Carbon sequestration pathway selection method and system based on assimilation pathway evaluation

Abstract

The invention belongs to the technical field of biological carbon sequestration, and discloses a carbon sequestration pathway selection method and system based on assimilation pathway evaluation.The method comprises the steps that all reactions with carbon dioxide, carbonate and a one-carbon compound as substrates are obtained, carbon dioxide and bicarbonate radicals are used as carbon sources, and a plurality of carbon sequestration reactions are obtained through screening; carrying out pathway calculation on the carbon sequestration reaction by utilizing a combinatorial algorithm in combination with a simple flux equilibrium analysis technology, and determining a core carbon sequestration reaction according to a calculation result; based on the enzyme catalysis efficiency, simplicity, energy and reduction equivalent consumption and metabolite conversion relation in the core carbon sequestration reaction, the carbon sequestration pathway is evaluated, and the corresponding carbon sequestration pathway is selected according to the evaluation result. According to the invention, a plurality of novel carbon dioxide assimilation approaches with prospects are obtained, and a valuable alternative scheme is provided for bioconversion of industrial carbon dioxide in the future.

Description

Technical Field

[0001] The present invention relates to the field of biological carbon fixation technology, and in particular to a method and system for selecting a carbon fixation pathway based on assimilation pathway evaluation. Background Art

[0002] The carbon fixation cycle is one of the most critical metabolic pathways in the biological field. Through plants or microorganisms, inorganic carbon sources such as carbon dioxide are converted into important biological compounds, which promote the flow of carbon in organisms and constitute the basic components of life. There are six widely recognized natural carbon fixation pathways in nature. Among them, the Calvin-Benson-Bassham (CBB) cycle is the first natural carbon fixation pathway discovered, accounting for 90% of the total carbon fixation in nature, and is widely present in plants, algae and some prokaryotes. From the discovery of the CBB cycle in the 1950s to the sixth 3-hydroxypropionic acid / 4-hydroxybutyric acid cycle reported in 2007, the discovery and verification of these six natural carbon fixation pathways took nearly 60 years.

[0003] In recent years, advances in systems biology have enabled the scientific community to accelerate the discovery of carbon fixation pathways using data-driven approaches. In 2018, researchers discovered a seventh carbon assimilation pathway—the reductive glycine pathway—in anaerobic phosphate bacteria (Phosphitivorax anaerolimi) through metagenomic and 16S rRNA analysis of wastewater sludge microbial communities. That same year, researchers combined omics analysis with biochemical experiments to validate an eighth carbon fixation cycle—the reverse oxidative tricalcium oxidase (TCA) cycle. This brings the total number of natural carbon fixation pathways to eight.

[0004] The evolutionary drivers of natural carbon fixation pathways are constrained by factors such as the ecological niche of their hosts and natural selection. Their metabolic pathways represent only local optimal solutions in specific environments, far from being globally optimal. The continuous accumulation of biological data and the interdisciplinary integration of computer science and biology have made it possible to design new artificial carbon fixation pathways that can overcome the limitations of natural pathways. Researchers collected and compared the enzymatic activity and oxygen sensitivity data of reported carbon-fixing carboxylases, screening for the most efficient carbon-fixing and oxygen-tolerant crotonyl-CoA carboxylase / reductase. Based on the catalytic reaction of this enzyme, they reverse-engineered a new carbon fixation cycle—the CETCH cycle—with a carbon dioxide conversion rate of 5 nmol min-1·mg-1 carbon-fixing enzyme, exceeding the in vivo carbon fixation efficiency of the natural CBB cycle (3 nmol min-1·mg-1 CBB cycle protein). Other researchers have developed a machine learning framework, METIS, which uses minimal experimental data to increase the efficiency of the CETCH cycle by sixfold. Other researchers have employed similar approaches, leveraging existing carboxylase activity data to strategically select crotonyl-CoA carboxylase / reductase and phosphoenolpyruvate carboxylase as building blocks. Using a retrosynthetic design approach and in vitro pathway optimization within the METIS machine learning framework, this team successfully constructed the THETA cycle, achieving a carbon fixation efficiency of 2.7 nmol min-1·mg-1, comparable to the natural CBB cycle. The POAP cycle, a simplified synthetic carbon dioxide fixation pathway consisting of four reactions, achieves a carbon dioxide fixation rate of 8.0 nmol min-1·mg-1 under anaerobic conditions at 50°C.

[0005] In recent years, a variety of artificial carbon sequestration pathways have been designed. Experimental evidence shows that these pathways often outperform natural pathways in many aspects. Over the past few years, databases have grown exponentially, and new carbon-fixing enzymes have been expressed and constructed. Therefore, identifying and selecting efficient artificial carbon sequestration pathways remains a pressing challenge. Summary of the Invention

[0006] The embodiments of the present invention provide a method and system for selecting a carbon sequestration pathway based on assimilation pathway evaluation to solve the above-mentioned technical problems in the prior art.

[0007] According to a first aspect of an embodiment of the present invention, a method for selecting a carbon sequestration pathway based on assimilation pathway evaluation is provided.

[0008] In one embodiment, the method for selecting a carbon sequestration pathway based on assimilation pathway assessment includes:

[0009] Obtain all reactions with carbon dioxide, carbonates, and one-carbon compounds as substrates, and screen out several carbon fixation reactions using carbon dioxide and bicarbonate as carbon sources;

[0010] The carbon fixation pathways were calculated using a combination algorithm combined with simplified flux balance analysis technology, and the core carbon fixation reactions were determined based on the calculation results.

[0011] Based on the enzyme catalytic efficiency, simplicity, energy and reducing equivalent consumption, and metabolite conversion relationship in the core carbon fixation reaction, the carbon fixation pathway is evaluated, and the corresponding carbon fixation pathway is selected based on the evaluation results.

[0012] In one embodiment, the method of obtaining all reactions with carbon dioxide, carbonates, and one-carbon compounds as substrates and using carbon dioxide and bicarbonate as carbon sources to screen out several carbon fixation reactions includes:

[0013] A preset script was used to extract all reactions with carbon dioxide, carbonate, and one-carbon compounds as substrates from the MetaCyc database. Reactions involving the interconversion of carbon dioxide / bicarbonate with organic one-carbon compounds were filtered out, and reactions with carbon dioxide as the product were retained.

[0014] Among the reactions catalyzed by isoenzymes and similar reactions involving different cofactors, any one representative reaction is retained, and carbon dioxide and bicarbonate are used as carbon sources to screen and retain the reactions that directly fix carbon, and obtain several carbon fixation reactions.

