Method for improving carbon dioxide fixation and conversion performance of genetic engineering escherichia coli

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a method for improving carbon dioxide immobilization and conversion performance of genetic engineering escherichia coli. According to the invention, an adaptive evolutionary strategy based on cooperation of gradient carbon source limitation and CO2 partial pressure regulation designed on the basis of genetically engineered bacteria is adopted; gradient carbon source limitation is to gradually reduce the proportion of an external carbon source LB in an M9 culture medium so as to weaken the dependence of strains on exogenous organic carbon; cO2 partial pressure regulation is characterized in that CO2 partial pressure is synchronously increased in a gradient carbon source limiting process, and carbon flux is promoted to flow to an autotrophic module reductive glycine path by enhancing thermodynamic driving force of carboxylation reaction. According to the invention, autotrophic growth verification and metabolic dependency verification are carried out on the obtained adaptive evolution strain, which proves that the initial genetically engineered bacterium has been converted from autotrophic growth to heterotrophic growth after adaptive evolution, and can take CO2 and formic acid as the unique carbon source and energy.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to a method for optimizing and transforming genetically engineered Escherichia coli. Background Art

[0002] Carbon dioxide (CO2), as the most important greenhouse gas on Earth, the problem of its excessive emission has become a key factor triggering climate change and global warming, posing a severe challenge to the ecosystem and human society. On the other hand, CO2 itself is also the most abundant one-carbon resource in nature and can be converted into high-value-added products through chemical, biological or physical means. Therefore, realizing the efficient fixation and conversion of CO2 can not only alleviate global climate problems but also expand new raw materials for the production of chemical products.

[0003] In recent years, the rapid development of synthetic biology technology has shown great potential in microbial carbon fixation and conversion. On the one hand, researchers are committed to improving natural autotrophic microorganisms, including cyanobacteria, microalgae, and acetogenic bacteria, etc., to solve the defects such as slow growth, low conversion efficiency, low production efficiency, and limited carbon yield. On the other hand, they also focus on the genetic modification of heterotrophic microorganisms, especially model bacteria, including Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris, etc. By introducing natural or artificially designed carbon fixation pathways, the transformation from heterotrophic growth to autotrophic growth using CO2 as a carbon source is achieved. The genetic modification of heterotrophic microorganisms can well make up for the limitations of autotrophic microorganisms such as slow growth, low production efficiency, occupation of land resources, and lack of gene editing tools, greatly promoting the practical application of microbial carbon fixation technology in CO2 emission reduction and resource utilization. The genetic modification of heterotrophic microorganisms has been able to use CO2 as the sole carbon source and produce high-value-added products such as biofuels, bioplastics, and organic acids.

[0004] Although the autotrophic transformation of heterotrophic microorganisms has shown great potential at the theoretical level, its practical application is still limited by problems such as metabolic network conflicts, insufficient energy supply, and poor environmental adaptability. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for improving the performance of genetically engineered Escherichia coli in fixing and converting carbon dioxide, so as to achieve efficient energy utilization and stable growth of Escherichia coli in the process of carbon dioxide fixation and conversion.

[0006] Based on sophisticated genetic engineering, the present invention gradually regulates CO2 concentration and other key culture conditions to continuously optimize the metabolic network of E. coli during the evolution process, thereby improving the efficiency of carbon dioxide fixation and the ability to synthesize conversion products. After multiple rounds of evolutionary screening, the present invention effectively alleviates the problems of metabolic conflict and insufficient energy supply, and provides reliable technical support for genetically engineered microorganisms in the field of CO2 capture and its resource utilization.

[0007] The method provided by the present invention for improving the performance of genetically engineered Escherichia coli in fixing and converting carbon dioxide adopts an adaptive evolution strategy driven by the synergistic effects of "gradient carbon source limitation" and "CO2 partial pressure regulation" designed based on genetically engineered bacteria, specifically:

[0008] (1) Gradient carbon source limitation, i.e. gradually reducing the proportion of the added carbon source LB in the M9 medium, specifically from 5% to 2% (v / v), to weaken the strain's dependence on exogenous organic carbon;

[0009] (2) CO2 partial pressure regulation, that is, during the gradient carbon source limitation process, the CO2 partial pressure is simultaneously increased, specifically from 5% to 10%, to enhance the thermodynamic driving force of the carboxylation reaction and promote the carbon flux to the reductive glycine pathway (rGly).

