Preparation method and application of blue light regulated escherichia coli

Abstract

The invention belongs to the technical field of biological engineering, and discloses a preparation method and application of Escherichia coli regulated by blue light, and the preparation method comprises the steps of blue light response element construction, strain engineering transformation, fermentation system design, culture condition optimization and product purification work.The blue light response element construction comprises the steps that a, a core light sensing assembly is selected; b, designing and synthesizing an EL222 coding sequence, c, constructing and synthesizing a promoter, and d, optimizing an EL222 binding site; the engineering modification of the strain comprises the following steps: a, integrating a photosensitive promoter and a target gene, and b, optimizing the expression level of EL222 protein. According to the invention, directional regulation and control of metabolic pathways can be realized at a subcellular level through the blue light response element, and through accurate control of illumination intensity and time, metabolic flux can be dynamically adjusted according to requirements, accumulation of metabolic intermediates is reduced, cytotoxicity is reduced, purity of a target product is improved, and the method is suitable for industrial production. The yield and the quality of a target product are improved through precise space-time regulation and control on escherichia coli metabolism.

Description

Technical Field

[0001] The present invention belongs to the field of biological engineering technology, and specifically relates to a preparation method and application of Escherichia coli regulated by blue light. Background Art

[0002] Escherichia coli is a commonly used host bacterium in bioengineering and is widely used in recombinant protein expression and metabolite production. Its advantages lie in its fast growth rate, clear genetic background, ease of genetic manipulation, and the availability of a wealth of molecular biology tools.

[0003] However, traditional gene expression regulation methods have some limitations in E. coli:

[0004] 1. Dependence on chemical inducers: Commonly used induction systems such as IPTG (isopropyl-β-D-thiogalactoside) or arabinose induction systems require the addition of exogenous chemicals to activate target gene expression, which not only increases production costs but may also introduce potential contaminants and affect the downstream purification process;

[0005] 2. Limited regulation precision: Chemical inducers are usually difficult to achieve precise dosage control, resulting in large variability in gene expression levels. There are only two regulation modes: "all or none", and it is difficult to meet the needs of fine metabolic engineering;

[0006] 3. Difficulty in spatiotemporal control: Traditional methods have difficulty in achieving precise temporal and spatial control of gene expression, which limits the optimization and dynamic regulation of complex metabolic networks;

[0007] 4. Poor reversibility: Once expression is initiated, it is usually difficult to quickly shut down or regulate, which poses certain problems in applications that require precise control of metabolic flux;

[0008] 5. Metabolic burden: Some chemical inducers impose additional burden on cell metabolism, affecting overall production efficiency. Summary of the invention

[0009] The purpose of the present invention is to provide a preparation method and application of Escherichia coli regulated by blue light to solve the problems raised in the above background technology.

[0010] In order to achieve the above-mentioned object, the present invention provides the following technical solutions: a preparation method and application of blue light-regulated Escherichia coli, including constructing a blue light response element, engineering and transforming the strain, designing a fermentation system, optimizing culture conditions and establishing a product purification process,

[0011] The blue light response element is constructed by: a selecting a core light sensing component, b designing and synthesizing an EL222 coding sequence, c constructing a synthetic promoter, and d optimizing an EL222 binding site;

[0012] The engineering modification of the strain: a integrates a light-sensitive promoter and a target gene, b optimizes the expression level of the EL222 protein, and c deletes endogenous genes that interfere with light response;

[0013] The fermentation system design includes: a. designing the fermentation tank, b. developing the lighting control system, and c. integrating the online monitoring system;

[0014] The optimization of culture conditions includes: a determining the best illumination parameters, b optimizing the culture medium composition, and c establishing a control strategy;

[0015] The establishment of product purification process: a. Developing purification process, b. Establishing quality control system,

[0016] Preparation steps:

[0017] S1. Material preparation:

[0018] Select bacterial hosts and procure necessary reagents and equipment;

[0019] S2. Gene construction:

[0020] Design the EL222 coding sequence and synthesize the promoter, and clone into the vector;

