliferation and maintained a relatively high dissolved oxygen level.

[0037] 2. Control system settings Online monitoring: Equipped with an online analysis system capable of sampling and rapidly determining approximate intracellular RNR values ​​per minute, preferably using sensors based on specific fluorescent probes.

[0038] Decision Model and Threshold: Based on preliminary studies of strain VL-06, the threshold for NADH / NAD ratio was determined. + The value exceeding the threshold T1=0.95 for 5 minutes, and this threshold T1 is obtained through historical data correlation analysis, indicates that the cell has accumulated sufficient reducing power and is ready to massively shift the carbon flow to L-valine synthesis, triggering the phase transition.

[0039] Actions executed: Upon triggering, the system automatically executes the following: a) reducing the stirring speed from 750 rpm to 350 rpm and the aeration rate from 2.0 L / min to 1.0 L / min (target dissolved oxygen <5%); b) switching the glucose feeding rate from exponential growth feeding mode to constant-rate limiting feeding mode; c) fine-tuning the pH to 6.8.

[0040] 3. Fermentation Comparison Experiment Experimental group (in this invention): The dynamic control method of L-valine fermentation process of this invention is adopted.

[0041] Control group (traditional method): (1) Fermentation strain The same L-valine-producing high-yield Escherichia coli engineered strain VL-06 as in the previous example was used.

[0042] (2) Seed culture The activated L-valine-producing high-yield Escherichia coli engineered strain VL-06 was inoculated into seed culture medium and cultured with shaking at 37 ℃ and 200 r / min for 8-10 h to obtain seed liquid.

[0043] (3) Initial culture in fermenter The seed culture was inoculated into a bioreactor containing 1.2 L of fermentation broth at a 10% inoculation rate. Initial fermentation conditions were set as follows: temperature 37 ℃, pH controlled at 6.7, aeration rate 2.0 L / min, and stirring speed adjusted in conjunction with dissolved oxygen (350-800 r / min) to maintain a dissolved oxygen concentration above 25%. The initial glucose concentration was 10 g / L. Once the glucose was largely consumed, an 80% (w / v) glucose solution was added, with the feeding rate dynamically adjusted based on real-time glucose concentration measurements to maintain a glucose concentration in the reactor not exceeding 1.5 g / L.

[0044] (4) Stage transition conditions During fermentation, the OD600 value (cell density) of the fermentation broth is continuously monitored. When the OD600 value reaches 80±5, it is determined that the cell growth has reached a suitable density, and the process parameters are switched to proceed to the product synthesis stage.

[0045] (5) Second stage process parameters After the phase transition, the following process parameters should be adjusted simultaneously: Stirring speed: Reduced from dissolved oxygen linkage adjustment mode to 350 r / min and maintained constant; Ventilation rate: reduced from 2.0 L / min to 1.0 L / min; Dissolved oxygen level: No longer actively controlled, it decreases naturally with bacterial metabolism, and is usually maintained below 5%; Temperature: Maintain at 37 ℃; pH: Maintain at 6.7; Feeding strategy: Continue to feed dynamically based on glucose concentration, maintaining the glucose concentration in the tank at no more than 1.5 g / L.

[0046] (6) Fermentation endpoint Fermentation was continued until the L-valine concentration reached saturation (approximately 85-90 g / L), or fermentation was terminated when the product accumulation rate decreased significantly. Using this method, strain VL-06 achieved an L-valine yield of 88.63 g / L within 32 h, with a sugar-acid conversion rate of 40.01% and a production intensity of 2.77 g / (L·h).

[0047] The aforementioned traditional two-stage control method with a fixed OD value has the following technical limitations: (1) The timing of conversion depends on the empirical threshold: the conversion threshold of OD600=80 is determined based on previous experimental experience and cannot adapt to the subtle differences in bacterial activity between different batches; (2) Ignoring the state of cell metabolism: The OD value only reflects the number of cells and cannot indicate whether the intracellular metabolism is ready to enter a state of efficient synthesis. This may result in mismatches such as "the number of cells is sufficient but the metabolism is not ready" or "the metabolism is ready but the number of cells is not sufficient". (3) Coarse parameter switching: After the stage transition, each process parameter is adjusted in one go, lacking a fine-tuning mechanism based on real-time feedback from cells; (4) Unable to adapt to optimization: It lacks the ability to adapt to the individual differences of each batch of fermentation, and the robustness of the process is limited.

