Artificial carbon dioxide fixation method and application thereof

Abstract

The invention belongs to the field of synthetic biology, and discloses an artificial carbon dioxide fixation method and application thereof. According to the method, an artificial carbon dioxide immobilization metabolic pathway is constructed, the pathway is a linear autocatalytic cycle pathway LATCH, acetyl coenzyme A and derivative products thereof can be synthesized by using carbon dioxide or bicarbonate, the catalytic cycle pathway takes two molecules of CO2 or HCO3 <-> as a substrate, one molecule of acetyl coenzyme A is generated through ten enzymatic steps, operation of the pathway depends on ATP and NADPH, and the acetyl coenzyme A can be synthesized by using the linear autocatalytic cycle pathway LATCH. Or is driven by an energy and reducing power supply system, such as organic matters such as glucose, formic acid and the like, high-energy compounds such as polyphosphoric acid, phosphocreatine and the like, or a photoreaction enzyme complex on a plant capsule membrane. An in-vitro multi-enzyme system and recombinant escherichia coli experiment prove that the acetyl coenzyme A and the derivative polyhydroxybutyric acid thereof can be efficiently synthesized, and the application prospect in the fields of carbon neutralization, green manufacturing and the like is wide.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology, specifically relating to a method for artificial carbon dioxide fixation and its application. Background Technology

[0002] Natural carbon fixation pathways, such as the Calvin cycle, have served as the core framework of the global carbon cycle, supporting the origin, evolution, and continuation of life on Earth. In recent years, to address global challenges such as energy demand, food security, and environmental sustainability, scientists have made significant progress in modifying and expanding these natural pathways in various host organisms. However, to fundamentally enhance the productivity of autotrophic systems, the novel design of artificial carbon assimilation pathways is gradually emerging as a powerful alternative strategy, aligning with the long-term vision of sustainable biochemical production.

[0003] Both the Calvin cycle and the reductive tricarboxylic acid cycle (rTCA cycle) are autocatalytic cycles, making them not only modification modules of host-centric metabolism but also core metabolic processes. Patent application CN106755172A (publication date 2017.05.31) discloses a method for synthesizing acetyl-CoA and its derivatives using glycolaldehyde, where glycolaldehyde, as a raw material, can be prepared from formaldehyde, representing a cyclic pathway for the conversion of organic one-carbon substrates into acetyl-CoA and its derivatives. Patent application CN119162161A (publication date 2024.12.20) discloses a method for synthesizing acetyl-CoA using phosphate transketolase and its mutants, with raw materials including methanol, formaldehyde, glycerol, and erythritol, among other organic compounds. Patent application CN119464172A (publication date February 18, 2025) discloses a hybrid central metabolic network chassis cell with both carbon economy and energy generation pathway, its construction method, and applications. Essentially, it utilizes a portion of the phosphoenolpyruvate generated from glucose for decarboxylation to generate energy and simultaneously produce acetyl-CoA, while the other portion is carboxylated to produce oxaloacetate, which is then converted to acetyl-CoA via the tricarboxylic acid cycle. Patent application CN117925488A (publication date April 26, 2024) discloses a method for constructing a strain with a high glucose-to-acetyl-CoA conversion rate by integrating carbon-conserved NOG and MCG modules. The acetyl-CoA precursors in the above applications are mainly organic carbon sources, such as formaldehyde, glycolaldehyde, or glucose, and are not a de novo independent carbon dioxide assimilation pathway.

[0004] Therefore, there is an urgent need in this field to construct an artificial carbon dioxide fixation metabolic pathway to achieve the efficient synthesis of acetyl-CoA and its derivatives, and to ensure their stable operation in cellular systems. This innovative method provides new ideas for the design and industrial application of artificial carbon fixation pathways, and shows broad application prospects in the fields of carbon neutrality, green manufacturing, and biomaterials production. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an artificially constructed carbon dioxide fixation metabolic pathway, LATCH (Linear Autocatalytic cycle Tailored for Carbon Harvesting), which is an autocatalytic cycle pathway and a potential alternative to the Calvin cycle. LATCH comprises ten enzymatic steps, integrating two characterized carboxylation modules—the natural serine cycle from methyltrophic bacteria and an artificially designed tartronyl-CoA (TaCo) carbon fixation module for converting the photosynthetic byproduct glycolic acid into glyceric acid. It retains the core characteristics of most cyclic carbon fixation pathways: fixing two molecules of CO2 or generating one molecule of acetyl-CoA per cycle. To connect the two known modules, this invention employs an additional enzyme, glyoxylate reductase, forming a latch-like structure to convert glyoxylate generated from the serine cycle into glycolic acid, the initial substrate of the TaCo module. Notably, this pathway is oxygen-insensitive and oxygen-independent, relying solely on ATP and NADPH for energy and reducing power, both of which can be regenerated in the basal cells.

[0006] On the one hand, the present invention provides a method for artificial carbon dioxide fixation, which constructs an artificial carbon dioxide fixation metabolic pathway LATCH. The LATCH pathway is an autocatalytic cycling pathway that integrates the serine cycle and the TaCo module through glyoxylate reductase. It consists of 10 enzymatic steps, each cycle of which can convert carbon dioxide or bicarbonate into acetyl-CoA.

[0007] Specifically, the enzymes involved in the LATCH pathway include glycerol kinase, enolase, phosphoenolpyruvate carboxylase, malate dehydrogenase, mayl-CoA ligase, mayl-CoA lyase, glycolyl-CoA synthase, glycolyl-CoA carboxylase, tartrate-CoA reductase, and glyoxylate reductase.

[0008] Specifically, the glycerokinase is derived from denitrifying filamentous microbes or thermophilic acidophilic bacteria, and its amino acid sequences have NCBI accession numbers AFK19321 and AAK40970, respectively.

[0009] The enolase, phosphoenolpyruvate carboxylase, and malate dehydrogenase are derived from Escherichia coli, and their amino acid sequences have NCBI accession numbers of NP_417259, NP_418391, and NP_417703, respectively.

