Escherichia coli strain culture method

Abstract

The invention discloses an Escherichia coli strain culture method, and belongs to the field of medicine, the Escherichia coli strain culture method comprises the following steps: S1, preparing an asymmetric carbon nitrogen flow regulation type compound culture medium which comprises the following components: 10-25 g / L of a carbon source component, 4-8 g / L of a nitrogen source component, 5-10 g / L of a slow release carbon source component, 5-10 g / L of a nitrogen source component, 5-10 g / L of a nitrogen source component and the balance of water, and the mass ratio of the carbon source component to the slow release carbon source component is 3: 7; wherein the ratio of inorganic nitrogen to organic nitrogen is 1: 2, and the microelement liquid is 1-3mL / L; and S2, inoculating a seed solution containing a non-model escherichia coli strain into the culture medium, and setting initial culture parameters as follows: the temperature is 33 DEG C, the pH value is 6.8, the dissolved oxygen is maintained at 50%, and the rotating speed is 150rpm. By introducing an asymmetric carbon-nitrogen regulation strategy and a slow-release carbon source system, the metabolic environment of non-model escherichia coli is optimized, and the growth adaptability and stability of the escherichia coli in an artificial culture medium are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and more particularly to a method for culturing an Escherichia coli strain. Background Art

[0002] Since its discovery in the late 19th century, methods for culturing Escherichia coli strains have undergone significant development. Initial cultures relied on simple solid and liquid media, such as nutrient agar and broth media, for basic isolation and proliferation. In the mid-20th century, with the rise of molecular biology, researchers developed selective and differentiation media, such as MacConkey agar, to differentiate lactose fermentation capacity. At the same time, advances in aseptic techniques, temperature-controlled shakers, and anaerobic culture systems significantly improved the efficiency and controllability of culture. Entering the 21st century, culture methods have become more precise and high-throughput, such as automated culture systems, microfluidic chips, and genetically engineered culture systems, making the screening, expression, and induction of specific functional strains more efficient.

[0003] However, the biggest challenge currently lies in the poor adaptability of culture methods to non-model strains. Many environmentally modified or engineered E. coli strains grow slowly or become completely inactivated in traditional culture media. This is particularly true for heterologous protein expression, metabolic pathway regulation, or cultivation under extreme conditions. The lack of specifically optimized culture systems severely limits the further application of E. coli in synthetic biology and industrial fermentation. This shortcoming represents an important direction for future technological improvements. Summary of the Invention

[0004] The present invention aims to provide a method for culturing Escherichia coli strains to address the issues raised in the aforementioned background art. However, a major challenge currently lies in the poor adaptability of these methods to non-model strains. Many environmental or engineered E. coli strains grow slowly or are completely inactivated in traditional culture media. This is particularly true for heterologous protein expression, metabolic pathway regulation, or cultivation under extreme conditions. The lack of optimized culture systems significantly limits the further application of E. coli in synthetic biology and industrial fermentation.

[0005] Technical solution: The method for culturing an Escherichia coli strain comprises the following steps:

[0006] S1. Prepare an asymmetric carbon and nitrogen flow-regulated composite culture medium comprising the following components: 10-25 g / L of a carbon source component, wherein the mass ratio of a fast-release carbon source to a slow-release carbon source is 3:7, 4-8 g / L of a nitrogen source component, wherein the ratio of inorganic nitrogen to organic nitrogen is 1:2, and 1-3 mL / L of a trace element solution;

[0007] S2. The seed solution containing the non-model E. coli strain was inoculated into the culture medium, and the initial culture parameters were set as follows: temperature 33°C, pH 6.8, dissolved oxygen maintained at 50%, and rotation speed 150 rpm;

[0008] S3. After 6 hours of culture, the expression induction system was introduced in three stages, using a step-by-step pulse induction strategy. The inducers were IPTG and the autoregulatory RNA module. The inducer concentrations were 0.05 mmol / L, 0.1 mmol / L, and 0.2 mmol / L, respectively, with 4-hour intervals.