[0021] S3. Strain transformation:

[0022] The promoter vector was transformed into E. coli and clones that successfully integrated the light-sensitive promoter and target gene were selected;

[0023] Identify and delete endogenous genes that interfere with light response;

[0024] S4. Fermentation system design and culture conditions optimization:

[0025] The fermentation tank is equipped with a blue LED array, and the designed programmable light controller is integrated with a light intensity sensor and installed in the fermentation tank for real-time light intensity monitoring and feedback adjustment;

[0026] Determine the culture medium, screen and obtain the culture medium formula suitable for light-induced conditions, determine the illumination parameters, and establish the culture parameter control strategy;

[0027] S5. Fermentation and product acquisition:

[0028] The positive clones were inoculated into the optimized culture medium for preliminary fermentation culture;

[0029] Blue LED light was used for light induction to activate EL222 and downstream gene expression;

[0030] Use an online monitoring system to track cell growth and target product expression levels in real time;

[0031] S6. Product purification:

[0032] Collect cells by centrifugation, remove culture medium and other impurities, and use high-pressure homogenization or other cell disruption methods to maximize the release of target protein and obtain the released target protein;

[0033] Perform optimized chromatography and use affinity chromatography to improve the purity of the obtained target protein;

[0034] The target protein test was qualified, confirming that the blue light-regulated Escherichia coli was prepared.

[0035] Preferably, the EL222 is a photosensitive protein derived from marine bacteria, which undergoes conformational changes under blue light irradiation, thereby activating downstream gene expression, and the EL222 photosensitive protein system is selected as the core light sensing component.

[0036] Preferably, in the integration of the photosensitive promoter and the target gene, the target gene is a recombinant protein gene, the promoter comprises an EL222 coding sequence, and the recombinant protein gene is accurately integrated into the genome using the λRed recombination system.

[0037] Preferably, in S2, the endogenous genes include transcription factor genes and protease genes. The transcription factor genes are genes that compete with EL222 for binding, and the protease genes are genes that affect the stability of photosensitive proteins.

[0038] Preferably, in S4, a modular LED panel is developed and flexibly assembled according to the size of the fermentation tank, and the programmable lighting controller includes a light intensity gradient system, an irradiation time system and a feedback adjustment system.

[0039] Preferably, in S4, the optimal light intensity, frequency and duration are determined experimentally, and a multi-factor orthogonal experiment is used to obtain data, and the culture parameter control strategy includes: a dynamic lighting strategy to adjust the lighting mode according to the fermentation stage; a feeding strategy to maintain the optimal nutrient supply; and an optimal control strategy for temperature, pH and dissolved oxygen parameters.

[0040] Preferably, in S5, the online monitoring system includes a fluorescent protein reporter system and a metabolite analysis module.

[0041] The application of the prepared blue light-regulated Escherichia coli is to prepare high-value compounds using the blue light-regulated Escherichia coli.

[0042] The application of the prepared blue light-regulated Escherichia coli is to prepare recombinant protein using the blue light-regulated Escherichia coli.

[0043] The beneficial effects of the present invention are as follows:

[0044] 1. The present invention realizes directional regulation of metabolic pathways at the subcellular level through blue light response elements. Through precise control of light intensity and time, metabolic flux can be dynamically adjusted according to demand, and the accumulation of metabolic intermediates is reduced, cytotoxicity is reduced, and the purity of the target product is improved. The precise spatiotemporal regulation of Escherichia coli metabolism improves the yield and quality of the target product.

[0045] 2. The present invention simplifies the downstream purification process, reduces raw material costs, improves the economic efficiency of production, and reduces chemical residues by avoiding the addition of chemical inducers such as IPTG, which is conducive to the production of green and safe biological products, eliminates the use of chemical inducers, reduces production costs, and reduces potential pollution risks.

[0046] 3. The blue light LED fermentation tank developed by the present invention can achieve uniform illumination for large-scale cultivation. The illumination parameter optimization strategy established can be extended and applied to other photosensitive systems. It also lays the foundation for building a complex multi-gene light-regulated network and establishes an expandable industrial light-regulated fermentation system, providing a model for light regulation of other microorganisms.