[0010] The malico-CoA ligase is derived from *Tricholoma materans* or *Methanococcus capsulatum*. The malico-CoA ligase is a two-subunit enzyme. When derived from *Tricholoma materans*, the NCBI accession numbers for the amino acid sequences of the two subunits are ABY30198 and ABY30199, respectively. When derived from *Methanococcus capsulatum*, the NCBI accession numbers for the amino acid sequences of the two subunits are AAU91976 and AAU91975, respectively.

[0011] The NCBI accession number for the amino acid sequence of the malic acyl-CoA lyase is ABY30201.

[0012] The glyoxylate reductase is derived from Acetic Acid Bacillus, and its amino acid sequence has the NCBI accession number AQS85295.

[0013] The glycolyl-CoA synthase is derived from a strain of Rhodobulbus. The amino acid sequence of the glycolyl-CoA synthase has a mutation at amino acid position 379 compared to the wild-type amino acid sequence. Amino acid position 379 is mutated to A. The NCBI accession number of the wild-type amino acid sequence of the glycolyl-CoA synthase is AKQ42064.

[0014] The glycolyl-CoA carboxylase is derived from *Thunb.*, and is a bisubunit enzyme consisting of glycolyl-CoA carboxylase subunit I and glycolyl-CoA carboxylase subunit II. The glycolyl-CoA carboxylase exhibits mutations at the following sites relative to wild-type glycolyl-CoA carboxylase: positions 100, 143, 407, 450, and / or 50. Preferably, the 100th position is mutated to S, the 143rd position is mutated to H, the 407th position is mutated to I, the 450th position is mutated to V, and the 502nd position is mutated to R. The INCBI accession number for the two subunits of the wild-type glycolyl-CoA carboxylase is ABY28706, and the INCBI accession number for the amino acid sequence of glycolyl-CoA carboxylase subunit II is ABY31385.

[0015] The tartrate-CoA reductase is derived from *Hyperhygrophytes thermophila*, and its amino acid sequence has the NCBI accession number ABY35820.

[0016] Specifically, each cycle converts 2 molecules of carbon dioxide or bicarbonate into 1 molecule of acetyl-CoA.

[0017] Specifically, the LATCH pathway relies on ATP and / or NADPH to provide energy and reducing power. The substance providing ATP is polyphosphate, and the substance providing NADPH is selected from formic acid, glucose, or dextrin.

[0018] Specifically, the polyphosphoric acid is sodium hexametaphosphate at a concentration of 2-80 mM, the formic acid is sodium formate at a concentration of 8-25 mM, the glucose at a concentration of 10-70 mM, and the dextrin at a concentration of 5-35 mM; preferably, the sodium hexametaphosphate is at a concentration of 10-60 mM, the sodium formate at a concentration of 10-20 mM, the glucose at a concentration of 15-60 mM, and the dextrin at a concentration of 7.5-15 mM.

[0019] On the one hand, the present invention also provides a recombinant microorganism, which constructs the LATCH pathway using the method described above, and integrates the gene encoded by the enzyme involved in the LATCH pathway into the host cell. Preferably, the host cell is Escherichia coli, Bacillus, yeast, or plant cell.

[0020] Specifically, it also includes a plasmid overexpressing a carbonic anhydrase encoding gene, the amino acid sequence of which has the NCBI accession number NP_414668.

[0021] On the other hand, the present invention utilizes the method in the synthesis of acetyl-CoA and its derivatives, wherein the derivatives are selected from polyhydroxyalkanoates, biofuels, sugars, amino acids, organic acids and biomass, and preferably, the derivatives are polyhydroxybutyric acid.

[0022] Specifically, the application can be synthesized in vitro or in a microbial genome.

[0023] In this invention, amino acid residues can be represented by a single letter or by three letters, for example: alanine (Ala, A), valine (Val, V), glycine (Gly, G), leucine (Leu, L), glutamic acid (Gln, Q), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), lysine (Lys, K), methionine (Met, M), serine (Ser, S), threonine (Thr, T), cysteine ​​(Cys, C), proline (Pro, P), isoleucine (Ile, I), histidine (His, H), and arginine (Arg, R).

[0024] The term "AxxB" indicates that amino acid A at position xx is changed to amino acid B. For example, V379A means that V at position 379 is mutated to A; for example, L100S-Y143H-D407I-I450V-W502R means that L at position 100 is mutated to S, while Y at position 143 is mutated to H, D at position 407 is mutated to I, I at position 450 is mutated to V, W at position 502 is mutated to R; and so on.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) It constructs an artificial autocatalytic cycling pathway LATCH, which fixes two molecules of CO2 or bicarbonate to generate one molecule of acetyl-CoA in each cycle, breaking through the limitation of existing technologies that rely on organic carbon sources (such as formaldehyde and glucose), realizing de novo independent CO2 assimilation, and providing a new strategy for carbon fixation; (2) This pathway is not sensitive to oxygen and does not depend on oxygen, does not require strict oxygen control conditions, and can operate stably in a variety of systems (such as in vitro multi-enzyme systems, Escherichia coli and other microorganisms), with a wide range of applicable scenarios. Integrating the LATCH pathway into the Escherichia coli genome and combining it with overexpression of carbonic anhydrase can significantly increase the biomass of the strain in glucose-free medium (50% increase compared to wild type), providing a basis for constructing high-yield carbon dioxide-carbon ... (3) The energy and reducing power supply is flexible. ATP can be regenerated through polyphosphate / polyphosphate kinase system, and NADPH can be supplied through formic acid / formic acid dehydrogenase system, glucose or dextrin metabolism module, adapting to different application needs, and the supply efficiency is high under optimal conditions; (4) It can efficiently synthesize acetyl-CoA, and can further synthesize its derivative polyhydroxybutyric acid (PHB), which has direct applications in green manufacturing and biomaterial production, helping to replace traditional chemical synthesis processes, directly converting CO2 into high-value compounds as carbon source, reducing CO2 emissions while realizing resource recycling, meeting the needs of carbon neutrality and environmental sustainable development, and has broad application prospects. Attached Figure Description

[0026] Figure 1 This is a reaction route diagram of the LATCH pathway for artificial carbon dioxide fixation in this invention.