[0015] In one embodiment, the method of calculating pathways of carbon fixation reactions using a combined algorithm combined with a simplified flux balance analysis technique, and determining core carbon fixation reactions based on the calculation results includes:

[0016] Based on the carbon fixation reactions screened, pathways containing a single carbon fixation reaction or two different carbon fixation reactions were set; carbon dioxide and bicarbonate were set as substrates, and acetyl-CoA, glyoxylate, oxalate, phosphoglyceraldehyde, and pyruvate were used to define the target products;

[0017] The combinatorial mathematics method is used to randomly combine several carbon fixation reactions obtained by screening, and the simplified flux balance analysis technology is used to calculate each target product. The core carbon fixation reaction involved in the carbon fixation cycle is determined based on the calculation results.

[0018] In one embodiment, the core carbon fixation reaction includes a reaction between two one-carbon compounds, a reaction between a one-carbon compound and a two-carbon compound, a reaction between a one-carbon compound and a three-carbon compound, a reaction between a one-carbon compound and a four-carbon compound, and a reaction between a one-carbon compound and a five-carbon compound.

[0019] In one embodiment, the reaction between the two one-carbon compounds comprises:

[0020] Protein-bound S8-aminomethyldihydroacyllysine reacts with carbon dioxide to produce glycine and acylprotein.

[0021] In one embodiment, the reaction between the one-carbon compound and the two-carbon compound comprises:

[0022] Adenosine triphosphate reacts with acetyl-CoA and bicarbonate ions to generate adenosine diphosphate, phosphate, and malonyl-CoA;

[0023] Acetyl-CoA reacts with carbon dioxide, reduced Coenzyme II, and hydrogen ions to generate pyruvate, Coenzyme A, and oxidized Coenzyme II.

[0024] In one embodiment, the reaction between the one-carbon compound and the three-carbon compound comprises:

[0025] Acryloyl-CoA reacts with carbon dioxide, reduced coenzyme II, and hydrogen ions to generate (R)-methylmalonyl-CoA and oxidized coenzyme II;

[0026] Adenosine triphosphate, propionyl-CoA and bicarbonate ion react to produce adenosine diphosphate, phosphate and (S)-methylmalonyl-CoA;

[0027] Water, phosphoenolpyruvate, and carbon dioxide react to produce phosphoric acid and oxaloacetic acid;

[0028] Adenosine triphosphate, pyruvate, and bicarbonate ions react to produce adenosine diphosphate, phosphoric acid, and oxaloacetic acid;

[0029] Two reduced ferredoxins, propionyl-CoA, carbon dioxide, and two hydrogen ions react to produce two oxidized ferredoxins, 2-oxobutyrate, and CoA.

[0030] In one embodiment, the reaction between the one-carbon compound and the four-carbon compound comprises:

[0031] Crotonyl-CoA reacts with carbon dioxide, reduced coenzyme II, and hydrogen ions to generate (2S)-ethylmalonyl-CoA and oxidized coenzyme II;

[0032] Succinyl-CoA reacts with carbon dioxide, reduced Coenzyme II, and hydrogen ions to generate 2-oxoglutarate, Coenzyme A, and oxidized Coenzyme II.

[0033] Butyryl-CoA reacts with carbon dioxide to produce (2S)-ethylmalonyl-CoA;

[0034] Crotonyl-CoA reacts with carbon dioxide to produce glutaconyl-1-CoA.

[0035] In one embodiment, the reaction between the one-carbon compound and the five-carbon compound comprises:

[0036] Ribulose 1,5-bisphosphate reacts with carbon dioxide and water to produce two molecules of 3-phosphoglycerate;

[0037] Adenosine triphosphate, 2-oxoglutarate and bicarbonate ion react to produce adenosine diphosphate, phosphoric acid and oxalosuccinic acid;

[0038] 2-Oxoglutarate reacts with carbon dioxide, reduced coenzyme II, and hydrogen ions to generate isocitrate and oxidized coenzyme II.

[0039] According to a second aspect of an embodiment of the present invention, a carbon sequestration pathway selection system based on assimilation pathway evaluation is provided.

[0040] In one embodiment, the carbon sequestration pathway selection system based on assimilation pathway assessment includes a carbon sequestration reaction screening module, a core carbon sequestration reaction determination module, and a carbon sequestration pathway selection module;

[0041] The carbon fixation reaction screening module is used to obtain all reactions with carbon dioxide, carbonates and one-carbon compounds as substrates, and to screen several carbon fixation reactions with carbon dioxide and bicarbonate as carbon sources;

[0042] The core carbon fixation reaction determination module is used to calculate the pathways of carbon fixation reactions using a combination algorithm combined with a simplified flux balance analysis technique, and determine the core carbon fixation reaction based on the calculation results;

[0043] The carbon fixation pathway selection module is used to evaluate carbon fixation pathways based on the enzyme catalytic efficiency, simplicity, energy and reducing equivalent consumption, and metabolite conversion relationship in the core carbon fixation reaction, and select the corresponding carbon fixation pathway based on the evaluation results.

[0044] According to a third aspect of an embodiment of the present invention, a computer device is provided.

[0045] In some embodiments, the computer device includes a memory and a processor, the memory stores a computer program, and the processor implements the steps of the above method when executing the computer program.

[0046] According to a fourth aspect of embodiments of the present invention, a computer-readable storage medium is provided.

[0047] In one embodiment, the computer-readable storage medium stores a computer program, and when the computer program is executed by a processor, the steps of the above method are implemented.

[0048] The technical solution provided by the embodiment of the present invention may have the following beneficial effects:

[0049] The present invention uses 6578 reaction data sets from the MetaCyc database to explore innovative carbon fixation pathways that can directly assimilate carbon dioxide and / or bicarbonate instead of organic one-carbon compounds (such as methanol, formaldehyde, and formic acid). Through systematic evaluation, 172 carbon fixation routes within 20 steps were identified from 15 core fixation reactions. Subsequently, the main carbon fixation modes, energy consumption and number of steps of various pathways were evaluated, and a variety of promising new carbon dioxide assimilation pathways were obtained, providing valuable alternatives for future industrial carbon dioxide bioconversion.

[0050] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0052] Figure 1 is a flow chart of a method for selecting a carbon sequestration pathway based on assimilation pathway evaluation according to an exemplary embodiment;

[0053] Figure 2 is a structural block diagram of a carbon sequestration pathway selection system based on assimilation pathway evaluation according to an exemplary embodiment;

[0054] Figure 3 This is a workflow diagram for calculating artificial carbon assimilation pathways using comb-FBA;

[0055] Figure 4 is a box plot showing the distribution of reaction steps in the computational pathway grouped by carbon fixation reaction;

[0056] Figure 5 This is a diagram of pathway calculation results from 15 carbon fixation reaction combinations;

[0057] Figure 6 This is a schematic diagram of the CO2 assimilation pathway calculated by comb-FBA;

[0058] Figure 7 It is a modular analysis of the CO2 assimilation pathway and a schematic diagram of alternative reactions;

[0059] Figure 8 The figure is a schematic diagram showing the structure of a computer device according to an exemplary embodiment. DETAILED DESCRIPTION

[0060] The following description and accompanying drawings sufficiently illustrate the specific embodiments herein to enable those skilled in the art to practice them. Portions and features of some embodiments may be included in or substituted for portions and features of other embodiments. The scope of the embodiments herein includes the entire scope of the claims, including all available equivalents thereof. Herein, the terms "first," "second," and the like are used solely to distinguish one element from another and do not require or imply any actual relationship or order between these elements. In practice, the first element can also be referred to as the second element, and vice versa. Furthermore, the terms "comprise," "comprising," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a structure, device, or apparatus comprising a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such structure, device, or apparatus. Without further limitation, an element defined by the phrase "comprising a..." does not preclude the presence of other identical elements in the structure, device, or apparatus comprising the element. The various embodiments herein are described in a progressive manner, with each embodiment focusing on its differences from the other embodiments. Similar or identical parts between the various embodiments can be referenced to each other.