[0047] 4. The present invention can be used in conjunction with online detection equipment through the illumination system to achieve closed-loop control. Compared with chemical induction, light induction has a faster response and more precise regulation. The programmable illumination scheme helps to standardize the production process, improve the consistency between batches, improve the automation level and controllability of the production process, and is conducive to the consistency control of product quality.

[0048] 5. The present invention can be used to construct metabolite-responsive biosensors through photosensitive promoters. The combination of multiple light-controlled systems can realize complex logic gate functions, providing new ideas for designing smart cell factories with adaptive regulation capabilities, and providing a technical platform for the development of new biosensors and smart cell factories. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 It is a schematic diagram of the preparation process of the present invention. DETAILED DESCRIPTION

[0050] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] like Figure 1As shown, the embodiment of the present invention provides a preparation method and application of Escherichia coli regulated by blue light, including construction of a blue light response element, strain engineering, fermentation system design, optimization of culture conditions and establishment of a product purification process, construction of a blue light response element: a selecting a core light sensing component, b designing and synthesizing an EL222 coding sequence, c constructing a synthetic promoter, d optimizing an EL222 binding site; strain engineering: a integrating a light-sensitive promoter with a target gene, b optimizing the expression level of EL222 protein, c deleting endogenous genes that interfere with light response; fermentation system design: a designing a fermentation tank, b developing a light control system, c integrating an online monitoring system; optimizing culture conditions: a determining the optimal light parameters, b optimizing the culture medium composition, c establishing a control strategy; establishing a product purification process: a developing a purification process, b establishing a quality control system,

[0052] Design and synthesize the EL222 coding sequence to ensure efficient expression in E. coli and optimize the number and arrangement of EL222 binding sites to obtain the best light response efficiency and gene expression level;

[0053] Select appropriate integration sites to ensure that the important functions of the host bacteria are not affected.

[0054] Preparation steps:

[0055] S1. Material preparation:

[0056] Select bacterial hosts and procure necessary reagents and equipment;

[0057] S2. Gene construction:

[0058] Design EL222 coding sequence and synthesize promoter, and clone into vector;

[0059] S3. Strain transformation:

[0060] The promoter vector was transformed into E. coli and clones that successfully integrated the light-sensitive promoter and target gene were selected;

[0061] Identify and delete endogenous genes that interfere with light response;

[0062] S4. Fermentation system design and culture conditions optimization:

[0063] The fermentation tank is equipped with a blue LED array, and the designed programmable light controller is integrated with a light intensity sensor and installed in the fermentation tank for real-time light intensity monitoring and feedback adjustment;

[0064] Determine the culture medium, screen and obtain the culture medium formula suitable for light-induced conditions, determine the illumination parameters, and establish the culture parameter control strategy;

[0065] S5. Fermentation and product acquisition:

[0066] The positive clones were inoculated into the optimized culture medium for preliminary fermentation culture;

[0067] Blue LED light was used for light induction to activate EL222 and downstream gene expression;

[0068] Use an online monitoring system to track cell growth and target product expression levels in real time;

[0069] S6. Product purification:

[0070] Collect cells by centrifugation, remove culture medium and other impurities, and use high-pressure homogenization or other cell disruption methods to maximize the release of target protein and obtain the released target protein;

[0071] Perform optimized chromatography and use affinity chromatography to improve the purity of the obtained target protein;

[0072] The target protein test was qualified, confirming that the blue light-regulated Escherichia coli was prepared.

[0073] Among them, EL222 is a photosensitive protein derived from marine bacteria. It will undergo conformational changes under blue light irradiation, thereby activating downstream gene expression. The EL222 photosensitive protein system was selected as the core light-sensing component, and the photosensitive promoter and target gene were integrated. The target gene is a recombinant protein gene, and the promoter contains the EL222 coding sequence. The recombinant protein gene uses the λRed recombination system for precise genome integration. Endogenous genes include transcription factor genes and protease genes. Transcription factor genes are genes that compete with EL222 for binding, and protease genes are genes that affect the stability of photosensitive proteins.