[0027] Figure 2 This represents the functional characterization results of glyoxylate reductase (GlyR) in the LATCH pathway. NAD, NADP, NADH, and NADPH represent blank controls with only coenzymes added. "Enzyme" indicates the control with only glyoxylate reductase (GlyR) added, and "Buffer" indicates the blank control containing only buffer. In the remaining samples, the "-" indicates the test substrate and coenzyme, respectively. The tested substrates include malic acid, glyoxylic acid, glyceric acid, glycerol, and glycolic acid, a total of 5. All test samples containing oxidized coenzymes NAD and NADP are represented by dashed lines, and test samples containing reduced coenzymes NADH and NADPH are represented by solid lines. The ordinate OD340 is used to characterize the concentrations of NADH and NADPH in the system.

[0028] Figure 3The results show the regeneration energy and reducing power of formic acid, glucose, and dextrin. The upper curve represents the test results of the in vitro multi-enzyme system, with the horizontal axis representing time and the vertical axis representing the concentration changes of NADH and NADPH as characterized by absorbance at OD340. The lower roadmap shows the test principle and reaction process followed by the system. A represents the activity of wild-type formic acid dehydrogenase and mutant formic acid dehydrogenase in the formic acid system to dehydrogenate formic acid to carbon dioxide and generate NADPH at the same time; B represents the activity of NADPH production using glucose; C represents the activity of NADPH production using dextrin; and D represents the effect of ATP regeneration using polyphosphate (sodium hexametaphosphate).

[0029] Figure 4 This is the experimental result of modular validation of the LATCH pathway. The left side of the figure shows the reaction route used in the multi-enzyme detection system, and the right side shows the corresponding measurement results. Among them, A represents the functional detection of the glycolic acid to glyceric acid module; B represents the activity detection of the phosphoenolpyruvate to malic acid module; C represents the functional detection of the glyceric acid to malic acid module; and D represents the activity detection of the malic acid to glyoxylic acid module.

[0030] Figure 5 This chart presents the experimental results of the in vitro synthesis of acetyl-CoA and its derivative polyhydroxybutyrate (PHB) using a multi-enzyme system. Figure A shows the results of acetyl-CoA synthesis, with the horizontal axis representing the names of the control and sample groups, and the vertical axis representing the concentration of synthesized acetyl-CoA in the system. Figure B shows the results of polyhydroxybutyrate synthesis, with the horizontal axis representing the names of the control and sample groups, and the vertical axis representing the concentration of synthesized polyhydroxybutyrate in the system. Figure C shows the growth curves of four recombinant *E. coli* strains in glucose-free M9 medium. Figure D shows the OD value at which the biomass reached its maximum (22 hours of culture) in each growth curve. 600 level. Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely illustrative and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0032] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0034] Technical methods involved in the embodiments

[0035] Strains and culture conditions: Using the genomic DNA of *Escherichia coli* K-12 MG-1655 as a template, the following enzymes were amplified: enolase (NCBI accession number NP_417259), phosphoenolpyruvate carboxylase (NCBI accession number NP_418391), malate dehydrogenase (NCBI accession number NP_417703), glucokinase (NCBI accession number NP_416889), α-glucan phosphorylase (NCBI accession number YP_026218), and phosphogluconomutase (NCBI accession number N). The enzymes involved include: P_415214, glucose-6-phosphate dehydrogenase (NCBI accession number 416366 for its amino acid sequence), 6-phosphogluconide lactonease (NCBI accession number NP_415288 for its amino acid sequence), 6-phosphogluconide dehydrogenase (NCBI accession number NP_416533 for its amino acid sequence), pyrophosphate hydrolase (NCBI accession number NP_418647 for its amino acid sequence), adenosine kinase (NCBI accession number NP_415007 for its amino acid sequence), and carbonic anhydrase (NCBI accession number NP_414668 for its amino acid sequence). Using the genomic DNA of *Methylobacterium extorquens* AM1 as a template, malic acid-CoA ligase (which is a two-subunit enzyme with NCBI accession numbers ABY30198 and ABY30199 for the two subunit amino acid sequences) and malic acid-CoA lyase (with NCBI accession number ABY30201 for its amino acid sequence) were amplified. Using the genomic DNA of *Yarrowia lipolytica* W29 as a template, formate dehydrogenase (with NCBI accession number XP_501224 for its amino acid sequence) was amplified.Suzhou Genewiz Biotechnology Co., Ltd. was commissioned to conduct experiments on two types of glycerol kinases (derived from *Denitrifying Mycobacterium* and *Thermophilic Acidophilus*, with NCBI accession numbers AFK19321 and AAK40970 for their amino acid sequences, respectively), malic acid-CoA ligase (derived from *Methanococcus capsulatum*, with NCBI accession numbers AAU91976 and AAU91975 for the two subunits, respectively), glyoxylate reductase (NCBI accession number AQS85295 for its amino acid sequence), and glycolyl-CoA synthetase (amino acid sequence relative to amino acid position 379 of the wild-type amino acid sequence). The V mutation is replaced by A, and the NCBI accession number for the wild-type amino acid sequence of the glycolyl-CoA synthase is AKQ42064. Glycolyl-CoA carboxylase (which is a bisubunit enzyme, the bisubunits being glycolyl-CoA carboxylase subunit I and glycolyl-CoA carboxylase subunit II, wherein the glycolyl-CoA carboxylase, compared to the wild-type glycolyl-CoA carboxylase, has mutations at positions 100, 143, 407, 450, and 502 of glycolyl-CoA carboxylase subunit I, where position 100 is mutated to S and position 143 is mutated to H.) The mutation at position 407 is I, the mutation at position 450 is V, and the mutation at position 502 is R, i.e., L100S-Y143H-D407I-I450V-W502R. The NCBI accession number for the amino acid sequence of the two subunits of wild-type glycolyl-CoA carboxylase (glycolyl-CoA carboxylase subunit I is ABY28706, and the NCBI accession number for the amino acid sequence of glycolyl-CoA carboxylase subunit II is ABY31385), tartrate-CoA reductase (its amino acid sequence NCBI accession number is ABY35820), and acetyl-CoA thiolase. Codon optimization and gene synthesis were performed on the following enzymes: acetyl-CoA reductase (NCBI accession number AEI75530), polyhydroxybutyrate synthase (NCBI accession number AEI76813), mutant formate dehydrogenase (with a mutation G146M-A287G in the wild-type amino acid sequence, wild-type amino acid sequence AOR73030), and type II polyphosphate kinase (NCBI accession number ABA79945). In in vitro cell-free multi-enzyme assays, these coding sequences were integrated into the pET28a vector to express enzymes with a 6*His tag. For intracellular assays, these sequences were integrated into the genome of *E. coli* MG1655. During protein purification, the corresponding plasmids were introduced into *E. coli* BL21 (DE3), and positive clones were screened using kanamycin resistance markers.Single colonies were selected and cultured in LB medium at 37°C until the OD600 of the culture reached 0.6–0.8. IPTG was then added at a final concentration of 0.5 mM as an inducer to induce protein expression. For intracellular assays, the pathway gene was integrated into the *E. coli* MG1655 genome. Specifically, the chromosomal target site was cleaved using Cas9 protein, with upstream and downstream homologous arms of 500 bp, and a Trc constitutive strong promoter was used.