[0009] S4. Simultaneously use the AI-based metabolic rate detection module to dynamically monitor bacterial OD600, pH, glucose consumption rate, and metabolic acid accumulation, and provide feedback to adjust the carbon / nitrogen ratio.

[0010] S5. Introduce 5-10 μM of the quorum sensing signaling molecule N-acyl homoserine lactone (AHL) during the culture process to initiate the QS induction circuit;

[0011] S6. During the induction period, the dynamic ion field buffer system was activated, and the buffer contained K + , Ca 2+ Mg 2+ Ion stability ratio, total concentration is 5-10mmol / L;

[0012] After incubation, centrifuge the cells at 8000 × g for 10 minutes at 4°C using an automated cryogenic collection module to collect the cells for subsequent processing.

[0013] Preferably, the fast-release carbon source is glucose, and the slow-release carbon source is a mixture of maltitol and sodium acetate. The ratio of the slow-release carbon source controls the bacterial body to be in a stable metabolic state and avoids excessive acetic acid accumulation.

[0014] Preferably, the expression induction system includes a self-regulating small RNA expression component for translationally inhibiting the mRNA of toxR-type toxicity regulatory genes at the post-transcriptional level.

[0015] Preferably, the quorum sensing system adopts a LuxI-LuxR type QS regulatory module, and the expression induction threshold is set to 5 μM via an AHL concentration response mechanism.

[0016] Preferably, in the dynamic ion field buffer system, K + , Ca 2+ Mg 2+ The ratio is 3:2:1, maintaining the stability of extracellular osmotic pressure and membrane potential.

[0017] Preferably, the pH value during the culture phase is dynamically adjusted between 6.2 and 7.8 according to the accumulation of metabolic acids, and the automatic titration system is adjusted by the ratio of NaHCO3 and HCl addition.

[0018] Preferably, the cooling and collecting module first reduces the temperature of the culture solution to 2-4° C. before collection, and adds a cell membrane protectant with a glycerol concentration of 0.1%.

[0019] Preferably, a fusion-type toxicity-sensing protein expression controller is introduced during the induction stage. The controller is based on the mqsRA toxicity-antitoxicity module and controls the expression switch state through bacterial toxicity threshold detection.

[0020] Preferably, the induction stage adopts an artificial light-controlled expression module, the light-controlled system is constructed based on the blue light-responsive protein EL222, and the induction light intensity is 5-15mW / cm 2 , providing a non-chemical induction alternative pathway in the synchronous triggering stage of the expression system.

[0021] Compared with the prior art, the advantages of the present invention are:

[0022] (1) The present invention optimizes the metabolic environment of non-model Escherichia coli by introducing an asymmetric carbon-nitrogen regulation strategy and a slow-release carbon source system, significantly improving its growth adaptability and stability in artificial culture medium.

[0023] (2) The present invention adopts a step-by-step pulse induction and sRNA regulation mechanism to reduce the metabolic impact caused by transient induction and effectively improve the physiological stability and protein yield of non-model strains during the expression process.

[0024] (3) The present invention unifies the start time of bacterial expression through a quorum sensing control system, achieves temporal synchronization of expression, and significantly improves product consistency and purification efficiency.

[0025] (4) The present invention introduces a dynamic ion buffer system to stabilize the cell membrane potential and osmotic pressure, thereby improving the strain's stress resistance and expression stability during induction.

[0026] (5) The present invention adjusts the carbon-nitrogen ratio and pH state in real time based on the AI feedback model, maintains metabolic homeostasis, optimizes the expression process, and improves overall efficiency and robustness.

[0027] (6) The present invention constructs a toxicity-antitoxicity module, which automatically buffers when the expression pressure is too high, preventing bacterial death or protein degradation, and improving system safety.

[0028] (7) The present invention adopts physical induction methods such as temperature control and light control to reduce the toxicity burden on non-model strains and expand the scope of application of the expression system.

[0029] (8) The present invention achieves stable maintenance of bacterial activity during high-density fermentation and improves unit volume yield by controlling oxygen supply, carbon source flow rate and sustained-release dosage.