[0074] The core function of EL222: It can undergo conformational changes under blue light irradiation and activate the expression of downstream genes, making it possible to precisely regulate the timing and intensity of target gene expression. It can achieve rapid and reversible gene expression regulation through light irradiation, avoiding the potential side effects of chemical induction and is suitable for dynamic experimental design.

[0075] The key points to note are: by selecting appropriate integration sites, ensure that the important functions of the host bacteria are not affected; by designing constitutive promoters of different strengths, regulate the expression of EL222; by screening the optimal expression level, strike a balance between light response efficiency and metabolic burden.

[0076] Among them, in S4, a modular LED panel was developed, which can be flexibly assembled according to the size of the fermentation tank. The programmable light controller includes a light intensity gradient system, an irradiation time system and a feedback adjustment system. In S4, the optimal light intensity, frequency and duration were determined experimentally, and multi-factor orthogonal experiments were used to obtain data. The culture parameter control strategies include: dynamic lighting strategy, adjusting the lighting mode according to the fermentation stage; feeding strategy, maintaining the optimal nutrient supply; optimal control strategy for temperature, pH and dissolved oxygen parameters.

[0077] In this embodiment, modular LED panels can be assembled flexibly according to the size of different fermentation tanks to ensure that the light evenly covers the fermentation liquid and adapt to fermentation processes of different scales, from laboratories to industrial production;

[0078] By adjusting the light intensity, suitable lighting conditions can be provided for different growth stages, promoting the effect of blue light activating EL222, thereby regulating the expression of downstream genes;

[0079] The irradiation time system can automatically adjust the illumination time according to the fermentation process to ensure that the target gene is activated within the optimal time period, thereby improving the synthesis efficiency of the recombinant protein;

[0080] The feedback regulation system monitors the fermentation conditions (such as light intensity, temperature, pH, etc.) in real time and dynamically adjusts the lighting parameters based on the feedback information. The adaptive control can maximize the optimization of culture conditions and enhance growth and product synthesis.

[0081] In addition, by experimentally determining the optimal light intensity, frequency, and duration, we can reduce resource waste and improve the expression and quality of recombinant proteins. By systematically analyzing the impact of different factors, we can effectively identify the factors that have the greatest impact on the target product and guide the subsequent optimization of the fermentation process.

[0082] In the culture parameter control strategy:

[0083] Dynamic lighting strategy can adjust the lighting mode according to the needs of different fermentation stages, so that the most suitable lighting conditions can be applied at different stages of cell growth, metabolism and product synthesis. Dynamic regulation can increase cell growth rate and product accumulation.

[0084] The feeding strategy can maintain the optimal nutrient supply, ensuring that the cells will not be affected in growth and protein synthesis due to nutrient deficiency during the fermentation process, thereby increasing the final yield;

[0085] Temperature, pH and dissolved oxygen control can provide an ideal growth environment for E. coli, improving cell health and product synthesis efficiency.

[0086] Among them, in S5, the online monitoring system includes a fluorescent protein reporter system and a metabolite analysis module.

[0087] The fluorescent protein reporter system can track the expression level of the target product in real time; the integrated metabolite analysis module can monitor the concentration of key intermediates and by-products.

[0088] Example 2: Preparation of high-value compounds using Escherichia coli regulated by blue light.

[0089] The prepared blue light-regulated Escherichia coli was used to produce bioplastic precursors.

[0090] Example 3: Preparation of recombinant protein using Escherichia coli regulated by blue light.

[0091] The prepared blue light-regulated Escherichia coli was used in combination with an improved fermentation platform to obtain the output of recombinant trypsin regulated by blue light.

