Construction method of photosynthetic engineering bacteria for producing succinic acid by using industrial waste gas COs
By constructing photosynthetic engineering bacteria, integrating the CO2 concentration mechanism and succinic acid synthesis pathway, the problem of relying on petrochemical resources and low photosynthetic microbial production is solved, and efficient and green succinic acid production is achieved.
Patent Information
- Application Number
- CN202510624050.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-15
- Publication Date
- 2025-08-08
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
In the prior art, the production of succinic acid depends on petrochemical resources and has serious pollution problems. At the same time, the yield of natural photosynthetic microorganisms is low and there is a lack of a coordinated regulation mechanism for efficient CO2 fixation and product synthesis.
Photosynthetic engineering bacteria were constructed, the CO2 concentration mechanism was integrated with the succinic acid synthesis pathway, and the selection of specific chassis strains, strengthening Rubisco enzyme activity and carbonic anhydrase expression, introducing heterologous enzyme systems, constructing carboxylates to increase the local CO2 concentration, and using CRISPR-Cas9 technology for gene editing, achieving efficient CO2 fixation and succinic acid synthesis.
High-efficiency succinic acid production with industrial waste gas as the only carbon source was achieved. The succinic acid concentration reached 12.3g/L in 72 hours, the fixed CO2 volume was 22.1g/L, and the conversion rate was increased by 200%, achieving carbon negative biomanufacturing.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of synthetic biology and biomanufacturing, and specifically relates to a method for constructing an engineered strain based on photosynthetic autotrophic microorganisms, which realizes efficient fixation of industrial waste gas CO2 and biosynthesis of succinic acid through metabolic engineering transformation. Background Art
[0002] As a C4 platform compound, succinic acid is widely used in biodegradable materials (such as PBS), pharmaceutical intermediates and green solvents. Traditional chemical synthesis methods rely on petrochemical resources and are highly polluting.
[0003] However, in the existing technology, the existing heterotrophic fermentation method requires the addition of sugar substrates, which is costly and competes with grain for land; natural photosynthetic microorganisms (such as cyanobacteria) have low succinic acid production and lack a coordinated regulatory mechanism for efficient CO2 fixation and product synthesis. Summary of the Invention
[0004] The present invention provides a method for constructing photosynthetic engineering bacteria that utilizes industrial waste gas CO2 to produce succinic acid. By constructing photosynthetic engineering bacteria, the CO2 concentration mechanism (CCM) and the succinic acid synthesis pathway are integrated to achieve "one-carbon biomanufacturing", achieving both carbon emission reduction and economic efficiency.
[0005] In order to achieve the above object, the present invention adopts the following technical solutions: The method for constructing photosynthetic engineering bacteria for producing succinic acid using industrial waste gas CO2 includes the following specific steps: The first step is the selection of chassis strains: Synechocystis PCC6803 or Synechococcus PCC7942 containing the Rubisco gene cluster are selected as chassis strains; The second step is to strengthen the CO2 fixation module: enhance Rubisco enzyme activity, overexpress carbonic anhydrase to accelerate CO2 hydration, and construct carboxylase to increase the local CO2 concentration; The third step is to strengthen the succinate synthesis module: heterologous phosphoenolpyruvate carboxylase (PEPC, GenBank accession number: NP_417498) from Escherichia coli and malate dehydrogenase (MDH, GenBank accession number: NP_013324) from Saccharomyces cerevisiae are introduced to carboxylate phosphoenolpyruvate carboxylase to oxaloacetate and reduce it to malate; Overexpression of fumarase and fumarate reductase catalyzes the dehydrogenation of malate to generate fumarate and then reduce it to succinate; Knocking out pyruvate kinase PYK blocks lactate and ethanol production.
[0006] As a further improvement of this technical solution: Synechocystis PCC6803 or Synechococcus PCC7942 both have high photosynthesis efficiency and can effectively treat SO2 (≤50ppm) and NO in industrial waste gas. x (≤100ppm) impurities are tolerant and the tolerance time is ≥72h.
[0007] As a further improvement of this technical solution: enhancing Rubisco enzyme activity, specifically: introducing a Form I Rubisco mutant, whose encoding gene mutates from GCT to GCC at codon 123 of the wild-type sequence (GenBank accession number: NC_000921), and the carboxylation efficiency is increased by 35% compared with the wild-type.
[0008] As a further improvement of this technical solution: the overexpression of carbonic anhydrase accelerates CO2 hydration, specifically: carbonic anhydrase can accelerate the hydration of CO2 to HCO3 - , providing sufficient substrate for Rubisco, and combined with Rubisco mutants can increase the CO2 fixation rate by 50%.