[0048] In response to the aforementioned limitations, this invention proposes an adaptive control method based on real-time monitoring of intracellular NADH / NAD+ physiological signals for dynamic decision-making.

[0049] Conditions: Run two fermentation processes for 50 h under the same initial conditions.

[0050] 4. Results Timing of conversion: In the experimental group, the RNR reached T1 9.5 h after the start of fermentation (at which time OD600 was approximately 68), and the system automatically triggered the conversion. In the control group, the conversion was performed at 11.2 h (when OD600 reached 80).

[0051] Fermentation performance comparison (50 h endpoint data): Fermentation performance such as Figure 2 (As shown in the comparison graph of L-valine concentration versus time between the dynamic RNR feedback control group and the traditional fixed OD value control group in the example), the experimental group monitored the intracellular NADH / NAD ratio online. +The ratio reached a preset threshold 9.5 hours after fermentation, automatically triggering a stage switch; while the control group only switched after the cell density grew to OD600=80 (approximately 11.2 hours). The experimental group switched 1.7 hours earlier, indicating that the method of this invention can detect the intrinsic need for cell metabolism to shift from "growth-driven" to "synthesis-driven" earlier.

[0052] from Figure 2 It is evident that the product concentration increase rate in the experimental group after switching was significantly greater than that in the control group, demonstrating a more efficient metabolic flow orientation. At 25 h of fermentation, the L-valine concentration in the experimental group reached 85 g / L, 1 h shorter than the control group (26 h), effectively improving equipment turnover. At the 50 h fermentation endpoint, the L-valine concentration in the experimental group reached 91.3 g / L, a 5.7% increase compared to the control group (86.4 g / L); the corresponding production intensity increased from 1.73 g / (L·h) to 1.83 g / (L·h), an increase of 5.8%. Using 85 g / L as the standard for tank discharge, the production intensity of the experimental group reached 3.40 g / (L·h), a 4.0% increase compared to the control group's 3.27 g / (L·h). The sugar-acid conversion rate in the experimental group was 0.46 g / g, a 4.5% increase compared to the control group (0.44 g / g), indicating that less carbon source was used for non-product synthesis pathways, significantly improving raw material utilization efficiency.

[0053] In summary, the dynamic fermentation regulation method and system based on cellular physiological state feedback (especially RNR) provided by this invention creatively upgrades process control from "external observation-driven" to "internal signal-driven." By scientifically defining and precisely quantifying the upward trend of RNR values, the physiological signal traditionally representing "reduction pressure" is transformed into a positive process switching trigger command, achieving adaptive capture of metabolic transition windows. Experimental data strongly demonstrate the significant effect of this method in improving key economic indicators of L-valine fermentation. This method possesses clear uniqueness, distinguishing it from all previously disclosed control strategies based on fixed parameters. Furthermore, the online monitoring scheme can employ high-frequency automatic sampling—enzyme labeling, fluorescence sensing, etc., providing a clear industrial implementation path and offering a reproducible and scalable new technology solution for the intelligent upgrading of the microbial fermentation industry.

[0054] When numerical ranges are given in the embodiments of this invention, it should be understood that, unless otherwise stated in this invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention, as well as the prior art known to those skilled in the art and the description of this invention, may be implemented using any prior art methods, devices, and materials similar to or equivalent to those described, used, or made in the embodiments of this invention.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for dynamic regulation of L-valine fermentation process, characterized in that, By monitoring the real-time physiological state signals of cells during fermentation online, and making dynamic decisions and switching process parameters based on the feedback of physiological state signals; The cellular physiological state signal includes at least one characteristic indicator that can characterize the intracellular reducing power level and metabolic flow reversal. This characteristic indicator is the real-time ratio of the oxidized to reduced state concentrations of intracellular nicotinamide adenine dinucleotide, i.e., NADH / NAD. + Real-time ratio.