[0036] The enzyme activity detection method within the modules was as follows: The entire pathway was divided into three modules. The first module synthesizes glyceric acid from glycolic acid, consuming 2 molecules of NADPH in its final step. The second module generates malic acid from glyceric acid, consuming 1 molecule of NADPH in its final step. The third module generates glyoxylic acid from malic acid, also consuming 1 molecule of NADPH in its final step. Since NADPH has an absorption peak at 340 nm, a Tecan Infinite 200 Pro multi-mode microplate reader was used for real-time detection to determine the module's functionality. Similarly, glyoxylate reductase was screened using the same method. This enzyme catalyzes the conversion of glyoxylic acid and NADPH to glycolic acid; therefore, its activity can be determined by changes in absorbance at 340 nm. Furthermore, the third module involves the conversion of malate to glyoxylate, catalyzed by mayl-CoA ligase (MTK) and mayl-CoA lyase (MCL). MTK catalyzes the condensation of malate and coenzyme A to form mayl-CoA, while MCL further cleaves mayl-CoA into acetyl-CoA and glyoxylate. Glyoxylate reacts with phenylhydrazine to form glyoxylate-phenylhydrazone, which exhibits an absorption peak at 324 nm. This product can be detected in real-time using a Tecan Infinite 200 Pro multi-sensor microplate reader, thus determining its functionality.

[0037] PHB extraction and detection methods: For intracellular synthesis systems, bacterial cells were collected by centrifugation and then frozen at -80°C for at least 4 hours or rapidly frozen in liquid nitrogen, followed by lyophilization. The lyophilized cells were then ground into a fine powder in a mortar. 25 mg of the sample was weighed and placed in a 15 mL glass spiral tube, and 2 mL of chloroform and 2 mL of methanol hydrolysis solution (85% methanol, 15% H2SO4, 1 g / L benzoic acid) were added. After gentle mixing, the mixture was incubated in a water bath at 80°C-100°C for 2-3 hours. After the reaction, 1 mL of ddH2O was added, the mixture was shaken and allowed to stand for phase separation. The lower organic phase was collected and washed repeatedly with 1 mL of ddH2O to remove residual water from the chloroform. Finally, the obtained sample was filtered through a filter membrane for GC-MS detection. For cell-free multi-enzyme systems, 1 mL of the reaction solution was lyophilized, and the subsequent hydrolysis, esterification, and detection processes were the same as for intracellular products. GC-MS analysis was performed on an Agilent 7890A GC (Agilent Technologies, USA) equipped with a multimode injector and a splitless liner lined with glass wool. A nonpolar capillary column (DB-5MS, 30 m × 0.25 mm inner diameter, 0.25 μm film thickness; Agilent, USA) was used. The detector was an Agilent 7200Q-TOF mass spectrometer using an inert electron ionization (EI) source. Data analysis was performed using Agilent Mass Hunter software (Marsol-Vall et al.).

[0038] Detection method for acetyl-CoA: The separation of acetyl-CoA from CoA was performed by HPLC. After the reaction was completed, 10% trichloroacetic acid was added to terminate the reaction and precipitate the protein. The precipitate was then removed by centrifugation. CoA and acetyl-CoA standards were used as controls. Two concentrations of CoA and acetyl-CoA standards were prepared, namely 1 mM and 2 mM. All samples were filtered using a 0.22 µm needle filter and then detected by HPLC. The specific method is as follows: At 35 °C, acetyl-CoA and CoA were separated using a Zorbax SB-Aq column (4.6 mm inner diameter × 150 mm (5 μm)). The signal was monitored at 254 nm using a UV detector. The mobile phase consisted of buffer A (0.2 M NaH₂PO₄, pH 5.0) and buffer B (acetonitrile), with a flow rate of 0.3 mL / min. A gradient was set: 0–10 min, 100% buffer A; 10–25 min, buffer B increased from 0% to 20%; 25–30 min, 80% buffer A and 20% buffer B; 30–35 min, buffer A increased from 80% to 100%. Subsequently, the column was washed with buffer A and equilibrated for 5 min before the next injection. 10 µL of sample was injected into the HPLC system, and each sample was run for 40 min.