[0010] The adaptive evolution process designed in the present invention is mainly divided into two stages: the initial adaptation stage and the enhanced selection stage; wherein:

[0011] Initial adaptation phase (cycle 1 to cycle 4), with carbon source and energy conditions: 5-10% CO2, 5-10% LB and 20-100 mM formic acid, 28-30°C and 200-250 rpm; this phase aims to maintain the basic growth of bacteria while activating the formate oxidation and CO2 assimilation pathways;

[0012] Intensive selection stage (cycle 5-cycle 12), the carbon source and energy conditions are: 10-20% CO2, 0-5% LB and 20-100mM formic acid, 28-30℃ and 200-250rpm. This stage mainly forces the metabolic network to be reconstructed to the autotrophic mode through the synergistic effect of carbon source limitation and CO2 partial pressure regulation, and obtains the evolved strains that have undergone the adaptive evolution process.

[0013] Table 1 Culture conditions for each cycle during adaptive evolution

[0014]

[0015] A single cycle is defined as the complete process from seed culture to subculture.

[0016] Cycles 1 - 4 refer to the M9 medium system containing 5 - 10% CO2, 5 - 10% (v / v) LB nutrient components, and 20 - 100 mM formic acid, with the culture parameters controlled as: temperature 28 - 30 °C, shaker speed 200 - 250 rpm. A total of 4 consecutive subculture cycles are performed in this stage, and each cycle ends with subculture.

[0017] Similarly, Cycles 5 - 12 refer to the M9 medium system containing 10 - 20% CO2, 1 - 5% LB, and 20 - 100 mM formic acid, with the culture parameters controlled as: temperature 28 - 30 °C, shaker speed 200 - 250 rpm. A total of 8 consecutive subculture cycles are performed in this stage, and each cycle ends with subculture.

[0018] For the adaptive evolution process, see Figure 1 .

[0019] An evolved strain obtained through the adaptive evolution process is denoted as A.

[0020] Furthermore, the adaptively evolved strain A is verified; specifically:

[0021] (1) Through a two - factor carbon - limited growth experiment, verify whether the evolved strain obtained after adaptive evolution can achieve autotrophic growth using CO2 and formic acid as the sole carbon and energy sources. The culture conditions for the two - factor carbon - limited growth experiment are shown in Table 2.

[0022] (2) Further analyze the metabolic dependence of the evolved strain A through an orthogonal substrate - deletion experiment. The orthogonal substrate - deletion experiment is divided into 4 groups, and the experimental conditions are shown in Table 3.

[0023] After two verification experiments, it can be seen that the initial genetically engineered bacterium has completed the transformation from autotrophic growth to heterotrophic growth after the laboratory adaptive evolution process and can use CO2 and formic acid as the sole carbon and energy sources.

[0024] Table 2 Culture conditions for the two - factor carbon - limited growth experiment

[0025]

[0026] Table 3 Conditions for the orthogonal substrate - deletion experiment

[0027]

[0028] Preparation of M9 medium (1 L): 7.098 g of disodium hydrogen phosphate, 2.72 g of potassium dihydrogen phosphate, 0.058 g of sodium chloride, 1.07 g of ammonium chloride, 0.492 g of magnesium sulfate heptahydrate, 0.0146 g of calcium chloride dihydrate, 0.24 g of isopropyl-β-D-thiogalactoside, 0.03912 g of ethylenediaminetetraacetic acid, 0.002 g of thiamine hydrochloride, 4.43 g of sodium formate, and 1 mL of trace metal solution, sterilized by filtration through a 0.22 μm filter membrane. Trace metal element solution (100 mL): 0.3512 g of ferric chloride hexahydrate, 0.0844 g of zinc chloride, 0.01294 g of copper chloride dihydrate, 0.0099 g of cobalt chloride hexahydrate, 0.01 g of boric acid, and 0.0016 g of manganese chloride tetrahydrate, adjusted to pH 4.0 with NaOH and sterilized by filtration through a 0.22 μm filter membrane.

[0029] Preparation of LB medium (1 L): 10 g of tryptone, 5 g of yeast extract, 5 g of sodium chloride, adjusted to pH 7.2 - 7.4 with NaOH, and sterilized at 121 °C for 15 min.

[0030] The technical features and functional advantages of the present invention mainly include:

[0031] (1) Stepwise adaptive evolution strategy: Gradient pressure culture combined with nutrient limitation is adopted to drive the metabolic adaptive evolution of strains in a high CO2 environment, enhancing the tolerance and catalytic stability of the cells.