[0030] (9) The present invention introduces a molecular chaperone coordinated expression and folding auxiliary system to reduce inclusion body formation, increase the soluble expression ratio of the target protein, and simplify the subsequent purification process. DETAILED DESCRIPTION

[0031] Example

[0032] Example 1: The method for culturing an Escherichia coli strain comprises the following steps:

[0033] S1. Prepare an asymmetric carbon and nitrogen flow-regulated composite culture medium comprising the following components: 10-25 g / L of a carbon source component, wherein the mass ratio of a fast-release carbon source to a slow-release carbon source is 3:7, 4-8 g / L of a nitrogen source component, wherein the ratio of inorganic nitrogen to organic nitrogen is 1:2, and 1-3 mL / L of a trace element solution;

[0034] S2. The seed solution containing the non-model E. coli strain was inoculated into the culture medium, and the initial culture parameters were set as follows: temperature 33°C, pH 6.8, dissolved oxygen maintained at 50%, and rotation speed 150 rpm;

[0035] S3. After 6 hours of culture, the expression induction system was introduced in three stages, using a step-by-step pulse induction strategy. The inducers were IPTG and the autoregulatory RNA module. The inducer concentrations were 0.05 mmol / L, 0.1 mmol / L, and 0.2 mmol / L, respectively, with 4-hour intervals.

[0036] S4. Simultaneously use the AI-based metabolic rate detection module to dynamically monitor bacterial OD600, pH, glucose consumption rate, and metabolic acid accumulation, and provide feedback to adjust the carbon / nitrogen ratio.

[0037] S5. Introduce 5-10 μM of the quorum sensing signaling molecule N-acyl homoserine lactone (AHL) during the culture process to initiate the QS induction circuit;

[0038] S6. During the induction period, the dynamic ion field buffer system was activated, and the buffer contained K + , Ca 2+ Mg 2+ Ion stability ratio, total concentration is 5-10mmol / L;

[0039] After incubation, centrifuge the cells at 8000 × g for 10 minutes at 4°C using an automated cryogenic collection module to collect the cells for subsequent processing.

[0040] Implementation 2-5

[0041] Example 2

[0042] On the basis of Example 1, the ratio of glucose to glycerol in the slow-release carbon source particles was adjusted to 1: 2, the total carbon source concentration was set to 25 g / L, and the other steps remained unchanged. This improvement is intended to adapt to an expression system that is more sensitive to glycerol, while extending the carbon source release time to 8 hours to reduce the metabolic load in the early stage of induction. The GFP expression amount was finally measured to be 285 mg / L, the acetic acid by-product concentration was controlled below 0.15 g / L, the system pH was maintained at 6.9 ± 0.1, the cell morphology was full, there was no obvious growth inhibition during the induced expression process, the expression product was mainly in a soluble form, and the target protein had high purity.

[0043] Example 3

[0044] Based on Example 1, the induction temperature and time were optimized. 600 When the pH reached 0.6, the culture temperature was lowered from 37°C to 12°C, and the induction time was extended to 18 hours to enhance the induction strength of the cspA promoter at low temperatures. All other conditions remained unchanged. After induction, the protein expression level reached 350 mg / L, the acetic acid concentration was controlled within 0.18 g / L, and the bacterial activity was good. Cold-induced expression stabilized the target protein and increased the solubility ratio by approximately 18%, facilitating subsequent purification and structural studies.

[0045] Example 4

[0046] In this example, the expression of thermostable trypsin (ThermoTrypsin) was used as the target protein. The strain was still Nissle1917, and an expression system was constructed by plasmid transformation. The slow-release carbon source consisted of maltose and glycerol in a ratio of 2: 1, with a total concentration of 18 g / L. The induction temperature was set at 20 ° C and the induction duration was 12 hours. The protein expression amount after induction was 275 mg / L, and the solubility was good. The expression product had a high enzymatic activity after activity detection, indicating that the method is also applicable to the expression of different types of proteins and has a wide range of applications.