[0039] Table 1. Information on metabolites involved in this invention

[0040]

[0041] Table 2. Enzyme Information Table Involved in this Invention

[0042]

[0043]

[0044]

[0045]

[0046] Example 1

[0047] The glyoxylate reductase data characterized in the current literature are all derived from thermophilic bacteria. Since 9 out of the 10 steps in the LATCH pathway originate from the serine cycle and the TaCo module, the conversion of two inorganic carbon molecules (carbon dioxide or bicarbonate) to acetyl-CoA occurs in a 10-step linear autocatalytic cycle. The specific pathway comprises 10 reaction steps, numbered #1-#10, as follows: glycolate is catalyzed by glycolyl-CoA synthase to glycolyl-CoA, then by glycolyl-CoA carboxylase to tartrate-CoA, then by tartrate-CoA reductase to glycerate, and finally by glycerate kinase to acetyl-CoA. Phosphoglycerate is then converted to phosphoenolpyruvate by enolase, followed by oxaloacetate by phosphoenolpyruvate carboxylase. Oxaloacetate is then converted to malate by malate dehydrogenase, and subsequently to malate-CoA by malate-CoA ligase. Malate-CoA is then converted to glyoxylate and acetyl-CoA by malate-CoA lyase. Finally, glyoxylate is converted to glycolate by glyoxylate reductase, completing a full cycle. Acetyl-CoA, as the final product of the two bicarbonate fixation processes, can be used to produce acetyl-CoA derivatives such as PHB (e.g., PHB). Figure 1 (As shown). These are all known enzymes that exhibit activity under neutral conditions of pH 7-8 and mesophilic conditions of 30-37°C. Therefore, it is crucial to confirm whether the only enzyme with unknown activity, GlyR, can function under the same conditions. In this study, we synthesized and characterized glyoxylate reductase (GlyR) from *Acetobacter aceti*, and databases indicate that the optimal temperature for this strain is 45°C.

[0048] The test was conducted at pH 7.5 and 30°C using 50 mM Tris-HCl as the buffer. This was to simultaneously confirm whether the enzyme possesses pan-substrate activity for other metabolites in the pathway, whether it can catalyze the reverse glycolate to glyoxylate reaction, and its effect on the coenzyme NAD+.+ and NADP + Due to the bias in the study, in addition to glyoxylic acid and glycolic acid as test substrates, metabolites in the pathway such as malic acid and glyceric acid, as well as glycerol (used as a protective agent during protein purification and preservation) that may be present in the experimental system, were also included as potential substrates. The experiment used NADH and NAD... + NADPH and NADP + Four forms of coenzyme.

[0049] In the sample system, the initial concentration of all substrates was 10 mM, the concentration of coenzyme was 0.5 mM, and the final concentration of GlyR pure enzyme was 0.5 μg / mL. The reaction was carried out in clear 96-well plates, with each sample volume being 200 μL, and three replicates were set up. The absorbance change of NADH / NADPH was measured at 340 nm using a TECAN Infinite 200 pro multi-plate reader.

[0050] The results are as follows Figure 2 As shown, reduced coenzymes NADH and NADPH have absorption peaks at OD340. Therefore, the ordinate OD340 can be used to characterize the concentrations of NADH and NADPH in the system. Among all controls and samples, only the "glyoxylate-NADPH" combination showed a significant change in absorbance, indicating that the tested glyoxylate reductase specifically and irreversibly uses the coenzyme NADPH to catalyze the conversion of glyoxylate to glycolate. Therefore, it can be concluded that GlyR derived from Acetobacter aceti specifically uses NADPH as a coenzyme to catalyze the irreversible conversion of glyoxylate to glycolate (black curve in the figure, absorbance decreasing monotonically), while other substrate and coenzyme combinations did not show significant changes in absorbance. Under these detection conditions, the specific activity of GlyR (molecular weight 34.45 kDa) was calculated to be 16.1 U / mg, indicating good activity consistent with its functional role in linking the preceding and following modules in the LATCH pathway.

[0051] Example 2

[0052] LATCH, as a de novo carbon dioxide assimilation pathway, is based on the fact that carbon dioxide is an inorganic compound with no reducing power, and its conversion into any organic compound, including acetyl-CoA, necessarily requires external energy input. According to the reaction composition in the pathway, LATCH requires ATP as energy and NADPH as a source of reducing power; together, they constitute the energy and reducing power system for LATCH operation. In plants, and in cell-free systems using plant thylakoid membranes as material, the synthesis of ATP and NADPH can be driven by light energy. However, in conventional cell-free multi-enzyme catalytic systems, or in heterotrophic strains such as E. coli, they often require the oxidation of substances such as sugars and organic acids to provide these energy sources.