[0061] The terms "longitudinal", "transverse", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like used herein to indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, are intended only to facilitate the description of this document and simplify the description, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation on the present invention. In the description herein, unless otherwise specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, they can be mechanical or electrical connections, or they can be internal connections between two elements, they can be directly connected, or they can be indirectly connected through an intermediate medium. For those of ordinary skill in the art, the specific meanings of the above terms can be understood according to the specific circumstances.

[0062] As used herein, unless otherwise specified, the term "plurality" means two or more.

[0063] In this document, the character " / " indicates that the preceding and following objects are in an "or" relationship. For example, A / B means: A or B.

[0064] In this article, the term "and / or" is used to describe the association relationship between objects, indicating that three relationships can exist. For example, A and / or B means: A or B, or, A and B.

[0065] It should be understood that, although the various steps in the flowchart are shown in sequence as indicated by the arrows, these steps are not necessarily performed in the order indicated by the arrows. Unless otherwise specified herein, there is no strict order restriction on the execution of these steps, and these steps may be performed in other orders. Moreover, at least a portion of the steps in the figure may include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily performed at the same time, but may be performed at different times. The execution order of these sub-steps or stages is not necessarily to be performed in sequence, but may be performed in turn or alternately with other steps or at least a portion of the sub-steps or stages of other steps.

[0066] Each module in the device or system of the present application can be implemented in whole or in part by software, hardware, or a combination thereof. The above modules can be embedded in or independent of the processor in the computer device in the form of hardware, or can be stored in the memory of the computer device in the form of software so that the processor can call and execute the operations corresponding to the above modules.

[0067] In the absence of conflict, the embodiments of the present invention and the features thereof may be combined with each other.

[0068] Figure 1 An embodiment of a carbon sequestration pathway selection method based on assimilation pathway evaluation according to the present invention is shown.

[0069] In this optional embodiment, the method for selecting a carbon sequestration pathway based on assimilation pathway assessment includes:

[0070] Step S101: Obtain all reactions using carbon dioxide, carbonates, and one-carbon compounds as substrates, and screen several carbon fixation reactions using carbon dioxide and bicarbonate as carbon sources;

[0071] Step S102: Calculate the pathways of carbon fixation reactions using a combination algorithm combined with a simplified flux balance analysis technique, and determine the core carbon fixation reaction based on the calculation results;

[0072] Step S103: Evaluate the carbon fixation pathway based on the enzyme catalytic efficiency, simplicity, energy and reducing equivalent consumption, and metabolite conversion relationship in the core carbon fixation reaction, and select the corresponding carbon fixation pathway based on the evaluation results.

[0073] In this optional embodiment, all reactions using carbon dioxide, carbonates, and one-carbon compounds as substrates are obtained, and carbon dioxide and bicarbonate are used as carbon sources, and several carbon fixation reactions are screened, including:

[0074] A preset script was used to extract all reactions with carbon dioxide, carbonate, and one-carbon compounds as substrates from the MetaCyc database. Reactions involving the interconversion of carbon dioxide / bicarbonate with organic one-carbon compounds were filtered out, and reactions with carbon dioxide as the product were retained.

[0075] Among the reactions catalyzed by isoenzymes and similar reactions involving different cofactors, any one representative reaction is retained, and carbon dioxide and bicarbonate are used as carbon sources to screen and retain the reactions that directly fix carbon, and obtain several carbon fixation reactions.

[0076] In this optional embodiment, the method of calculating the pathways of carbon fixation reactions using a combined algorithm combined with a simplified flux balance analysis technique, and determining the core carbon fixation reaction based on the calculation results includes:

[0077] Based on the carbon fixation reactions screened, pathways containing a single carbon fixation reaction or two different carbon fixation reactions were set; carbon dioxide and bicarbonate were set as substrates, and acetyl-CoA, glyoxylate, oxalate, phosphoglyceraldehyde, and pyruvate were used to define the target products;

[0078] The combinatorial mathematics method is used to randomly combine several carbon fixation reactions obtained by screening, and the simplified flux balance analysis technology is used to calculate each target product. The core carbon fixation reaction involved in the carbon fixation cycle is determined based on the calculation results.

[0079] In this optional embodiment, a combinatorial mathematics method is used to randomly combine the screened carbon fixation reactions, and a simplified flux balance analysis technique is used to calculate each target product. The core carbon fixation reactions involved in the carbon fixation cycle are determined based on the calculation results, including:

[0080] 1) Random carbon fixation reaction combination generation:

[0081] From the 48 CO2 / HCO3 - In the reaction, an exhaustive method is used to generate all possible combinations:

[0082] (a) Independent combinations of single carbon fixation reactions (48 types);

[0083] (b) Pairwise combinations of two different carbon fixation reactions (1128 in total), generating a total of 1176 combinations (48 + 1128).

[0084] 2) Target product pathway calculation:

[0085] Set the substrate to CO2 / HCO3 -, the target product is a C2 / C3 metabolite (such as acetyl-CoA, pyruvate, etc.), and an ATP / NADH supply reaction is added (to simulate energy input). Using the comb-FBA algorithm, combined with MetaCyc's 6530 background reaction networks, a flux balance analysis is performed for each combination:

[0086] (a) Minimize the total flux through linear programming and screen the simplest pathway that satisfies carbon, energy, and redox balances;

[0087] (b) For each target product (e.g., acetyl-CoA), pFBA was calculated 1176 times to generate a set of feasible pathways.

[0088] 3) Determination of core carbon fixation reaction:

[0089] The participation frequency of each reaction in the feasible pathway was counted, and 15 reactions that could form a complete carbon fixation cycle were screened out;

[0090] Performance indicators for evaluating reactions:

[0091] (a) Catalytic efficiency: Based on the Kcat / Km value ranking of the BRENDA database, efficient enzymatic reactions (such as 2_CCR and 5_PCC) were retained;

[0092] (b) Pathway efficiency: Prioritize combinations with fewer steps (e.g., 8_PYC requires only 3 steps) and lower ATP consumption (e.g., POAP requires only 2 ATP / CO2).