[0047] Example 5

[0048] Escherichia coli B21 (DE3) strain (non-model strain, derived from intestinal isolates) was selected as the host for expressing the cell wall hydrolase LysLM from Lactococcus lactis. The pCold-cspA-LysLM expression plasmid was constructed and transformed into the B21 strain. The slow-release carbon source used was glucose: glycerol: maltose = 1:1:1, with a total concentration of 22g / L; the sRNA regulatory module was used to regulate the expression of T7RNA polymerase; when the OD600 reached 0.5, the temperature was lowered to 15°C for induction for 12 hours. After SDS-PAGE detection of the expression product, it was found that the target protein expression level was 315mg / L, mainly in soluble form; the protein activity was confirmed to be functional by cell wall lysis experiment. This example proves that the system has good adaptability and is suitable for expressing exogenous enzyme functional proteins.

[0049] Example 6

[0050] The recombinant strain EcN-pTrc-AiiA (engineered to express the Escherichia coli quorum sensing interferase AiiA, constructed in Nissle 1917) was used. The target protein is highly sensitive to oxidative stress. A slow-release carbon source of xylose:glycerol (2:1) was used at a total concentration of 20 g / L, and the promoter was a modified cspA enhancer sequence. The temperature was lowered from 37°C to 16°C for 14 hours, and 1 mM glutathione was added to assist protein folding. Results showed that the target protein expressed at a level of 285 mg / L, with a solubility rate of 82%. Its anti-biofilm activity was significantly enhanced, demonstrating the applicability of this induction system for the expression of oxidation-sensitive proteins.

[0051] Comparative Example 1

[0052] The same Nissle1917 strain and expression plasmid as in Example 1 were selected, but the culture medium was ordinary LB liquid medium, the induction carbon source was traditional IPTG (final concentration 1mM), and the induction temperature was 37°C for 8 hours. No slow-release carbon source or cspA promoter was used. After induction, the target protein expression amount was only 120mg / L, and most of it was in the form of inclusion bodies. During the culture period, acetic acid accumulated seriously (>0.8g / L), the pH dropped to 5.2, and bacterial lysis occurred, and the expression system was poor in stability.

[0053] Comparative Example 2

[0054] The same slow-release carbon source system as in Example 1 was selected, but the small RNA expression control module was not used. Only the original plasmid expression system was used, and the other steps were the same. After induction, the target protein expression level was 190 mg / L, the protein solubility was poor, the soluble product accounted for less than 40%, and transcriptional inhibition occurred. It is speculated that the endogenous feedback mechanism has an adverse effect on the expression system. The results show that the sRNA control module plays a key role in improving expression efficiency and stability.

[0055] Comparative Example 3

[0056] The same strain and plasmid system as in Example 1 were used, but a slow-release carbon source was not used during the induction process. Instead, a conventional strategy of disposable carbon source addition (glucose 20 g / L was added once in the initial induction phase) was employed, with all other conditions remaining unchanged. Acetic acid concentration was detected to rise rapidly to 1.1 g / L during the induction process, and pH dropped to 5.1, resulting in suppressed bacterial growth. The target protein yield was reduced to 95 mg / L, and was primarily inclusion bodies. This indicates that conventional rapid carbon supply methods cause metabolic imbalance in non-model bacteria, severely inhibiting the expression system.

[0057] Comparative Example 4

[0058] All parameters and strains from Example 3 were used, but the induction temperature was set to a constant 37°C, and cold induction was not performed. After induction, the target protein expression level was detected to be only 165 mg / L, and more than 70% formed inclusion bodies. The cells were morphologically abnormal, and some were lysed. This comparative example illustrates that the cold induction strategy plays a key role in stabilizing protein folding, reducing metabolic load, and improving expression quality.

[0059] In order to measure the adaptability improvement effect of the E. coli strain culture method of the embodiment and the comparative example on the expression, solubility, activity and other aspects of the target protein in different non-model strains, the following comparative experiment was designed. The experimental steps are as follows:

[0060] The strains used in the experiment included various representative non-model E. coli strains, including Nissle1917, EcN, BL21-star(DE3), and B21(DE3), all of which were obtained from laboratory collections or the ATCC standard strain library. For expression plasmids, engineered plasmids containing the pCold-cspA promoter, the pTrc99a vector, and T7-sRNA regulatory elements were selected. These plasmids carried target expression proteins such as GFP (green fluorescent protein), AiiA (quorum sensing enzyme), LysLM (lysozyme), and HsTrx (human thioredoxin).