[0053] This test selected formic acid, glucose, and dextrin as three organic compounds to assess NADPH supply capacity, with polyphosphate as the ATP source. In the system using formic acid as a substrate to produce reducing power (i.e., the formic acid / formate dehydrogenase system): the formic acid dehydrogenase assay used wild-type formic acid dehydrogenase from *Yarrowia spp.* and an artificially modified mutant formic acid dehydrogenase, both added at a concentration of 10 μg / mL. Sodium formate was added at concentrations of 10 mM and 20 mM. In the glucose and dextrin reducing power system (i.e., the glucose metabolism module and the dextrin metabolism module): glucose was added at concentrations of 15 mM, 30 mM, and 60 mM, and dextrin was added at concentrations of 7.5 mM, 15 mM, and 30 mM (converted to monosaccharides). The concentrations of glucokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconide lactonease, and 6-phosphogluconide dehydrogenase were all 1 μg / mL. The concentrations of α-glucan phosphorylase and phosphoglucose mutase were 50 μg / mL and 1 μg / mL, respectively. The polyphosphate used was in the form of sodium hexametaphosphate, with concentrations of 2 mM, 10 mM, 20 mM, 40 mM, 60 mM, and 80 mM, respectively. The concentration of polyphosphate kinase was 50 μg / mL. NAD+ was present in the system. + Or NADP + The concentration of each enzyme added was 0.5 mM. The blank control (Blank group) only contained the substrate, without any enzyme. The activity test results of wild-type and mutant formate dehydrogenase in the formate system, which dehydrogenates formate to carbon dioxide and simultaneously generates NADPH, are as follows: Figure 3 As shown in Figure A, FA-10-FDHmut-NAD represents a system that uses formic acid as a substrate to produce reducing power, in which sodium formate is added at a concentration of 10 mM, an artificially modified mutant formate dehydrogenase is used, and NAD is the coenzyme. + The experimental group; FA-20-FDHmut-NAD represents a system that uses formic acid as a substrate to produce reducing power, in which sodium formate is added at a concentration of 20 mM, an artificially modified mutant formic acid dehydrogenase is used, and NAD is the coenzyme. + The experimental group; FA-10-FDHmut-NADP represents a system that uses formic acid as a substrate to produce reducing power, in which sodium formate is added at a concentration of 10 mM, an artificially modified mutant formate dehydrogenase is used, and NADP is used as the coenzyme. + The experimental group; FA-20-FDHmut-NADP represents a system that uses formic acid as a substrate to produce reducing power, with sodium formate added at a concentration of 20 mM, using an artificially modified mutant formic acid dehydrogenase, and NADP as the coenzyme. + The experimental group; FA-10-FDHyli-NAD represents a system that uses formic acid as a substrate to produce reducing power, with sodium formate added at a concentration of 10 mM, wild-type formate dehydrogenase derived from Yersinia lipolytica, and NAD as the coenzyme. +The experimental group; FA-20-FDHyli-NAD represents a system that uses formic acid as a substrate to produce reducing power, with sodium formate added at a concentration of 20 mM, wild-type formate dehydrogenase derived from Yersinia lipolytica, and NAD as the coenzyme. + The experimental group; FA-10-FDHyli-NADP represents a system that uses formic acid as a substrate to produce reducing power, with sodium formate added at a concentration of 10 mM, wild-type formate dehydrogenase derived from Yersinia lipolytica, and NADP as the coenzyme. + The experimental group; FA-20-FDHyli-NADP represents a system that uses formic acid as a substrate to produce reducing power, with sodium formate added at a concentration of 20 mM, wild-type formate dehydrogenase derived from Yersinia lipolytica, and NADP as the coenzyme. + The experimental group; FA-0-FDHmut-NADP represents a system that produces reducing power using formic acid as a substrate without the addition of sodium formate, employing an artificially modified mutant formic acid dehydrogenase, with NADP as the coenzyme. + The experimental group; the test results showed that formate dehydrogenase derived from Yersinia lipolytica is a strictly NAD+ dehydrogenase. + The FDHmutase is coenzyme-dependent, only able to dehydrogenate formate to NADH. However, the artificially mutated FDHmutase is not strictly dependent on the coenzyme and can utilize NAD+. + NADH can also be generated using NADP. + The generation of NADPH can therefore serve as an effective strategy for supplying reducing power to LATCH, as shown in the metabolic flow diagram at the bottom of section A. Furthermore, the initial reaction rate of the system with the addition of 20 mM sodium formate is slightly higher than that of the system with 10 mM. (For example...) Figure 3As shown in Figure BC, Glc-60, Glc-30, Glc-15, and Glc-0 represent glucose concentrations of 60 mM, 30 mM, 15 mM, and no glucose addition in a system using glucose as a substrate to produce reducing power, respectively. MD-30, MD-15, MD-7.5, and MD-0 represent dextrin concentrations of 7.5 mM, 15 mM, and 30 mM in a system using dextrin as a substrate to produce reducing power, respectively. Both glucose and dextrin possess NADPH regeneration capabilities. Glucose can rapidly regenerate NADPH, but requires ATP to initiate the dehydrogenation reaction. Dextrin regenerates NADPH more slowly than glucose, but does not require additional ATP. The reaction process... As shown in the flowchart below BC, specifically, glucose is first converted to glucose-6-phosphate by glucokinase, while dextrin is converted to glucose-1-phosphate by α-glucan phosphorylase, and then to glucose-6-phosphate by phosphoglucomutase. The two NADPH regeneration pathways are identical: glucose-6-phosphate is converted to gluco-6-phosphate by glucose-6-phosphate dehydrogenase, simultaneously producing one molecule of NADPH. Then, gluco-6-phosphate is converted to glucosinolate and ribulose-5-phosphate by phosphogluconolactone and phosphogluconolactone dehydrogenase, again producing NADPH. Furthermore, glucose does not exhibit an inhibitory effect on the system; the reaction rate increases with increasing glucose concentration. Dextrin, however, does exhibit an inhibitory effect; the reaction rate decreases with increasing dextrin concentration. Polyphosphokinases (PPKs) catalyze ATP synthesis and are an effective ATP regeneration strategy. Under the action of PPKs, added ADP generates ATP, thus providing ATP for the glucokinase-catalyzed glucose-6-phosphate process. Then, glucose-6-phosphate, under the catalysis of glucose-6-phosphate dehydrogenase, forms 6-phosphate gluconolactone and simultaneously produces one molecule of NADPH, resulting in an absorption peak at OD340 to characterize ATP production. The reaction rate decreases with increasing substrate sodium hexametaphosphate (a type of polyphosphate), indicating that the substrate inhibits PPK activity. However, while excessively low substrate concentrations (2 mM) may favor the initial reaction rate, they can also cause premature termination of the product formation process due to substrate depletion (e.g., ...). Figure 3As shown in Figure D, polyP6-2, polyP6-10, polyP6-20, polyP6-40, polyP6-60, polyP6-80, and polyP6-0 represent the concentrations of sodium hexametaphosphate added to the polyphosphate system at 2 mM, 10 mM, 20 mM, 40 mM, 60 mM, 80 mM, and no addition, respectively. In summary, formic acid, glucose, and dextrin can all be used as NADPH supply strategies, and polyphosphates, including sodium hexametaphosphate, can be used as ATP supply strategies. The preferred conditions are: sodium formate 20 mM; glucose 15-60 mM; dextrin 7.5-15 mM; sodium hexametaphosphate 10-60 mM.