[0093] In this optional embodiment, the core carbon fixation reaction includes a reaction between two one-carbon compounds, a reaction between a one-carbon compound and a two-carbon compound, a reaction between a one-carbon compound and a three-carbon compound, a reaction between a one-carbon compound and a four-carbon compound, and a reaction between a one-carbon compound and a five-carbon compound;

[0094] Among them, the reaction between two one-carbon compounds includes:

[0095] Protein-bound S8-aminomethyldihydroacyllysine reacts with carbon dioxide to produce glycine and acylprotein;

[0096] Reactions between one-carbon compounds and two-carbon compounds include:

[0097] Adenosine triphosphate reacts with acetyl-CoA and bicarbonate ions to generate adenosine diphosphate, phosphate, and malonyl-CoA;

[0098] Acetyl-CoA reacts with carbon dioxide, reduced Coenzyme II and hydrogen ions to generate pyruvate, Coenzyme A and oxidized Coenzyme II;

[0099] Reactions between one-carbon compounds and three-carbon compounds include:

[0100] Acryloyl-CoA reacts with carbon dioxide, reduced coenzyme II, and hydrogen ions to generate (R)-methylmalonyl-CoA and oxidized coenzyme II;

[0101] Adenosine triphosphate, propionyl-CoA and bicarbonate ion react to produce adenosine diphosphate, phosphate and (S)-methylmalonyl-CoA;

[0102] Water, phosphoenolpyruvate, and carbon dioxide react to produce phosphoric acid and oxaloacetic acid;

[0103] Adenosine triphosphate, pyruvate, and bicarbonate ions react to produce adenosine diphosphate, phosphoric acid, and oxaloacetic acid;

[0104] Two reduced ferredoxins, propionyl-CoA, carbon dioxide, and two hydrogen ions react to produce two oxidized ferredoxins, 2-oxobutyrate, and CoA;

[0105] Reactions between one-carbon compounds and four-carbon compounds include:

[0106] Crotonyl-CoA reacts with carbon dioxide, reduced coenzyme II, and hydrogen ions to generate (2S)-ethylmalonyl-CoA and oxidized coenzyme II;

[0107] Succinyl-CoA reacts with carbon dioxide, reduced Coenzyme II, and hydrogen ions to generate 2-oxoglutarate, Coenzyme A, and oxidized Coenzyme II.

[0108] Butyryl-CoA reacts with carbon dioxide to produce (2S)-ethylmalonyl-CoA;

[0109] Crotonyl-CoA reacts with carbon dioxide to produce glutaconyl-1-CoA;

[0110] Reactions between one-carbon compounds and five-carbon compounds include:

[0111] Ribulose 1,5-bisphosphate reacts with carbon dioxide and water to produce two molecules of 3-phosphoglycerate;

[0112] Adenosine triphosphate, 2-oxoglutarate and bicarbonate ion react to produce adenosine diphosphate, phosphoric acid and oxalosuccinic acid;

[0113] 2-Oxoglutarate reacts with carbon dioxide, reduced coenzyme II, and hydrogen ions to generate isocitrate and oxidized coenzyme II.

[0114] In this optional embodiment, the carbon fixation pathway is evaluated based on the enzyme catalytic efficiency, simplicity, energy and reducing equivalent consumption, and metabolite conversion relationship in the core carbon fixation reaction, and the corresponding carbon fixation pathway is selected according to the evaluation results, including:

[0115] All carbon fixation reactions were subjected to comb-FBA combination calculations, and 15 core carbon fixation reactions involved in the carbon fixation pathway were identified. The enzymatic efficiency of the carbon fixation enzymes was determined based on database and literature searches and sorted in order and listed in Table 1. For example, the 1_CCR enzyme with serial number 1 has the highest carbon fixation efficiency and ranks first. Among the calculated carbon fixation pathways, the higher the carbon fixation enzyme activity, the higher the pathway feasibility. Further comprehensive consideration is given to the simplicity of the pathway (i.e., the shorter the reaction steps, the better), energy and reducing equivalent consumption (the less consumption, the lower the pathway modification cost), and metabolite conversion relationship (the higher the conversion rate from substrate to product, the better). The above conditions were used to screen out Figure 6 12 carbon sequestration pathways.

[0116] Figure 2 An embodiment of a carbon sequestration pathway selection system based on assimilation pathway evaluation according to the present invention is shown.

[0117] In this optional embodiment, the carbon sequestration pathway selection system based on assimilation pathway assessment includes:

[0118] The carbon fixation reaction screening module 201 is used to obtain all reactions using carbon dioxide, carbonates, and one-carbon compounds as substrates, and to screen several carbon fixation reactions using carbon dioxide and bicarbonate as carbon sources;

[0119] The core carbon fixation reaction determination module 202 is used to calculate the pathways of carbon fixation reactions using a combination algorithm combined with a simplified flux balance analysis technique, and determine the core carbon fixation reaction based on the calculation results;

[0120] The carbon fixation pathway selection module 203 is used to evaluate the carbon fixation pathway based on the enzyme catalytic efficiency, simplicity, energy and reducing equivalent consumption, and metabolite conversion relationship in the core carbon fixation reaction, and select the corresponding carbon fixation pathway according to the evaluation results.

[0121] In order to facilitate understanding of the above technical solutions of the present invention, the above technical solutions of the present invention are further explained from the perspective of architecture and principle as follows:

[0122] A method for selecting carbon sequestration pathways based on assimilation pathway assessment, including:

[0123] 1. Extraction and treatment of carbon fixation reaction:

[0124] A Python script was used to extract all reactions using carbon dioxide, carbonates, and one-carbon compounds (such as formaldehyde and methane) as substrates from the MetaCyc network model. This method focuses on direct fixation of carbon dioxide or bicarbonate, avoiding reactions that convert carbon dioxide / bicarbonate to other organic C1 compounds (i.e., organic one-carbon compounds such as formaldehyde, formic acid, and methanol). Therefore, for reactions involving the conversion of carbon dioxide to organic C1 substances, only the direction of carbon dioxide production was retained.

[0125] For reactions catalyzed by isozymes and similar reactions involving different cofactors, only one was retained to simplify the calculations without affecting the results. Carbon dioxide and bicarbonate were designated as carbon sources, and after screening, 48 carbon fixation reactions were finally obtained.