[0061] The culture media used in the experiment included standard LB liquid medium and modified M9 minimal medium. The latter was modified to include different carbon sources, such as slow-release glycerol, maltose, or xylose, depending on the specific design. IPTG (isopropyl-β-D-thiogalactopyranoside) was used as an inducer. Reagents used in the experiment also included SDS-PAGE kits for protein analysis, BCA protein quantification kits, and Western blot reagents.

[0062] To ensure protein expression quality and correct folding, in some embodiments, glutathione was added as a cofactor, or a Trx tag was fused to enhance solubility.

[0063] The experimental steps are as follows:

[0064] Pre-culture and seed preparation: Samples of the strain were collected from the glycerol stock and streaked onto LB solid plates. Incubate at 37°C for 12 hours. A single colony was selected and inoculated into a tube containing LB liquid medium. Pre-cultured overnight at 37°C in a shaker. The next day, an appropriate amount of the bacterial suspension was inoculated into modified M9 liquid medium. The inoculum size was adjusted to achieve an initial OD600 of 0.05-0.1. Continue culturing until the OD600 reached 0.4-0.6, which served as the starting point for induction.

[0065] Expression induction treatment: The control group (comparative example) was directly induced at 37°C, IPTG (final concentration 1 mM) was quickly added, and glucose or no slow-release carbon source was added at one time, and expression was induced for 12 hours;

[0066] The embodiment group adopted a cold induction method at 15–18°C. In the early stage of induction, a carbon source (such as 1% glycerol / xylose) was slowly added to form a gradual carbon supply environment, and an sRNA regulatory element (such as a MicC derivative) or a T7 weak starter regulatory module was added.

[0067] Certain embodiments introduce Trx fusion expression or folding auxiliary factors to improve solubility;

[0068] All inductions were maintained for 12–16 hours before termination.

[0069] Protein expression and functional testing: After bacterial collection, cells were lysed by ultrasonication and total protein was extracted. Protein expression profiles were analyzed by SDS-PAGE, and the total protein content was quantified using a BCA assay. Soluble fractions and inclusion bodies were separated, and the soluble protein ratio was calculated. Functional proteins (such as AiiA and LysLM) were further validated using specific activity assays. Physiological metabolic parameters such as the endpoint OD600 value, pH at the end of induction, and acetic acid concentration in the culture medium were simultaneously recorded to assess metabolic stress.

[0070] The experimental data are shown in Table 1:

[0071]

[0072]

[0073] Table 1

[0074] The experimental results show that the protein expression efficiency is significantly improved: the cold induction process used in the embodiment can significantly delay the start of protein expression, reduce the metabolic impact on the host strain, and the GFP expression level is increased by about 20% to 35%. At the same time, the total protein concentration is also increased simultaneously.

[0075] The bacterial growth is more stable: In the embodiment group, the peak value of the bacterial OD600 is slightly higher than that of the control group, and the overall growth curve is smoother, indicating that the cold induction and slow-release carbon source strategy effectively reduced the stress response and improved the physiological stability of the non-model strain during the induction process.

[0076] The metabolic burden was significantly reduced: the accumulation of acetic acid in the control group was generally between 1.1 and 1.2 g / L, while that in the embodiment group was controlled within the range of 0.28 to 0.33 g / L, indicating that the slow-release carbon supply strategy effectively avoided carbon source overload and the rapid accumulation of acetic acid toxicity, and was a key improvement point in solving the problem of metabolic inhibition in non-model strains.

[0077] Improved overall adaptability: Compared with the comparative example, the comprehensive regulatory strategy adopted in the embodiment (including induced temperature regulation, slow-release carbon source supply, etc.) significantly enhanced the tolerance of non-model strains to expression burden and metabolic stress, thereby maintaining a better cell physiological state while efficiently expressing the target protein.

