Construction method of photosynthetic engineering bacteria for producing succinic acid by using industrial waste gas CO2

Abstract

The invention discloses a construction method of photosynthetic engineering bacteria for producing succinic acid by using industrial waste gas CO2, and relates to the technical field of biology, the construction method of the photosynthetic engineering bacteria for producing succinic acid by using industrial waste gas CO2 comprises the following steps: S1, selection of culture strains: selecting natural microorganisms with photosynthetic carbon sequestration capability, such as blue-green algae or photosynthetic bacteria; s2, gene editing: performing target analysis on the genes of the strain, judging the genes for producing succinic acid from CO2 in the genes of the strain, strengthening or introducing the genes for producing succinic acid through gene binding engineering, optimizing carbon flow to succinic acid, and introducing the genes with acid-resistant characteristics to improve the acid-resistant characteristics of the strain; the method has the advantages that the acid resistance is remarkably improved, the method can adapt to different SO2 and NO2 concentrations in the industrial waste gas, the photosynthetic engineering bacteria grow healthily in a state, the treatment effect on CO2 in the industrial waste gas is high, and photosynthetic carbon sequestration can be effectively carried out.

Description

Technical Field

[0001] This invention relates to the field of biological technology, specifically to a method for constructing photosynthetic engineered bacteria that produce succinic acid using industrial waste gas CO2. Background Technology

[0002] Industrial production processes generate a large amount of CO2. In order to reduce carbon emissions, existing technologies use various methods to treat CO2, including but not limited to various physical, chemical and biological methods. Bioconversion has become a research hotspot due to its environmental protection and sustainability advantages.

[0003] In existing technologies, using genetically engineered photosynthetic bacteria (such as cyanobacteria) to synthesize succinic acid from light energy and CO2 is a mature technology that can effectively fix CO2. However, industrial waste gas not only contains a large amount of CO2 but also a certain amount of SO2 and NO2. When using photosynthetic bacteria to fix CO2 in industrial waste gas, the remaining waste gases in the industrial waste gas need to be effectively removed, which greatly increases the treatment cost of CO2 in industrial waste gas. Therefore, we propose a method for constructing photosynthetic bacteria to produce succinic acid from CO2 in industrial waste gas. Summary of the Invention

[0004] The purpose of this invention is to provide a method for constructing photosynthetic engineered bacteria that produce succinic acid using industrial waste gas CO2.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for constructing photosynthetic engineered bacteria for producing succinic acid using industrial waste gas CO2, comprising the following steps: S1. Selection of culture strains: Select microorganisms with natural photosynthetic carbon fixation capabilities, such as cyanobacteria or photosynthetic bacteria; S2. Gene editing: By analyzing the target points of the strain's genes, we can identify the genes in the strain that are used to produce succinic acid from CO2. We can then enhance or introduce genes that produce succinic acid through gene-binding engineering to optimize the carbon flow to succinic acid. At the same time, we can introduce genes with acid-resistant properties to improve the acid resistance of the strain. S3, Cultivation of photosynthetic engineered bacteria; By culturing the strains in S2, and simultaneously introducing industrial waste gas during the cultivation process, photosynthetic engineered bacteria with acid-resistant properties and strong succinic acid production are screened. S4, High-density cultivation: Cultivate the photosynthetic engineered bacteria screened in S3.

[0006] As a further aspect of the present invention, the gene that is enhanced or introduced in gene editing may be phosphoenolpyruvate carboxylase or succinate dehydrogenase, etc.

[0007] As a further aspect of the present invention, the acid-resistant gene introduced in gene editing needs to have the ability to resist sulfuric acid and nitric acid in order to resist the acidic environment generated by industrial waste gas dissolved in water.

[0008] As a further aspect of the present invention, during the gene editing process, genes that produce succinic acid from CO2 and are acid-resistant can be rapidly screened by querying a known ligand-target relationship database.

[0009] As a further aspect of the present invention, the acid-resistant genes introduced during the gene editing process can be multiple different gene sequences, each capable of resisting different acids.

[0010] As a further aspect of the present invention, gene editing involves precise editing of genes using CRISPR-Cas9 or homologous recombination technology.

[0011] As a further aspect of the present invention, during the cultivation of photosynthetic engineered bacteria, the concentration of succinic acid and the pH of the culture medium need to be monitored in real time.

[0012] As a further aspect of the present invention, a plate reactor is used in the cultivation and high-density culture of photosynthetic engineered bacteria.

[0013] Compared with the prior art, the beneficial effects of the present invention by adopting the above technical solution are as follows: 1. Significantly improved acid resistance, adaptable to different SO2 and NO2 concentrations in industrial waste gas, and the photosynthetic engineered bacteria grow healthily; 2. It has a strong effect on treating CO2 in industrial waste gas and can effectively carry out photosynthetic carbon fixation.

[0014] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0015] Figure 1 This is a flowchart of the method for constructing photosynthetic engineered bacteria for producing succinic acid using industrial waste gas CO2, as per the present invention. Detailed Implementation

[0016] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings. It should be noted that the description of these embodiments is for the purpose of helping to understand the present invention, but does not constitute a limitation of the present invention.