[0126] 2. Calculation of carbon sequestration pathways:

[0127] Using the 48 identified carbon fixation reactions, pathways containing a single carbon fixation reaction or two different carbon fixation reactions were designed. Combinatorial mathematics methods were used to exhaustively generate all possible combinations. In the pathway calculation, carbon dioxide and bicarbonate were set as substrates, while common metabolites in natural or engineered carbon fixation pathways - acetyl-CoA, glyoxylate, oxalate, 3-phosphoglyceraldehyde and pyruvate - were defined as target products. Products such as "acetyl-CoA", "acetaldehyde", "oxalate", "3-phosphoglyceraldehyde" and "pyruvate" were used as calculation targets. Since the carbon fixation pathway is a process that consumes energy and reducing equivalents, exogenous supply reactions of NADH (nicotinamide adenine dinucleotide) and ATP (adenosine triphosphate) were added, as shown in the following formula:

[0128] ATP c +WATER c →ADP c +Pi c

[0129] ATP c +WATER c →AMP c +PPI c

[0130] NADPH c →NADP c +2.0PROTON c

[0131] NADH c →NAD+2.0PROTON c

[0132] Among them, ATP cAdenosine triphosphate (ATP), WATER c Water (H2O), ADP c Adenosine diphosphate (ADP), AMP c Adenosine monophosphate (AMP), Pi c Inorganic phosphate (Pi), PPI c Inorganic pyrophosphate (PPI), NADPH c Nicotinamide adenine dinucleotide phosphate (NADPH), NADP c Nicotinamide adenine dinucleotide phosphate (NADP) + ), PROTON c For protons (H + ), NADH c Nicotinamide adenine dinucleotide (NADH) is a reduced form of NAD c Oxidized nicotinamide adenine dinucleotide (NAD + ).

[0133] During the pathway design phase, the developed comb-FBA method was utilized, which combines a combinatorial algorithm with parsimonious flux balance analysis (pFBA). Computational simulations were performed in Python using software packages such as COBRApy. Linear programming problems were solved using the CPLEX solver.

[0134] 3. Assessment of carbon sequestration pathways:

[0135] Comparison of enzyme parameters in carbon fixation reactions: Pathway calculations confirmed that 15 of the 48 carbon fixation reactions were involved in the carbon fixation cycle. Catalytic efficiency parameters for the enzymes involved in these 15 carbon fixation reactions were obtained based on data from the literature and the BRENDA database. However, some carbon fixation enzymes are strictly anaerobic or require specific extraction conditions, making it impossible to obtain activity data.

[0136] Comparison of Pathway Simplicity: Pathway simplicity was assessed by counting the number of reactions involved in each pathway. The reactions considered did not include transport reactions, exchange reactions, or demand reactions. Additionally, reactions added to supply ATP and NADH were not counted. Because the algorithm used minimizes flux, the resulting pathway represents one of the simplest combinations of carbon fixation reactions possible under the given conditions.

[0137] Comparison of energy and reducing equivalent consumption: For ATP consumption, the present invention mainly analyzes the net number of ATP molecules consumed in each pathway. When ATP is hydrolyzed to AMP, it is considered to consume 2 ATP molecules, because the energy released when ATP is hydrolyzed to AMP is greater than the energy released when it is hydrolyzed to ADP. For reactions that consume other energy carriers (such as GTP or UTP), they are uniformly converted to ATP equivalents. Reducing equivalent consumption (such as NADH or NADPH) is standardized to NADH equivalents. Since the substrates and products of all pathways are the same, the stoichiometry of electron transfer ensures that the consumption of NADH in each pathway is the same. The calculation results confirm this. For example, 4 NADH molecules are required to convert two carbon dioxide molecules into one acetyl-CoA, so NADH consumption is no longer compared separately.

[0138] Network analysis of metabolite transformation: To systematically elucidate the transformation relationships between metabolites, this paper uses the open-source bioinformatics platform Cytoscape to construct and analyze metabolite transformation networks. Leveraging its advanced network visualization and topology analysis capabilities, Cytoscape is particularly effective in studying interactions within metabolic pathways.

[0139] In addition, the present invention also includes the following contents:

[0140] 1. CO2 assimilation pathway calculation based on comb-FBA

[0141] The calculation of CO2 assimilation pathways was performed using the comb-FBA28 algorithm (e.g. Figure 3 comb-FBA is an extension of traditional flux balance analysis (FBA) that allows the exploration of multiple pathways by performing comprehensive combinatorial calculations on reactions in a combinatorial reaction set. In the present invention, 48 CO2 and HCO3 reactions were extracted from the MetaCyc database. - The reactions used as substrates constitute the combined reaction set SetCO2. The comb-FBA calculation uses SetCO2 as the combined reaction set and operates on SetMet, which contains 6530 (6578-48=6530) MetaCyc reactions, as the main reaction set.

[0142] 48 carbon fixation reactions were combined together, and 1176 pFBA calculations were performed for each target product (acetyl-CoA, glyoxylate, oxalate, glyceraldehyde-3-phosphate or pyruvate). The results of the combined calculations showed that among these 48 carbon fixation reactions, 15 reactions were able to participate in the carbon fixation cycle, and the remaining reactions could not form a complete carbon fixation cycle. These 15 core reactions were ranked from 1 to 15 according to the catalytic efficiency (Kcat / Km) of the enzymes that catalyze these reactions.

[0143] These 15 core carbon fixation reactions form 120 different carbon fixation pathways for each target product. This means that any single core carbon fixation reaction or any combination of two core reactions can constitute a carbon fixation cycle. This indicates that the carbon fixation products of these 15 reactions can be freely converted into C2 or C3 metabolites, such as acetyl-CoA and pyruvate, through the reaction network in MetaCyc. Of these 15 reactions, seven do not belong to known natural carbon fixation pathways, and three of them (1_CCR, 2_CCR, and 8_PYC) have been engineered into three new artificial carbon fixation pathways: CETCH (1_CCR+2_CCR)18, THETA (1_CCR+8_PYC)20, and POAP21. Notably, four of these reactions (7_CFI, 12_ECH, 13_GCD, and 15_OSR) have not been observed in natural or previously reported artificial carbon fixation pathways.

[0144] According to the number of carbon atoms in the main substrate molecules (excluding the CoA group), these carbon fixation reactions can be divided into five categories (Table 1): C1+C1 (one reaction: 14_CVP), C2+C1 (two reactions: 3_ACC and 9_POR), C3+C1 (five reactions: 2_CCR, 5_PCC, 6_PPC, 8_PYC and 15_OSR), C4+C1 (four reactions: 1_CCR, 11_KOR, 12_ECH and 13_GCD), and C5+C1 (three reactions: 4_RBC, 7_CFI and 10_ICC).

[0145] Table 1 Carbon fixation reaction types

[0146]

[0147] 2. Analysis of reaction steps in the computational pathway reveals significant variability in CO2 sequestration pathways

[0148] For any target metabolite, there is considerable variability in the carbon fixation pathways that consist of different reaction combinations. Since the difference in the number of reaction steps is the most significant difference between pathways, the number of steps was preferentially used for analysis. Figure 4As shown in Figure 2, most carbon fixation pathways exhibit significant variability in the number of steps. Pathways composed of specific carbon fixation reactions (such as 2_CCR, 5_PCC, 6_PPC, or 8_PYC) exhibit a more convergent pattern in the number of steps, with relatively few overall reaction steps ( Figure 4 These reactions are mainly concentrated in the C3+C1 category (Table 1), and their carbon fixation products are oxaloacetate or (R) / (S)-methylmalonate CoA.