[0092] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing blue light-regulated Escherichia coli, characterized in that: Including the construction of blue light response elements, strain engineering, fermentation system design, optimization of culture conditions and establishment of product purification process, The blue light response element is constructed by: a selecting a core light sensing component, b designing and synthesizing an EL222 coding sequence, c constructing a synthetic promoter, and d optimizing an EL222 binding site; The engineering modification of the strain: a integrates a light-sensitive promoter and a target gene, b optimizes the expression level of the EL222 protein, and c deletes endogenous genes that interfere with light response; The fermentation system design includes: a. designing the fermentation tank, b. developing the lighting control system, and c. integrating the online monitoring system; The optimization of culture conditions includes: a determining the best illumination parameters, b optimizing the culture medium composition, and c establishing a control strategy; The establishment of product purification process: a. Developing purification process, b. Establishing quality control system, Preparation steps: S1. Material preparation: Select bacterial hosts and procure necessary reagents and equipment; S2. Gene construction: Design the EL222 coding sequence and synthesize the promoter, and clone into the vector; S3. Strain transformation: The promoter vector was transformed into E. coli and clones that successfully integrated the light-sensitive promoter and target gene were selected; Identify and delete endogenous genes that interfere with light response; S4. Fermentation system design and culture conditions optimization: The fermentation tank is equipped with a blue LED array, and the designed programmable light controller is integrated with a light intensity sensor and installed in the fermentation tank for real-time light intensity monitoring and feedback adjustment; Determine the culture medium, screen and obtain the culture medium formula suitable for light-induced conditions, determine the illumination parameters, and establish the culture parameter control strategy; S5. Fermentation and product acquisition: The positive clones were inoculated into the optimized culture medium for preliminary fermentation culture; Blue LED light was used for light induction to activate EL222 and downstream gene expression; Use an online monitoring system to track cell growth and target product expression levels in real time; S6. Product purification: Collect cells by centrifugation, remove culture medium and other impurities, and use high-pressure homogenization or other cell disruption methods to maximize the release of target protein and obtain the released target protein; Perform optimized chromatography and use affinity chromatography to improve the purity of the obtained target protein; The target protein test was qualified, confirming that the blue light-regulated Escherichia coli was prepared.

2. The method for preparing blue light-regulated Escherichia coli according to claim 1, characterized in that: The EL222 is a photosensitive protein derived from marine bacteria. It undergoes conformational changes under blue light irradiation, thereby activating downstream gene expression. The EL222 photosensitive protein system is selected as the core light sensing component.

3. The method for preparing blue light-regulated Escherichia coli according to claim 2, characterized in that: In the integration of the photosensitive promoter and the target gene, the target gene is a recombinant protein gene, the promoter comprises an EL222 coding sequence, and the recombinant protein gene is accurately integrated into the genome using the λRed recombination system.

4. The method for preparing blue light-regulated Escherichia coli according to claim 3, characterized in that: In the S2, the endogenous genes include transcription factor genes and protease genes. The transcription factor genes are genes that compete with EL222 for binding, and the protease genes are genes that affect the stability of the photosensitive protein.

5. The method for preparing blue light-regulated Escherichia coli according to claim 4, characterized in that: In the S4, a modular LED panel is developed and flexibly assembled according to the size of the fermentation tank. The programmable light controller includes a light intensity gradient system, an irradiation time system and a feedback adjustment system.

6. The method for preparing blue light-regulated Escherichia coli according to claim 5, characterized in that: In S4, the optimal light intensity, frequency and duration are determined experimentally, and a multi-factor orthogonal experiment is used to obtain data. The culture parameter control strategy includes: a dynamic lighting strategy to adjust the lighting mode according to the fermentation stage; a feeding strategy to maintain the optimal nutrient supply; and an optimal control strategy for temperature, pH and dissolved oxygen parameters.

7. The method for preparing blue light-regulated Escherichia coli according to claim 6, characterized in that: In S5, the online monitoring system includes a fluorescent protein reporter system and a metabolite analysis module.

8. The use of the blue light-regulated Escherichia coli prepared according to claim 7, characterized in that: Application of blue light-controlled Escherichia coli to prepare high-value compounds.

9. The use of the blue light-regulated Escherichia coli prepared according to claim 8, characterized in that: The application of blue light-controlled Escherichia coli in the preparation of recombinant proteins.

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