[0009] As a further improvement of this technical solution: the carboxylation body is constructed to increase the local concentration of CO2, specifically: carboxylation body microregions are formed in the cell, the local CO2 concentration can reach more than 10 mM, which is 100 times the cytoplasmic concentration, effectively inhibiting the oxygenation side reaction of Rubisco.
[0010] As a further improvement of this technical solution: using the pCas9-Syne system independently constructed by the applicant, the system is based on CRISPR-Cas9 technology and contains 3 sgRNA expression cassettes and homologous recombination donor sequences.
[0011] A method for producing succinic acid using the photosynthetic engineered bacteria described in any of the above methods for producing succinic acid using industrial waste gas CO2, comprising placing a fermentation broth inoculated with the photosynthetic engineered bacteria into a 5L flat-plate gaslift reactor, and introducing industrial waste gas containing 5% CO2 at a ventilation rate of 5% v / v, with the gas-liquid ratio controlled at 1:5.
[0012] As a further improvement of the present technical solution: the process parameters of the flat-plate gaslift reactor are: the light intensity is automatically controlled to 500 μmol photons m⁻²s⁻¹ by an LED light source, and the temperature is maintained at 30°C ± 1°C by a constant temperature device.
[0013] As a further improvement of this technical solution: after the fermentation liquid inoculated with the photosynthetic engineering bacteria is placed in a flat-plate gaslift reactor, trace element mother solution (Fe³+ 0.1mM, Mn²+ 0.05mM, MoO4²⁻0.01mM) and vitamin B12 (final concentration 1μg / L) are added every 12 hours.
[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. Improved carbon conversion efficiency: This invention constructs photosynthetic engineered bacteria and integrates the CO2 concentration mechanism with the succinate synthesis pathway. After 72 hours, the succinate concentration reaches 12.3 g / L, the CO2 fixation amount reaches 22.1 g / L, and the conversion rate (succinate / CO2) is 0.48, which is a 200% increase in yield compared to the wild-type strain. 2. Green process: Using industrial waste gas as the only carbon source, 1.8 tons of CO2 are fixed for every ton of succinic acid produced, achieving negative carbon biomanufacturing.
[0015] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and to implement it according to the contents of the description, the following preferred embodiments of the present invention are described in detail with reference to the accompanying drawings. The specific implementation methods of the present invention are given in detail by the following embodiments and the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings: Figure 1 This is a schematic diagram of the method for constructing photosynthetic engineering bacteria to produce succinic acid using industrial waste gas CO2 proposed in the present invention; Figure 2 This is a schematic diagram of the process for constructing photosynthetic engineering bacteria for producing succinic acid using industrial waste gas CO2 proposed in the present invention. DETAILED DESCRIPTION
[0017] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples provided are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The following paragraphs describe the present invention in more detail by way of example with reference to the accompanying drawings. It should be noted that the drawings are all in a very simplified form and are not to exact scale, and are only used for the purpose of conveniently and clearly illustrating the embodiments of the present invention.
[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention pertains. The terms used in this specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.
[0019] The engineered strain Synechocystis PCC6803 was named Synechocystis sp. and was deposited on August 4, 2017, at the General Microbiology Center of China Culture Collection Administration (CGMCC), located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 14333.
[0020] The engineered strain Synechococcus PCC7942 was named Thermosynechococcus sp. and was deposited in the China Center for Type Culture Collection on June 27, 2024, with the deposit number CCTCC NO.20241317.
[0021] Example 1: Chassis strain selection and gene editing system construction Chassis strain selection: Synechocystis PCC6803 or Synechococcus PCC7942 containing Rubisco gene cluster were selected as chassis strains; Synechocystis PCC6803 or Synechococcus PCC7942 both have high photosynthesis efficiency and are sensitive to SO2 (≤50ppm) and NO in industrial waste gas. x (≤100ppm) impurities are tolerant, and the tolerance time is ≥72h; Example 2: Strengthening of CO2 Fixation Module and Succinate Synthesis Module Strengthening of CO2 fixation module: strengthening Rubisco enzyme activity, overexpressing carbonic anhydrase to accelerate CO2 hydration, and constructing carboxylation body to increase the local concentration of CO2; The activity of Rubisco enzyme was enhanced by introducing a Form I Rubisco mutant, in which the 123rd codon of the wild-type sequence (GenBank accession number: NC_000921) was mutated from GCT to GCC, resulting in a 35% increase in carboxylation efficiency compared to the wild-type.
[0022] Carboxylase: A subcellular structure surrounded by a protein shell that contains Rubisco and carbonic anhydrase. It increases the efficiency of Rubisco carboxylation by concentrating CO2. The local CO2 concentration can reach more than 100 times that of the cytoplasm.