[0149] Figure 4 Box plots show the distribution of reaction steps in the calculated pathways grouped by carbon fixation reaction, for acetyl-CoA (A), glyoxylate (B), and oxalate (C). The minimum (lower bar), median (black horizontal line), mean (black asterisk), maximum (upper bar), and outliers (black squares) are shown. The boxes represent data points between the first and third quartiles. The solid black line represents the length of the pathway step formed by the most core carbon fixation reactions alone.

[0150] When analyzing the combinatorial effects between carbon fixation reactions, an interesting trend was observed. For C3 products, the pathways formed by most core carbon fixation reactions alone were significantly shorter than those formed by their combination with other reactions. In contrast, for C2 products, the combination of most core carbon fixation reactions resulted in a significant increase or decrease in the number of reaction steps ( Figure 4 This suggests that the carbon sequestration pathways of C2 and C3 products may follow distinct patterns.

[0151] Both natural and artificially designed carbon fixation pathways consist of no more than 20 enzymatic steps. Shorter pathways are generally more feasible experimentally. Therefore, the present invention calculated the frequency of each carbon fixation reaction in pathways with less than 20 steps. The results showed that 2_CCR, 5_PCC, 6_PPC and 8_PYC appeared most frequently, while 14_CVP and 4_RBC appeared less frequently. This trend is consistent with the Figure 4 The average number of steps per reaction in the pathway is consistent with that in the Figure 1. Therefore, when designing shorter artificial carbon fixation pathways, it may be more advantageous to prioritize reactions such as 2_CCR, 5_PCC, 6_PPC, or 8_PYC.

[0152] 3. Analysis of carbon assimilation pathways within 20 steps reveals three carbon fixation modes

[0153] In this invention, computational pathways containing no more than 20 steps (59 pathways producing acetyl-CoA, 59 pathways producing glyoxylate, 43 pathways producing oxalate, 3 pathways producing 3-phospho-D-glycerate and 8 pathways producing pyruvate) were systematically analyzed ( Figure 5When pathways have the same initial substrate and the same product, reduction equivalents are balanced. Therefore, different carbon fixation pathways that utilize the same amount of CO₂ to synthesize the same product require the same number of reduction equivalents. For example, all pathways that convert 2 moles of CO₂ to 1 mole of acetyl-CoA consume 4 reduction equivalents. The main focus is on comparing the number of steps and ATP consumption in different pathways. Figure 5 The steps and ATP requirements of the pathway for converting CO2 to acetyl-CoA are shown. A total of 59 pathways with less than 20 steps were identified and analyzed. Among these 59 pathways, the carbon fixation combinations of 7 pathways overlapped with known carbon fixation pathways, including 4 natural pathways (CBB cycle, reversible TCA cycle, 3-HP cycle and DC / HB cycle) and 3 artificial pathways (CETCH, THETA and POAP). In all calculated combinations, ATP consumption was mainly distributed between 2 and 5 ATP molecules per mole of acetyl-CoA ( Figure 5 From the perspective of ATP efficiency alone, these pathways are almost all more energy-efficient than the CBB cycle, which requires 6 ATP molecules ( Figure 5 ).

[0154] Figure 5 Pathway calculation results from 15 carbon fixation reaction combinations for acetyl-CoA. The rows and columns represent the reaction combination type. In each box, the number on the left indicates the number of reaction steps required to complete the carbon fixation process, while the number on the right indicates the number of ATP molecules consumed in the specific pathway.

[0155] The most common natural carbon fixation pathway CBB cycle is a cycle involving only 4_RBC carbon fixation reactions. According to the calculation results of the present invention, this pathway consists of 14 reaction steps, from CO2 to acetyl-CoA, consuming 6 ATP molecules ( Figure 5 Many of the newly predicted pathways in the present invention have fewer reaction steps and / or lower energy consumption than the natural CBB cycle, such as pathways within 10 steps from 5_PCC+9_POR, 6_PPC+9_POR, 7_CFI+11_KOR and 10_ICC+11_KOR combinations ( Figure 5 ).

[0156] By analyzing the synthetic pathways of various products, three main carbon fixation modes were identified ( Figure 6 The first pattern involves the reactions: C1+C3→C4, C1+C4→C5, C5→C2+C3( Figure 6 A). The reported artificial carbon fixation pathways CETCH and THETA belong to this model. The CETCH cycle is an in vitro carbon fixation pathway that produces glyoxylate as a product ( Figure 6The thick black solid line and dashed line P1 in A). When the target is the central metabolic building block acetyl-CoA, the CETCH variant pathway P1 can be obtained. For the THETA pathway ( Figure 6 A thick black solid line and dashed line P2 in A) identified a theta variant (P2 pathway) that saves two ATP molecules compared to the original theta cycle. In addition to these known pathways, many previously unreported pathways, such as Figure 6 P3, P4, and P5 in A also belong to the first carbon fixation mode. Among them, the P3 pathway has a significant advantage due to its shorter path (8 steps) and lower energy consumption (only 1 ATP is required).

[0157] Figure 6 CO2 assimilation pathways calculated by comb-FBA. The thick black solid line indicates overlapping reactions between the reported pathway and the newly predicted pathway, the dashed line indicates reactions unique to the reported pathway, and the black solid line indicates reactions unique to the newly predicted pathway.

[0158] The second mode involves reactions C1+C2→C3, C1+C3→C4, and C4→C2+C2( Figure 6 B). The published POAP pathway belongs to this model. Carbon fixation occurs by combining 8_PYC and 9_POR reactions ( Figure 6 (Black thick solid and dashed lines for P6 in B), a POAP variant (P6 pathway) was obtained that reduced the consumption of 0.5 ATP molecules per mole of oxalate by adding four additional steps. This model also includes several pathways with strong experimental potential. For example, the P7 pathway combines the highly carbon-fixing enzymes 2_CCR and 3_ACC, requires only one ATP, and has fewer steps and lower energy consumption than the THETA cycle. The P8 pathway is another promising pathway that uses a third efficient carbon fixation reaction (3_ACC) together with the widely used cellular carbon fixation reaction 8_PYC. It is worth noting that the P9 pathway is a shorter pathway for producing glyoxylate under this model, but the main challenge lies in how to achieve the carbon fixation reaction of 9_POR under anaerobic conditions21.