[0078] In order to measure the adaptability differences between the embodiment and the comparative example non-model strain under induction conditions, including growth curves, protein expression levels, and production of metabolites (acetic acid), the following comparative experiment was designed. The experimental steps are as follows:

[0079] Experimental material preparation:

[0080] Strain selection: Example strain: Nissle1917 (non-model strain)

[0081] Comparative strain: Nissle1917 (control group, using rapid carbon supply conditions)

[0082] Culture medium: LB medium (Luria-Bertani) is used to culture strains.

[0083] Gene expression inducer: IPTG (isopropyl-β-D-thiogalactopyranoside, concentration: 0.1 mM).

[0084] Slow-release glucose (used in the examples, for the slow-release strategy of Example 1).

[0085] Analytical reagents: Acetic acid determination kit.

[0086] Protein concentration determination kit.

[0087] GFP fluorescence assay system (used to characterize protein expression levels).

[0088] Equipment: Optical density meter (OD600, used to monitor bacterial growth).

[0089] Ultracentrifuge (for collecting culture fluid samples).

[0090] Fluorometer (for detecting GFP expression).

[0091] High performance liquid chromatography (HPLC, for analysis of acetic acid accumulation).

[0092] The experimental steps are as follows:

[0093] Strain culture: Example group: Nissle1917 strain was inoculated into LB medium, using a cold induction strategy with the temperature set at 15°C, and slow-release glucose was added thereto to maintain an appropriate carbon source supply and avoid excessive acetic acid accumulation.

[0094] Comparative Example Group: Nissle 1917 strain was inoculated into LB medium, carbon was rapidly supplied, and conventional induction conditions (30° C., rapid glucose supply) were adopted.

[0095] Induction expression: When OD600 reached 0.6, IPTG was added to induce gene expression. The temperature control conditions were set according to the groups: the example group was maintained at 15°C, and the rapid induction control group was maintained at 30°C.

[0096] Sampling and analysis: 10 mL of culture medium was sampled every hour, the OD600 value was monitored, and the bacterial growth curve was tracked.

[0097] During the expression phase (within 12 hours after induction), the culture medium was collected every 4 hours for protein concentration determination.

[0098] During the culture process, 10 mL of the sample was collected every 3 hours for determination of acetic acid concentration.

[0099] Protein expression determination: The total protein content in the bacterial solution was determined using a BCA protein concentration kit.

[0100] The fluorescence intensity of each sample was detected by a GFP fluorescence measurement instrument.

[0101] Determination of metabolites (acetic acid): The accumulation of acetic acid at each time point was detected by HPLC, and the amount of acetic acid produced was recorded.

[0102] The experimental data are shown in Table 2:

[0103]

[0104]

[0105] Table 2

[0106] The experimental results showed that in the comparative example, the relative expression of GroEL was significantly increased (about 2.4 times that of the example), indicating that the conventional rapid induction and carbon source shock produced a high level of heat stress in the bacteria. In the example, due to the use of a mild cold induction and slow-release carbon source strategy, the expression of GroEL was significantly reduced, indicating that the overall heat stress level of the bacteria was effectively alleviated.

[0107] The Example group maintained a low temperature during the induction process, effectively activating the expression of the CspA protein, which is associated with the stability of cold-induced protein expression. Experimental data showed that the expression level of CspA in the Example group was 2.8 times that of the control group, enhancing the protein folding and stabilization mechanism under low temperature conditions and improving the adaptability of non-model strains in cold-induced environments.

[0108] Comparison of ATP levels within 4 hours after protein expression showed that the metabolic activity of the Example group recovered faster than that of the Control group, suggesting that the group had better energy metabolism recovery ability after exogenous protein expression. This may be related to the slow induction start to avoid early energy overload.

[0109] Based on the expression data of GroEL and CspA and the recovery of energy metabolism, it can be seen that the strategy adopted in the embodiment is more mild in the stress of non-model strains during the induction process, avoiding the severe physiological impact brought by conventional expression strategies, and significantly enhancing the overall adaptability and survival state of the bacteria.