[0032] (2) Optimization of continuous subculture process: Through multiple rounds of continuous subculture (a total of 12 cycles), highly active strains are screened to ensure the genetic stability of the target metabolic pathway and avoid functional degradation.

[0033] (3) Potential for multi-scenario application: The stepwise culture parameters (temperature, rotation speed, CO2 concentration) are standardized, which is easy to scale up to the fermenter scale, adapt to existing bioreactor equipment, and has the potential for multi-scenario application in the fields of emission reduction, biomanufacturing, and synthetic biology platforms. Description of the Drawings

[0034] Figure 1 It is an overall schematic diagram of the adaptive evolution process.

[0035] Figure 2 It is the adaptive evolution process based on the starting strain MFC7.

[0036] Figure 3 It is the change trend of (a) the maximum OD 600 value and (b) the doubling time in each cycle during the adaptive evolution process. Detailed Embodiments

[0037] The experimental operations in the adaptive evolution process are as follows:

[0038] (1) Pick a single colony and inoculate it into 4 mL of LB liquid medium containing Cm R and culture it with shaking at 30 °C and 200 rpm for 12 h to obtain a seed culture;

[0039] (2) Take 2 mL of the seed culture and centrifuge it at 3000 rpm for 10 min to collect the cell pellet. After collection, wash it twice with 2 mL of PBS buffer and resuspend it in 2 mL of PBS buffer;

[0040] (3) Inoculate 200 μL of the cell resuspension into 20 mL of M9 medium containing 5% LB and Cm R and culture it with shaking at 30 °C, 200 rpm and 10% CO₂. Take samples every 12 h and measure the OD 600 of the bacterial solution with a microplate reader;

[0041] (4) During the culture process, if the OD 600 values measured continuously for 36 h do not change significantly or the OD 600 values decrease, subculture is required, which also indicates the completion of one cycle;

[0042] (5) Take 2 mL of the cultured bacterial solution and centrifuge it at 3000 rpm for 10 min to collect the cell pellet. After collection, wash it twice with 2 mL of PBS buffer and resuspend it in 2 mL of PBS buffer;

[0043] (6) Inoculate 200 μL of the cell resuspension again into 20 mL of M9 medium containing 5% LB and Cm R (starting from the fourth cycle, the LB concentration in the M9 medium is reduced from 5% to 2%) and culture it with shaking at 30 °C, 200 rpm and 10% CO₂. Take samples every 12 h and measure the OD 600 with a microplate reader;

[0044] (7) After each cycle, the bacterial strain needs to be preserved and stored in a -20 °C refrigerator.

[0045] The constructed carbon fixation genetic engineering bacterium MFC7 was selected for the adaptive evolution process experiment. See Figure 2。Although the MFC7 strain that can theoretically use CO2 as a carbon source and formic acid as an energy source for autotrophic growth has been constructed through the CRISPR-Cas9 gene editing technology. However, through the verification of the two-factor carbon-limited growth experiment, it was found that MFC7 could not achieve strict autotrophic growth, that is, MFC7 could not use CO2 as a carbon source and formic acid as an energy source. Therefore, adaptive evolution was carried out based on the carbon-fixing genetically engineered bacterium MFC7, and finally the autotrophic carbon-fixing bacterium MEvo1 was obtained. The culture conditions for the adaptive evolution experiment are shown in Table 1. During the cycle, when the MEvo1 strain was supplied with 2% LB (cycle 12), the biomass of MEvo1 (OD 600 ) could reach 0.746 ± 0.020 (60 h), which was 262.1% higher than that of the starting strain MFC7 (OD 600 = 0.206 ± 0.004 under 5% LB condition, 144 h) ( Figure 3 a). In addition, the doubling time required for the strain during the adaptive evolution process was shortened from 146.35 ± 3.68 h (cycle 1) to 15.206 ± 0.084 h (cycle 12), and the efficiency was increased by 9.6 times ( Figure 3 b).

[0046] The above results indicate that the coupling strategy of "gradient carbon source limitation" and "CO2 partial pressure regulation" effectively drives the transformation of the metabolic network from heterotrophy (MFC7) to autotrophy (MEvo1). MEvo1 has achieved robust growth close to the mixotrophic metabolic mode under the condition of 2% LB added carbon source (OD 600 > 0.7), proving that the adaptability of its carbon assimilation module, the reductive glycine pathway, and the energy supply system has been significantly improved.