[0159] The third mode involves reactions C1+C4→C5, C1+C5→C6, and C6→C2+C4( Figure 6C). Currently, no known natural or artificial carbon fixation pathway has been reported to belong to this model. This model has relatively short steps and low ATP consumption. Notably, the P10 pathway requires only six steps and does not consume any ATP in the process of producing oxalate. It is more energy-efficient than the four steps and two ATP consumption of POAP. When glyoxylate is used as the target product, the pathway is further shortened to four steps (P11), making it the shortest glyoxylate synthesis pathway discovered to date. Compared with the CETCH pathway, the number of steps is reduced by four times and the energy consumption does not change. Overall, this model shows the promising potential of short and more energy-efficient carbon fixation pathways. However, the enzymes that catalyze these carbon fixation reactions (10_ICC, 11_KOR and 13_GCD) have not been fully explored or engineered, and the lack of kinetic data limits further evaluation and analysis. Future research needs to address the challenges associated with enzyme mining and optimization.

[0160] 4. Analysis of metabolite transformation relationships reveals interconnected reaction networks and alternative carbon fixation pathways

[0161] If the CO2 fixation pathway is divided into different modules, such as C1+C3→C4 and C1+C4→C5 modules, it can be found that alternative pathways often exist in different modules ( Figure 7 ). During the experimental construction of the CO2 fixation pathway, the pathway can be optimized by selecting alternative pathways with better thermodynamic and kinetic properties. During the assembly of the starch synthesis pathway, it was found that each module had multiple alternative pathways. Although the shortest path is theoretically optimal, its actual efficiency is lower than the alternative routes. Therefore, the final ASAP pathway includes two additional high-energy steps to address thermodynamic limitations. Therefore, in the actual construction process, it is necessary to search for alternative pathways between different carbon fixation reactions based on the connection relationship between metabolites. In order to identify alternative pathways, the carbon fixation reactions are modularized into a compact model that contains all carbon cycle pathways for graph theory analysis and visual representation.

[0162] Figure 7 A represents the transformation relationship network of high-frequency metabolites (appearing ≥150 times in the pathway results), where nodes represent metabolites, connecting lines represent transformation relationships, and arrows indicate transformation directions.

[0163] Figure 7 B is a schematic diagram of alternative pathways for the Cn→Cn+1 conversion module. The thermodynamic driving forces (ΔrG'° values) of each reaction were calculated using the eQuilibrator computing platform.

[0164] like Figure 7 As shown in Figure 2, it was found that there are multiple alternative pathways for the conversion of Cn metabolites to Cn+1 metabolites. Taking the C1+C3→C4 module as an example ( Figure 7 A), three C3 metabolites (pyruvate, phosphoenolpyruvate, propionyl-CoA) and two C4 metabolites ((S)-methylmalonyl-CoA, oxaloacetate) can form three alternative pathways ( Figure 7 B), based on reactions 5_PCC, 6_PPC and 8_PYC, respectively.

[0165] The 8_PYC pathway has the shortest number of steps (1 step), but the 5_PCC and 6_PPC pathways require additional reactions to complete the conversion of pyruvate to oxaloacetate.

[0166] Thermodynamic comparison: The 5_PCC and 8_PYC pathways have similar total driving forces (approximately -7 kJ / mol), while the 6_PPC pathway has a significantly higher total driving force (approximately -15 kJ / mol) due to the two-step thermodynamic driving forces of 25.0±0.8 and -40.3±6.1 kJ / mol, respectively. If the thermodynamic bottleneck of the first step can be overcome by regulating the metabolite concentration ratio, the 6_PPC pathway may achieve higher conversion efficiency.

[0167] In the C1+C4→C5 module, crotonyl-CoA can be converted to (2S)-ethylmalonyl-CoA via two pathways:

[0168] 1) Single-step 1_CCR pathway (ΔrG'° = -126.8 ± 4.7 kJ / mol);

[0169] 2) two-step 12_ECH pathway (ΔrG′° = -50.6 ± 5.2 kJ / mol and -76.1 ± 4.5 kJ / mol);

[0170] Although the 1_CCR pathway has higher enzyme activity, if a highly active 12_ECH enzyme is discovered or engineered in the future, this pathway may become a viable alternative to 1_CCR. Similarly, in the C1+C5→C6 module, the conversion of 2-ketoglutarate to D-threoisocitrate can be achieved by:

[0171] 1) Single-step 10-ICC pathway (ΔrG'° = -5.4 ± 6.2 kJ / mol);

[0172] 2) two-step 7_CFI pathway (total ΔrG′° = -27.0 kJ / mol);

[0173] Thermodynamically, the 7_CFI pathway is more advantageous, suggesting that prioritizing the development of its catalytic enzyme may lead to a dually thermodynamically and kinetically superior pathway.

[0174] 5. Natural carbon sequestration pathways are difficult to commercialize due to their lengthy steps, high energy consumption, and low efficiency. Only a few artificial carbon sequestration pathways have been reported in the past decade, highlighting the challenges of designing new pathways. This paper systematically explores the diversity of CO2 assimilation pathways using the comb-FBA algorithm and finds that:

[0175] 1) Core reactions dominate efficient pathways: Reactions such as 2_CCR, 5_PCC, 6_PPC, and 8_PYC constitute the shortest pathway skeleton, which is consistent with the optimal pathway that appears frequently in various products.

[0176] 2) Three carbon sequestration modes:

[0177] Mode 1 (such as CETCH and THETA cycles): short steps and low energy consumption, but limited by the core reaction enzyme activity.

[0178] Mode 2 (such as the POAP pathway): The synthesis of C2 products is energy-efficient, but the increase in steps may reduce experimental feasibility.

[0179] Mode 3 (P10-P11 novel pathway): The oxalate synthesis pathway can even achieve zero ATP consumption, but the enzyme activity needs to be optimized.

[0180] 3) Pathway modularity and interchangeability: For example, 5_PCC, 6_PPC, and 8_PYC can be interchanged according to enzyme activity, enhancing the flexibility of synthetic pathway design.

[0181] While computation provides a theoretical basis for pathway optimization, experimental efficiency still relies heavily on enzyme activity. For example, a research team developed the METIS machine learning framework, which screened the optimal solution from 1,025 conditions through 1,000 tests, resulting in a six-fold increase in carbon sequestration efficiency in the CETCH cycle.

[0182] In one embodiment, a computer device is provided. The computer device may be a server, and its internal structure diagram may be as follows: Figure 8 As shown. The computer device includes a processor, a memory, and a network interface connected via a system bus. The processor of the computer device is used to provide computing and control capabilities. The memory of the computer device includes a non-volatile storage medium and an internal memory. The non-volatile storage medium stores an operating system, a computer program, and a database. The internal memory provides an environment for the operation of the operating system and computer program in the non-volatile storage medium. The database of the computer device is used to store static information and dynamic information data. The network interface of the computer device is used to communicate with an external terminal via a network connection. When the computer program is executed by the processor, the steps of the above-mentioned method embodiment are implemented.

[0183] Those skilled in the art will understand that Figure 8The structure shown in the figure is merely a block diagram of a portion of the structure related to the solution of the present invention and does not constitute a limitation on the computer device to which the solution of the present invention is applied. The specific computer device may include more or fewer components than shown in the figure, or combine certain components, or have a different component arrangement.