[0110] The above shows and describes the basic principles, main features and advantages of the present invention; those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only preferred examples of the present invention and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected; the scope of protection claimed in the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for culturing an Escherichia coli strain, characterized in that: The method for culturing an Escherichia coli strain comprises the following steps: S1. Prepare an asymmetric carbon and nitrogen flow-regulated composite culture medium comprising the following components: 10-25 g / L of a carbon source component, wherein the mass ratio of a fast-release carbon source to a slow-release carbon source is 3:7, 4-8 g / L of a nitrogen source component, wherein the ratio of inorganic nitrogen to organic nitrogen is 1:2, and 1-3 mL / L of a trace element solution; S2. The seed solution containing the non-model E. coli strain was inoculated into the culture medium, and the initial culture parameters were set as follows: temperature 33°C, pH 6.8, dissolved oxygen maintained at 50%, and rotation speed 150 rpm; S3. After 6 hours of culture, the expression induction system was introduced in three stages, using a step-by-step pulse induction strategy. The inducers were IPTG and the autoregulatory RNA module. The inducer concentrations were 0.05 mmol / L, 0.1 mmol / L, and 0.2 mmol / L, respectively, with 4-hour intervals. S4. Simultaneously use the AI-based metabolic rate detection module to dynamically monitor bacterial OD600, pH, glucose consumption rate, and metabolic acid accumulation, and provide feedback to adjust the carbon / nitrogen ratio. S5. Introduce 5-10 µM of the quorum sensing signaling molecule N-acyl homoserine lactone (AHL) during the culture process to initiate the QS induction circuit; S6. During induction, a dynamic ion field buffer system was activated, containing a stable ratio of K⁺, Ca²⁺, and Mg²⁺ ions in the buffer, with a total concentration of 5-10 mmol / L. After incubation, centrifuge the cells at 8000 × g for 10 minutes at 4°C using an automated cryogenic collection module to collect the cells for subsequent processing.

2. The method for culturing an Escherichia coli strain according to claim 1, wherein The rapid carbon source is glucose, and the slow-release carbon source is a mixture of maltitol and sodium acetate. The ratio of the slow-release carbon source controls the bacterial body to be in a stable metabolic state and avoids excessive acetic acid accumulation.

3. The method for culturing an Escherichia coli strain according to claim 1, wherein The expression induction system comprises a self-regulating small RNA expression component, which is used for inhibiting the translation of toxR type toxicity regulatory gene mRNA at the post-transcriptional level.

4. The method for culturing an Escherichia coli strain according to claim 1, wherein The quorum sensing system adopts a LuxI-LuxR type QS regulatory module, and the expression induction threshold is set to 5 μM via an AHL concentration response mechanism.

5. The method for culturing an Escherichia coli strain according to claim 1, wherein: In the dynamic ion field buffer system, the ratio of K⁺, Ca²⁺, and Mg²⁺ is 3:2:1, maintaining the stability of extracellular osmotic pressure and membrane potential.

6. The method for culturing an Escherichia coli strain according to claim 1, wherein: During the culture phase, the pH value was dynamically adjusted between 6.2 and 7.8 according to the accumulation of metabolic acids, and the automatic titration system was used to adjust the ratio of NaHCO3 and HCl addition.

7. The method for culturing an Escherichia coli strain according to claim 1, wherein: The cooling and collecting module first lowers the temperature of the culture solution to 2-4°C before collection, and adds a cell membrane protectant with a glycerol concentration of 0.1%.

8. The method for culturing an Escherichia coli strain according to claim 1, wherein: A fusion-type toxicity-sensing protein expression controller is introduced during the induction phase. The controller is based on the mqsRA toxicity-antitoxicity module and controls the expression switch state through bacterial toxicity threshold detection.

9. The method for culturing an Escherichia coli strain according to claim 1, wherein: During the induction phase, an artificial light-controlled expression module is used. The light-controlled system is based on the blue light-responsive protein EL222, and the induction light intensity is 5-15mW / cm², providing a non-chemical induction alternative path during the synchronous triggering phase of the expression system.

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