[0017] Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0018] Example 1 Please see the appendix Figure 1 A method for constructing photosynthetic engineered bacteria for producing succinic acid using industrial waste gas CO2 includes the following steps: S1. Selection of culture strains: Select microorganisms with natural photosynthetic carbon fixation capabilities, such as cyanobacteria or photosynthetic bacteria; S2. Gene Editing: By querying known ligand-target relationship databases (including but not limited to Genecard, OMIM, and GAD), genes involved in CO2 production of succinic acid and acid resistance are rapidly screened to identify those in the bacterial strain. Genes for CO2 production of succinic acid are then enhanced or introduced through gene-linked engineering to optimize carbon flow towards succinic acid. Enhanced or introduced genes may include phosphoenolpyruvate carboxylase or succinate dehydrogenase. Simultaneously, genes with acid resistance are introduced to improve the strain's acid resistance. These acid-resistant genes must be resistant to sulfuric acid and nitric acid to withstand the acidic environment generated by dissolved industrial wastewater. Acid-resistant genes can be knocked out from *Curvobacterium tumefaciens*. The acid-resistant genes introduced during gene editing can be multiple different gene sequences, each resistant to different acids. Precise gene editing is achieved using CRISPR-Cas9 or homologous recombination technology. S3. Cultivation of photosynthetic engineered bacteria: The bacteria from S2 are cultivated while industrial waste gas is introduced during the cultivation process. The concentration of succinic acid and the pH of the culture medium are monitored in real time to screen for photosynthetic engineered bacteria with acid-resistant properties and strong succinic acid production capabilities. During cultivation, the light intensity is controlled between 40-180 μmol photons / m². 2 Between / s, intermittent light is introduced into the industrial waste gas to control the CO2 content in the industrial waste gas to 5%, SO2 content to 0.2% and NO2 content to 0.05%, maintain pH stability, and supplement bicarbonate as a buffer carbon source when necessary. S4, High-density cultivation: The photosynthetic engineered bacteria screened in S3 are cultivated using a plate reactor.

[0019] Example 2 During the cultivation of photosynthetic engineered bacteria, the light intensity was controlled between 40-180 μmol photons / m². 2 Between / s, intermittent light is introduced into the industrial waste gas to control the CO2 content in the industrial waste gas to 10%, SO2 content to 0.1% and NO2 content to 0.01%, and to maintain pH stability; Comparative Example 1 During the cultivation of photosynthetic engineered bacteria, the light intensity was controlled between 40-180 μmol photons / m². 2Between / s, intermittent light is introduced into the industrial waste gas to control the CO2 content in the industrial waste gas to 0%, SO2 content to 1%, and NO2 content to 0.1%, while maintaining pH stability.

[0020] The status and processing results are shown in the table below:

[0021] 1. Significantly improved acid resistance, adaptable to different SO2 and NO2 concentrations in industrial waste gas, and the photosynthetic engineered bacteria grow healthily; 2. It has a strong effect on treating CO2 in industrial waste gas and can effectively carry out photosynthetic carbon fixation.

[0022] The terms "front," "back," "left," "right," "top," and "bottom" all refer to the figures in the accompanying drawings. Figure 1 Based on.

[0023] In the description of this invention, it should be understood that the terms "center", "longitudinal", "lateral", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting the scope of protection of this invention.

[0024] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings, but the present invention is not limited to the described embodiments.

[0025] For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A method for constructing photosynthetic engineered bacteria for producing succinic acid using industrial waste gas CO2, characterized in that: Includes the following steps: S1. Selection of culture strains: Select microorganisms with natural photosynthetic carbon fixation capabilities, such as cyanobacteria or photosynthetic bacteria; S2. Gene editing: By analyzing the target points of the strain's genes, we can identify the genes in the strain that are used to produce succinic acid from CO2. We can then enhance or introduce genes that produce succinic acid through gene-binding engineering to optimize the carbon flow to succinic acid. At the same time, we can introduce genes with acid-resistant properties to improve the acid resistance of the strain. S3, Cultivation of photosynthetic engineered bacteria; By culturing the strains in S2, and simultaneously introducing industrial waste gas during the cultivation process, photosynthetic engineered bacteria with acid-resistant properties and strong succinic acid production are screened. S4, High-density cultivation: Cultivate the photosynthetic engineered bacteria screened in S3.

2. The method for constructing photosynthetic engineered bacteria for producing succinic acid using industrial waste gas CO2 according to claim 1, characterized in that: Genes that can be enhanced or introduced during gene editing include phosphoenolpyruvate carboxylase or succinate dehydrogenase.

3. The method for constructing photosynthetic engineered bacteria for producing succinic acid using industrial waste gas CO2 according to claim 2, characterized in that: The acid-resistant genes introduced in gene editing need to be resistant to sulfuric acid and nitric acid in order to resist the acidic environment generated by dissolved water from industrial waste gas.

4. The method for constructing photosynthetic engineered bacteria for producing succinic acid using industrial waste gas CO2 according to claim 3, characterized in that: During gene editing, genes that produce succinic acid from CO2 and are acid-resistant can be rapidly screened by querying known ligand-target relationship databases.

5. The method for constructing photosynthetic engineered bacteria for producing succinic acid using industrial waste gas CO2 according to claim 4, characterized in that: The acid-resistant genes introduced during gene editing can be multiple different gene sequences, each capable of resisting different acids.

6. The method for constructing photosynthetic engineered bacteria for producing succinic acid using industrial waste gas CO2 according to claim 5, characterized in that: Gene editing involves the precise editing of genes using CRISPR-Cas9 or homologous recombination technology.

7. The method for constructing photosynthetic engineered bacteria for producing succinic acid using industrial waste gas CO2 according to claim 1, characterized in that: During the cultivation of photosynthetic engineered bacteria, it is necessary to monitor the succinic acid concentration and the pH of the culture medium in real time.

8. The method for constructing photosynthetic engineered bacteria for producing succinic acid using industrial waste gas CO2 according to claim 1, characterized in that: Plate reactors are used in the cultivation and high-density breeding of photosynthetic engineered bacteria.

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