[0184] The present invention is not limited to the structures described above and shown in the drawings, and various modifications and changes can be made without departing from the scope thereof. The scope of the present invention is limited only by the appended claims.

Claims

1. A method for selecting carbon sequestration pathways based on assimilation pathway assessment, characterized in that: include: Obtain all reactions with carbon dioxide, carbonates, and one-carbon compounds as substrates, and screen out several carbon fixation reactions using carbon dioxide and bicarbonate as carbon sources; The carbon fixation pathways were calculated using a combination algorithm combined with simplified flux balance analysis technology, and the core carbon fixation reactions were determined based on the calculation results. Based on the enzyme catalytic efficiency, simplicity, energy and reducing equivalent consumption, and metabolite conversion relationship in the core carbon fixation reaction, the carbon fixation pathway is evaluated, and the corresponding carbon fixation pathway is selected based on the evaluation results.

2. The method for selecting carbon sequestration pathways based on assimilation pathway assessment according to claim 1, characterized in that: The method obtains all reactions with carbon dioxide, carbonate and one-carbon compounds as substrates, and uses carbon dioxide and bicarbonate as carbon sources, and screens out several carbon fixation reactions including: A preset script was used to extract all reactions with carbon dioxide, carbonate, and one-carbon compounds as substrates from the MetaCyc database. Reactions involving the interconversion of carbon dioxide / bicarbonate with organic one-carbon compounds were filtered out, and reactions with carbon dioxide as the product were retained. Among the reactions catalyzed by isoenzymes and similar reactions involving different cofactors, any one representative reaction is retained, and carbon dioxide and bicarbonate are used as carbon sources to screen and retain the reactions that directly fix carbon, and obtain several carbon fixation reactions.

3. The method for selecting carbon sequestration pathways based on assimilation pathway assessment according to claim 1, characterized in that: The combined algorithm is combined with the simplified flux balance analysis technology to calculate the pathway of the carbon fixation reaction, and the core carbon fixation reaction is determined based on the calculation results, including: Based on the carbon fixation reactions screened, pathways containing a single carbon fixation reaction or two different carbon fixation reactions were set; carbon dioxide and bicarbonate were set as substrates, and acetyl-CoA, glyoxylate, oxalate, phosphoglyceraldehyde, and pyruvate were used to define the target products; The combinatorial mathematics method is used to randomly combine several carbon fixation reactions obtained by screening, and the simplified flux balance analysis technology is used to calculate each target product. The core carbon fixation reaction involved in the carbon fixation cycle is determined based on the calculation results.

4. The method for selecting carbon sequestration pathways based on assimilation pathway assessment according to claim 3, characterized in that: The core carbon fixation reaction includes a reaction between two one-carbon compounds, a reaction between a one-carbon compound and a two-carbon compound, a reaction between a one-carbon compound and a three-carbon compound, a reaction between a one-carbon compound and a four-carbon compound, and a reaction between a one-carbon compound and a five-carbon compound.

5. The method for selecting carbon sequestration pathways based on assimilation pathway assessment according to claim 4, characterized in that: The reaction between the two one-carbon compounds includes: Protein-bound S8-aminomethyldihydroacyllysine reacts with carbon dioxide to produce glycine and acylprotein.

6. The method for selecting carbon sequestration pathways based on assimilation pathway assessment according to claim 4, characterized in that: The reaction between the one-carbon compound and the two-carbon compound comprises: Adenosine triphosphate reacts with acetyl-CoA and bicarbonate ions to generate adenosine diphosphate, phosphate, and malonyl-CoA; Acetyl-CoA reacts with carbon dioxide, reduced Coenzyme II, and hydrogen ions to generate pyruvate, Coenzyme A, and oxidized Coenzyme II.

7. The method for selecting carbon sequestration pathways based on assimilation pathway assessment according to claim 4, characterized in that: The reaction between the one-carbon compound and the three-carbon compound comprises: Acryloyl-CoA reacts with carbon dioxide, reduced coenzyme II, and hydrogen ions to generate (R)-methylmalonyl-CoA and oxidized coenzyme II; Adenosine triphosphate, propionyl-CoA and bicarbonate ion react to produce adenosine diphosphate, phosphate and (S)-methylmalonyl-CoA; Water, phosphoenolpyruvate, and carbon dioxide react to produce phosphoric acid and oxaloacetic acid; Adenosine triphosphate, pyruvate, and bicarbonate ions react to produce adenosine diphosphate, phosphoric acid, and oxaloacetic acid; Two reduced ferredoxins, propionyl-CoA, carbon dioxide, and two hydrogen ions react to produce two oxidized ferredoxins, 2-oxobutyrate, and CoA.

8. The method for selecting carbon sequestration pathways based on assimilation pathway assessment according to claim 4, characterized in that: The reaction between the one-carbon compound and the four-carbon compound comprises: Crotonyl-CoA reacts with carbon dioxide, reduced coenzyme II, and hydrogen ions to generate (2S)-ethylmalonyl-CoA and oxidized coenzyme II; Succinyl-CoA reacts with carbon dioxide, reduced Coenzyme II, and hydrogen ions to generate 2-oxoglutarate, Coenzyme A, and oxidized Coenzyme II. Butyryl-CoA reacts with carbon dioxide to produce (2S)-ethylmalonyl-CoA; Crotonyl-CoA reacts with carbon dioxide to produce glutaconyl-1-CoA.

9. The method for selecting carbon sequestration pathways based on assimilation pathway assessment according to claim 4, characterized in that: The reaction between the one-carbon compound and the five-carbon compound comprises: Ribulose 1,5-bisphosphate reacts with carbon dioxide and water to produce two molecules of 3-phosphoglycerate; Adenosine triphosphate, 2-oxoglutarate and bicarbonate ion react to produce adenosine diphosphate, phosphoric acid and oxalosuccinic acid; 2-Oxoglutarate reacts with carbon dioxide, reduced coenzyme II, and hydrogen ions to generate isocitrate and oxidized coenzyme II.

10. A carbon sequestration pathway selection system based on assimilation pathway evaluation, characterized in that: It includes carbon fixation reaction screening module, core carbon fixation reaction determination module and carbon fixation pathway selection module; The carbon fixation reaction screening module is used to obtain all reactions with carbon dioxide, carbonates and one-carbon compounds as substrates, and to screen several carbon fixation reactions with carbon dioxide and bicarbonate as carbon sources; The core carbon fixation reaction determination module is used to calculate the pathways of carbon fixation reactions using a combination algorithm combined with a simplified flux balance analysis technique, and determine the core carbon fixation reaction based on the calculation results; The carbon fixation pathway selection module is used to evaluate carbon fixation pathways based on the enzyme catalytic efficiency, simplicity, energy and reducing equivalent consumption, and metabolite conversion relationship in the core carbon fixation reaction, and select the corresponding carbon fixation pathway based on the evaluation results.

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