[0047] In summary, through the dynamic adaptive evolution strategy for improving the performance of carbon-fixing genetically engineered Escherichia coli in fixing and transforming carbon dioxide constructed in the present invention, the carbon-fixing genetically engineered bacterium MFC7 that could not achieve autotrophic growth has been successfully evolved into the MEvo1 strain that can achieve autotrophic growth.

Claims

1. A method for improving the performance of genetically engineered Escherichia coli in fixing and converting carbon dioxide, characterized in that, The adaptive evolution strategy is driven by the synergistic effect of "gradient carbon source limitation" and "CO2 partial pressure regulation" based on genetically engineered bacteria design. Specifically: (1) Gradient carbon source limitation, i.e. gradually reducing the proportion of the added carbon source LB in the M9 medium, specifically from 5% to 2% (v / v), to weaken the strain's dependence on exogenous organic carbon; (2) CO2 partial pressure regulation, that is, during the gradient carbon source limitation process, the CO2 partial pressure is simultaneously increased, specifically from 5% to 10%, to enhance the thermodynamic driving force of the carboxylation reaction and promote the carbon flux to the reductive glycine pathway.

2. The method according to claim 1, characterized in that, The adaptive evolution process is mainly divided into two stages: the initial adaptation stage and the enhanced selection stage; Initial adaptation phase, cycle 1 to cycle 4, with carbon source and energy conditions: 5-10% CO2, 5-10% LB and 20-100 mM formic acid, 28-30°C and 200-250 rpm; this phase aims to maintain the basic growth of bacteria while activating the formate oxidation and CO2 assimilation pathways; The intensive selection stage, cycle 5 to cycle 12, has the following carbon source and energy conditions: 10-20% CO2, 0-5% LB and 20-100mM formic acid, 28-30℃ and 200-250rpm. This stage mainly forces the metabolic network to be reconstructed towards the autotrophic mode through the synergistic effect of carbon source limitation and CO2 partial pressure regulation, thus obtaining an evolved strain that has undergone an adaptive evolution process.

3. The method according to claim 2, wherein The evolved strain A obtained through the adaptive evolution process was verified, specifically: (1) A two-factor carbon-limited growth experiment was conducted to verify whether the evolved strains obtained after adaptive evolution could achieve autotrophic growth using CO2 and formic acid as the sole carbon and energy sources; the culture conditions of the two-factor carbon-limited growth experiment are shown in Table 2; (2) The metabolic dependence of the evolved strain A was further analyzed by orthogonal substrate depletion experiments; the orthogonal substrate depletion experiments were divided into 4 groups, and the experimental conditions are shown in Table 3; After two verification experiments, it was found that the initial genetically engineered bacteria had completed the transformation from autotrophic growth to heterotrophic growth after the laboratory adaptive evolution process, and could use CO2 and formic acid as the only carbon source and energy source; Table 2 Culture conditions of the dual-factor carbon-limited growth experiment Table 3 Conditions for orthogonal substrate deletion experiments 4. The method according to claim 2 or 3, characterized in that: Preparation of the M9 medium (1 L): 7.098 g of disodium hydrogen phosphate, 2.72 g of potassium dihydrogen phosphate, 0.058 g of sodium chloride, 1.07 g of ammonium chloride, 0.492 g of magnesium sulfate heptahydrate, 0.0146 g of calcium chloride dihydrate, 0.24 g of isopropyl-β-D-thiogalactoside, 0.03912 g of ethylenediaminetetraacetic acid, 0.002 g of thiamine hydrochloride, 4.43 g of sodium formate, and 1 mL of trace metal solution, sterilized by filtration through a 0.22 μm filter membrane; Trace metal element solution (100 mL): 0.3512 g of ferric chloride hexahydrate, 0.0844 g of zinc chloride, 0.01294 g of copper chloride dihydrate, 0.0099 g of cobalt chloride hexahydrate, 0.01 g of boric acid, and 0.0016 g of manganese chloride tetrahydrate, adjusted to pH 4.0 with NaOH, and sterilized by filtration through a 0.22 μm filter membrane; Preparation of the LB medium (1 L): 10 g of tryptone, 5 g of yeast extract, 5 g of sodium chloride, adjusted to pH 7.2 - 7.4 with NaOH, and autoclaved at 121 °C for 15 min.

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