[0023] Overexpression of carbonic anhydrase accelerates CO2 hydration, specifically: carbonic anhydrase can accelerate the hydration of CO2 to HCO3 -, providing sufficient substrate for Rubisco, and combined with Rubisco mutants can increase the CO2 fixation rate by 50%.
[0024] Constructing carboxylases to increase the local concentration of CO2. Specifically, carboxylase microdomains are formed within the cell, where the local CO2 concentration can reach over 10 mM, 100 times the cytoplasmic concentration, effectively inhibiting the oxygenation side reaction of Rubisco. Example 3: Fermentation Verification and Product Separation Enhancement of the succinate synthesis module: heterologous phosphoenolpyruvate carboxylase (PEPC, GenBank accession number: NP_417498) from Escherichia coli and malate dehydrogenase (MDH, GenBank accession number: NP_013324) from Saccharomyces cerevisiae were introduced to carboxylate phosphoenolpyruvate carboxylase to oxaloacetate and reduce it to malate; Overexpression of fumarase and fumarate reductase catalyzes the dehydrogenation of malate to generate fumarate and then reduce it to succinate; To knock out pyruvate kinase (pyk) and block lactate and ethanol production, the present invention utilizes the applicant's independently constructed pCas9-Syne system, which is based on CRISPR-Cas9 technology and contains three sgRNA expression cassettes and a homologous recombination donor sequence. The pCas9-Syne system, based on CRISPR-Cas9 technology, uses sgRNAs to guide the Cas9 nuclease to cleave specific locations in the target genome, inducing DNA double-strand breaks (DSBs). Cells repair DSBs through non-homologous end joining (NHEJ) or homologous recombination (HDR). The former easily introduces insertion / deletion mutations to achieve gene knockout, while the latter can integrate exogenous DNA fragments to achieve gene knockin. Multi-gene editing capabilities: The pCas9-Syne system's modular design allows for simultaneous integration of multiple sgRNA expression cassettes and donor DNA templates, enabling simultaneous knockout and integration of multiple genes. Its advantage lies in the ability to manipulate multiple targets with a single vector, eliminating multiple transfection or transformation steps, reducing the risk of off-target effects, and improving experimental efficiency.
[0025] Fermentation validation Verification Example Initial conditions: BG11 medium (BG11 medium formula: NaNO3 1.5g / L, K2HPO4 0.04g / L, MgSO4・7H2O 0.075g / L, CaCl2・2H2O 0.036g / L, NaHCO3 5g / L, trace element solution 1mL / ), photosynthetic engineering bacteria inoculation amount OD 730 =0.2; Fermentation results: After 72 h, the succinic acid concentration reached 12.3 g / L, the CO2 fixation amount was 22.1 g / L, and the conversion rate (succinic acid / CO2) was 0.48.
[0026] A method for producing succinic acid using photosynthetic engineered bacteria using industrial waste gas CO2, comprising placing a fermentation broth inoculated with the photosynthetic engineered bacteria into a 5L flat-plate gaslift reactor, introducing industrial waste gas containing 5% CO2 at a ventilation rate of 5% v / v, with a gas-liquid ratio controlled at 1:5, controlling the light intensity (500 μmol photons m⁻²s⁻¹), temperature (30°C), and CO2 ventilation rate (5% v / v), while simultaneously removing O2 to avoid photoinhibition. Trace elements (Fe, Mn, Mo) and vitamin B6 are supplemented in stages every 12 hours. 12 , maintain enzyme activity; Product separation: The fermentation broth is acidified (pH 2.0) to precipitate succinic acid crystals, and combined with electrodialysis desalination, the purity reaches over 99%.
[0027] Comparative Example Initial conditions: BG11 medium (containing 0.5% NaHCO3), unmodified Synechocystis PCC6803 inoculum OD730 = 0.2; Fermentation results: After 72 h, the succinic acid concentration reached 12.3 g / L, the CO2 fixation amount was 22.1 g / L, and the conversion rate (succinic acid / CO2) was 0.48.
[0028] The fermentation broth inoculated with unmodified Synechocystis PCC6803 was placed in a 5-liter flat-plate gaslift reactor. Industrial waste gas containing 5% CO2 was introduced at a 5% v / v aeration rate, with a gas-liquid ratio of 1:5. The light intensity (500 μmol photons m⁻²s⁻¹), temperature (30°C), and CO2 aeration rate (5% v / v) were controlled. O2 was removed simultaneously to avoid photoinhibition. Trace elements (Fe, Mn, Mo) and vitamin B12 were added in stages every 12 hours to maintain enzyme activity. Compared to the unmodified Synechocystis PCC6803, the engineered strain Synechocystis SUCC-CO2 increased its succinic acid production by 200% (12.3 g / L vs 4.1 g / L) and its CO2 fixation rate by 150%, as shown in the table below.