2. The method for dynamic control of L-valine fermentation process according to claim 1, characterized in that, Specifically, the following steps are included: S1: Introduce L-valine-producing strains into the fermenter to start the first fermentation stage aimed at rapid cell proliferation; S2: Using an online monitoring system, continuously obtain real-time NADH / NAD levels in cells within the fermentation system. + ratio; S3: When the NADH / NAD is detected + When the ratio shows a continuous upward trend and reaches or exceeds the first preset threshold T1, the system determines that the cell metabolic flow has shifted from growth-dominated to product synthesis-dominated, and the automatic control system immediately triggers process switching. S4: The system enters the second fermentation stage, which focuses on maximizing L-valine synthesis. At least two of the following process parameters are adjusted simultaneously: a) reduce the stirring speed and / or aeration rate to lower the dissolved oxygen concentration; b) switch the feeding strategy from nutrient-rich feed to promote cell growth to restrictive feed to maintain metabolism; c) fine-tune the pH value to optimize the reactivity of key enzymes.

3. The method for dynamic control of L-valine fermentation process according to claim 2, characterized in that, It also includes step S5: continuous monitoring of NADH / NAD + Regarding the fluctuation of the ratio, when the ratio deviates from the second preset range R2, the process parameters of the second stage are fine-tuned to stabilize the ratio back within the second preset range R2. The second preset range R2 is determined through batch experiments, and the NADH / NAD ratio corresponding to the highest product synthesis rate is taken. + The range of ±15% of the ratio.

4. A method for dynamic control of L-valine fermentation process according to claim 2 or 3, characterized in that, The L-valine-producing strain is a metabolically engineered Escherichia coli strain, which is modified by knocking out genes related to the pyruvate competitive pathway and / or introducing key enzyme-encoding genes optimized by cofactor preference.

5. A method for dynamic control of L-valine fermentation process according to claim 2 or 3, characterized in that: The first preset threshold T1 is determined by conducting multiple batches of fermentation experiments using the same strain and culture medium to control NADH / NAD ratio. + The dynamic change curve of the ratio was correlated with the L-valine accumulation rate curve and the determination was made.

6. A method for dynamic control of L-valine fermentation process according to claim 2 or 3, characterized in that, The first fermentation stage in step S1 is the rapid growth phase of the microbial cells, and the specific fermentation conditions are as follows: a: Inoculate the activated L-valine-producing strain into a fermenter containing fermentation broth at an inoculation rate of 8% to 12%, with an initial glucose concentration of 15 g / L to 25 g / L; b: The fermentation temperature should be controlled between 36 ℃ and 38 ℃; c: The pH of the fermentation broth is controlled between 6.8 and 7.2 by automatically adding alkali solution; d: Maintain dissolved oxygen concentration between 25% and 40% by adjusting stirring speed and aeration rate; e: The feeding adopts the exponential feeding mode to control the glucose concentration in the fermentation broth to be maintained between 1.0 g / L and 3.0 g / L.

7. A method for dynamic control of L-valine fermentation process according to claim 2 or 3, characterized in that, The specific fermentation conditions for the second fermentation stage in step S4 are as follows: a: When the NADH / NAD+ ratio monitored online reaches the preset threshold T1 and remains so for more than 5 minutes, the control system automatically switches the process parameters. b: Reduce the dissolved oxygen concentration from 25% to 40% in the first fermentation stage to below 5%; c: Reduce the stirring speed from 700 r / min to 850 r / min to 300 r / min to 450 r / min; d: Reduce ventilation from 1.5 vvm to 2.0 vvm to 0.5 vvm to 1.0 vvm; e: Maintain the fermentation temperature at 36 ℃ to 38 ℃; f: Adjust the pH of the fermentation broth from 6.8 to 7.2 in the first stage to 6.5 to 6.9; g: The feeding mode was switched from exponential feeding mode to constant rate restrictive feeding mode, and the glucose concentration in the fermentation broth was maintained at 0.5 g / L to 1.5 g / L.

8. A dynamic control system for L-valine fermentation, employing the dynamic control method for L-valine fermentation according to any one of claims 1-6, characterized in that, include: Bioreactor body; The online physiological signal monitoring unit is used to measure the NADH / NAD ratio in cells of the fermentation system in real time. + ratio; The central control unit, which has a pre-stored decision model and preset thresholds, is responsible for receiving monitoring data, making logical judgments and issuing control commands. The process parameter execution unit, based on the instructions issued by the central control unit, performs coordinated control of the reactor's stirring system, aeration system, feeding system, and pH adjustment system.

9. The dynamic control system for L-valine fermentation process according to claim 7, characterized in that, The online physiological signal monitoring unit employs a non-invasive online fluorescence sensor based on fluorescence lifetime or a specific wavelength, or an online analysis device that combines high-frequency automatic sampling with rapid quenching and enzyme labeling detection.

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