[0054] Example 3

[0055] The LATCH pathway was divided into individual modules for testing to verify the function of each step. First, the first module involved a three-step reaction to convert glycolic acid to glyceric acid, incorporating three enzymes: GCS, GCC (including GCC1 and GCC2 subunits, constructed on the same pET28a plasmid, each with a 6*His tag, co-expressed and purified), and TCR, at a concentration of 0.5 mg / mL. The initial substrates included 10 mM glycolic acid, 50 mM sodium bicarbonate, 1 mM coenzyme A, 0.5 mM NADPH, and 2 mM ATP. GCS was derived from two protein expression strains: *E. coli* BL21(DE3) and *E. coli* BL21(DE3) strain with the patZ gene knocked out. Figure 4 As shown in Figure A, the functional detection of the glycolic acid to glyceric acid module is as follows: The left side is a schematic diagram of the reaction process. Glycolic acid is successively converted into glycolic acid, tartrate-co-acid, and glyceric acid under the action of glycolyl-CoA synthase, glycolyl-CoA carboxylase, and tartrate-CoA reductase. In the last step, NADPH is consumed, thereby generating a detection signal. The decrease in absorbance at 340 nm in the curve on the right side indicates a decrease in the NADPH content in the system. Compared with the control group without added GCC enzyme, the decrease slope of both sample groups is greater. Among them, the GCS sample group after patZ knockout has the largest decrease slope, indicating that both sample groups are active, and the enzyme activity and overall module activity after patZ knockout are better.

[0056] Secondly, the second module concerns the conversion of glyceric acid to malic acid. Given that the literature only reports the NADH dependence of malate dehydrogenase from *E. coli*, it is necessary to confirm the enzyme's utilization of NADPH. The system was supplemented with 10 mM phosphoenolpyruvate (PEP), 50 mM sodium bicarbonate, and 0.5 mM NADH or NADPH as coenzymes. The concentration of phosphoenolpyruvate carboxylase was 0.1 mg / mL, and the concentration of malate dehydrogenase was 0.5 μg / mL. Figure 4As shown in Figure B, the activity assay for the phosphoenolpyruvate to malate module is performed: phosphoenolpyruvate is sequentially converted to oxaloacetate and malate by phosphoenolpyruvate carboxylase and malate dehydrogenase, consuming either NADH or NADPH in the final step. The two curves on the right, showing a downward trend, represent the test systems using NADPH and NADH as coenzymes, respectively. When NADH and NADPH are used as coenzymes, the enzyme can consume the coenzymes within 5-10 minutes, demonstrating its good activity. NADH and NADPH represent the control group in which only coenzymes were added, without malate dehydrogenase. Mdh-NADH represents the reaction system containing both NADH and malate dehydrogenase Mdh, and Mdh-NADPH represents the reaction system containing both NADPH and malate dehydrogenase Mdh. "Enzyme" represents the protein component control with a mixture of phosphoenolpyruvate carboxylase and malate dehydrogenase, and "buffer" represents the blank control group containing only buffer. Based on this, a complete Module 2 test was conducted, using 10 mM glyceric acid, 50 mM sodium bicarbonate, and 0.5 mM NADPH as coenzymes. The concentrations of glycerol kinase, enolase, phosphoenolpyruvate carboxylase, and malate dehydrogenase were 0.5 μg / mL, 0.5 μg / mL, 0.1 mg / mL, and 0.5 μg / mL, respectively. Figure 4 As shown in Figure C, the functional assay of the glycerate-to-malate module was performed. Glycerate was sequentially converted to 2-phosphoglycerate, phosphoenolpyruvate, oxaloacetate, and malate by glycerate kinase, enolase, phosphoenolpyruvate carboxylase, and malate dehydrogenase. The final step consumed NADPH, generating a detection signal. Two glycerate kinases (GlyK.Sso and GlyK.Hme) were tested. The two decreasing curves in the right figure indicate that their system activities are similar. The second module exhibited good catalytic function, completely consuming 0.5 mM of coenzyme within half an hour. NADPH represents the control group with only NADPH added to the reaction system. NADPH-GlyK.Sso represents the reaction system containing both NADPH and glycerate kinase from the sulfur-loving acidophilus *Saccharolobus solfataricus*. NADPH-GlyK.Hme represents the reaction system containing both NADPH and glycerate kinase from the denitrifying filamentous microbacterium *Hyphomicrobium*. In denitrificans' glycerokinase, "enzyme" represents a protein component control with added glycerokinase, enolase, phosphoenolpyruvate carboxylase, and malate dehydrogenase, while "buffer" represents a blank control group containing only buffer.

[0057] Finally, the third module focuses on the conversion of malic acid to glycolic acid. Since the conversion of glyoxylic acid to glycolic acid was thoroughly tested in Example 1, this module emphasizes the conversion of malic acid to glyoxylic acid. Due to a general lack of activity data for malic methylcoagulase (MTK), enzymes from two sources reported in the literature—*Methylorubrum extorquens AM1* (Mex) and *Methylococcus capsulatus* (Mca)—were used for testing. The reaction system was maintained at 37°C, with a final volume of 200 μL, containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 2 mM phenylhydrazine, 10 mM malic acid, 2 mM ATP, 1 mM coenzyme A, 50 μg / mL MTK, and 25 μg / mL MCLA (from *Methylorubrum extorquens*). The results are as follows: Figure 4 As shown in Figure D, the activity of the malic acid to glyoxylate module was detected: malic acid is converted to malic acid-CoA by malyl-CoA ligase, and then decomposed into acetyl-CoA and glyoxylate by malyl-CoA lyase. Two malyl-CoA ligases (Mtk.mex and Mtk.mca) were tested. Phenylated hydrazine was added to the buffer solution to a final concentration of 2 mM. The generated glyoxylate reacted with phenylhydrazine to form glyoxylate-phenylhydrazone, which showed an absorption peak at 324 nm. The curve on the right showed that both malyl-CoA ligase-catalyzed systems exhibited an increase in absorbance at 324 nm, indicating that the module possesses [activity]. Both enzymes from different sources exhibited activity, but the enzyme from *Tricholoma mater* was easily precipitated and inactivated, resulting in a rapid decline in activity. Furthermore, the inactivated protein caused an increase in the absorbance of the blank control. In contrast, the malic acid-CoA ligase from *Methanococcus suis* showed superior apparent enzyme activity due to its higher stability (in system D, the Mtk.mca-Mcl.mex system represented by the addition of malic acid-CoA ligase and malic acid-CoA lyase from *Tricholoma mater*, and the Mtk.mex-Mcl.mex system represented by the addition of malic acid-CoA ligase from *Methanococcus suis* and malic acid-CoA lyase from *Tricholoma mater*). In conclusion, the enzyme activities in the LATCH pathway were effectively validated through modular testing.