[0029] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any ordinary technician in this industry can smoothly implement the present invention as shown in the drawings and described above. However, any equivalent changes, modifications and evolutions made by technicians familiar with this profession without departing from the scope of the technical solution of the present invention using the technical content disclosed above are all equivalent embodiments of the present invention. At the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the scope of protection of the technical solution of the present invention.
Claims
1. A method for constructing photosynthetic engineering bacteria for producing succinic acid using industrial waste gas CO2, characterized in that: The specific steps include: The first step is the selection of chassis strains: Synechocystis PCC6803 or Synechococcus PCC7942 containing the Rubisco gene cluster are selected as chassis strains; The second step is to strengthen the CO2 fixation module: enhance Rubisco enzyme activity, overexpress carbonic anhydrase to accelerate CO2 hydration, and construct carboxylase to increase the local CO2 concentration; The third step is to strengthen the succinate synthesis module: heterologous phosphoenolpyruvate carboxylase from Escherichia coli and malate dehydrogenase from Saccharomyces cerevisiae are introduced to carboxylate phosphoenolpyruvate carboxylase to oxaloacetate and reduce it to malate. Overexpression of fumarase and fumarate reductase catalyzes the dehydrogenation of malate to generate fumarate and then reduce it to succinate; Knocking out pyruvate kinase PYK blocks lactate and ethanol production.
2. The method for constructing photosynthetic engineering bacteria for producing succinic acid using industrial waste gas CO2 according to claim 1, characterized in that: Synechocystis PCC6803 and Synechococcus PCC7942 both have high photosynthesis efficiency and are very sensitive to SO2 (≤50ppm) and NO in industrial waste gas. x (≤100ppm) impurities are tolerant and the tolerance time is ≥72h.
3. The method for constructing photosynthetic engineering bacteria for producing succinic acid using industrial waste gas CO2 according to claim 2, characterized in that: The activity of Rubisco enzyme was enhanced by introducing a Form I Rubisco mutant, in which the 123rd codon of the wild-type sequence (GenBank accession number: NC_000921) was mutated from GCT to GCC, resulting in a 35% increase in carboxylation efficiency compared to the wild-type.
4. The method for constructing photosynthetic engineering bacteria for producing succinic acid using industrial waste gas CO2 according to claim 3, characterized in that: The overexpression of carbonic anhydrase accelerates CO2 hydration, specifically: carbonic anhydrase can accelerate the hydration of CO2 to HCO3 - , providing sufficient substrate for Rubisco, and combined with Rubisco mutants can increase the CO2 fixation rate by 50%.
5. The method for constructing photosynthetic engineering bacteria for producing succinic acid using industrial waste gas CO2 according to claim 4, characterized in that: The carboxylation body is constructed to increase the local concentration of CO2, specifically: a carboxylation body microregion is formed in the cell, the local CO2 concentration can reach above 10 mM, which is 100 times the cytoplasmic concentration, and effectively inhibits the oxygenation side reaction of Rubisco.
6. The method for constructing photosynthetic engineering bacteria for producing succinic acid using industrial waste gas CO2 according to claim 5, characterized in that: The independently constructed pCas9-Syne system was used, which is based on CRISPR-Cas9 technology and contains 3 sgRNA expression cassettes and homologous recombination donor sequences.
7. A method for producing succinic acid using the photosynthetic engineered bacteria according to claims 1-6, characterized in that: The fermentation broth inoculated with the photosynthetic engineered bacteria was placed in a 5 L flat-plate gaslift reactor, and industrial waste gas containing 5% CO2 was introduced at a ventilation rate of 5% v / v, with the gas-liquid ratio controlled at 1:
5.
8. The method for constructing photosynthetic engineering bacteria for producing succinic acid using industrial waste gas CO2 according to claim 7, characterized in that: The process parameters of the flat-plate gaslift reactor are as follows: the light intensity is automatically controlled to 500 μmol photons m⁻²s⁻¹ by an LED light source, and the temperature is maintained at 30°C ± 1°C using a constant temperature device.
9. The method for constructing photosynthetic engineering bacteria for producing succinic acid using industrial waste gas CO2 according to claim 7, characterized in that: After the fermentation liquid inoculated with the photosynthetic engineering bacteria is placed in a flat-plate gas lift reactor, trace element mother solution and vitamin B are added every 12 hours. 12 .