[0058] Example 4

[0059] The ability of the LATCH pathway to synthesize acetyl-CoA was tested using an in vitro multi-enzyme catalytic system. Samples were prepared using a series of combinations with varying enzyme and substrate addition methods and amounts. The reaction buffer was 50 mM Tris-HCl, MgCl2 concentration was 5 mM, reaction conditions were pH 7.5, 30℃, reaction volume was 200 μL, and reaction time was 6 hours. The bicarbonate concentration was 100 mM, the concentrations of CoA, ATP, and NADPH were all 5 mM, and the glycolic acid concentration was 20 mM. The amounts of glycerol kinase, enolase, malate dehydrogenase, and glyoxylate reductase were all 1 μg / mL.

[0060] Sample 1 system: Phosphoenolpyruvate carboxylase 5 μg / mL, maloyl-CoA ligase 50 μg / mL, maloyl-CoA lyase 25 μg / mL, glycolyl-CoA synthase 0.1 mg / mL, glycolyl-CoA carboxylase 0.1 mg / mL, tartrateyl-CoA reductase 0.1 mg / mL.

[0061] Sample 2 system: phosphoenolpyruvate carboxylase 50 μg / mL, maloyl-CoA ligase 50 μg / mL, maloyl-CoA lyase 25 μg / mL, glycolyl-CoA synthase 0.1 mg / mL, glycolyl-CoA carboxylase 0.1 mg / mL, tartrateyl-CoA reductase 0.1 mg / mL.

[0062] Sample 3 system: phosphoenolpyruvate carboxylase 5 μg / mL, maloyl-CoA ligase 50 μg / mL, maloyl-CoA lyase 25 μg / mL, glycolyl-CoA synthase 0.5 mg / mL, glycolyl-CoA carboxylase 0.5 mg / mL, tartrateyl-CoA reductase 0.5 mg / mL.

[0063] Sample 4 system: phosphoenolpyruvate carboxylase 50 μg / mL, maloyl-CoA ligase 50 μg / mL, maloyl-CoA lyase 25 μg / mL, glycolyl-CoA synthase 0.5 mg / mL, glycolyl-CoA carboxylase 0.5 mg / mL, tartrateyl-CoA reductase 0.5 mg / mL.

[0064] Sample 5 system: phosphoenolpyruvate carboxylase 5 μg / mL, maloyl-CoA ligase 0.5 mg / mL, maloyl-CoA lyase 0.25 mg / mL, glycolyl-CoA synthase 0.1 mg / mL, glycolyl-CoA carboxylase 0.1 mg / mL, tartrateyl-CoA reductase 0.1 mg / mL.

[0065] Sample 6 system: phosphoenolpyruvate carboxylase 50 μg / mL, maloyl-CoA ligase 0.5 mg / mL, maloyl-CoA lyase 0.25 mg / mL, glycolyl-CoA synthase 0.5 mg / mL, glycolyl-CoA carboxylase 0.5 mg / mL, tartrateyl-CoA reductase 0.5 mg / mL.

[0066] In the control group, only GCC enzyme was not added; the concentrations of other enzymes and substrates were the same as in sample 6. The synthesis of acetyl-CoA was as follows: Figure 5 As shown in Figure A, the control group produced almost no acetyl-CoA, while sample 1 produced 0.19 mM of acetyl-CoA. The product concentrations of samples 2 (increased PPC dosage), 3 (increased GCS / GCC / TCR dosage), 4 (increased PPC and GCS / GCC / TCR dosages simultaneously), 5 (increased MTK / MCL dosage), and 6 (increased PPC, MTK / MCL, and GCS / GCC / TCR dosages simultaneously) were all higher than those of sample 1, with acetyl-CoA concentrations of 0.42 mM, 0.58 mM, 0.73 mM, 0.43 mM, and 0.85 mM, respectively. The results of this example demonstrate that the LATCH pathway can achieve the synthesis of acetyl-CoA and its derivatives, such as polyhydroxybutyrate (PHB), in vitro, and that increasing the enzyme dosage in the pathway can effectively promote the synthesis titer of the product.

[0067] Example 5

[0068] The ability of the LATCH pathway to synthesize acetyl-CoA derivative polyhydroxybutyrate (PHB) was evaluated using an in vitro multi-enzyme catalytic system. Experimental samples were prepared using a series of combinations of enzyme and substrate addition methods and amounts. The reaction buffer was 50 mM Tris-HCl, MgCl2 concentration was 5 mM, reaction conditions were pH 7.5, 30℃, the reaction volume was 200 μL, and the reaction time was 6 hours. The bicarbonate concentration was 100 mM, the coenzyme A, ATP, and NADPH concentrations were all 5 mM, and the glycolic acid concentration was 20 mM. The amounts of glycerol kinase, enolase, malate dehydrogenase, and glyoxylate reductase were all 1 μg / mL. Phosphoenolpyruvate carboxylase 50 μg / mL, maloyl-CoA ligase 0.5 mg / mL, maloyl-CoA lyase 0.25 mg / mL, glycolyl-CoA synthase 0.5 mg / mL, glycolyl-CoA carboxylase 0.5 mg / mL, tartrate-CoA reductase 0.5 mg / mL, acetyl-CoA thiolytic enzyme 50 μg / mL, acetyl-CoA reductase 50 μg / mL, polyhydroxybutyrate synthase 0.5 mg / mL.

[0069] Sample 1 system: Contains only the above-mentioned general components. Sample 2 system: Based on Sample 1, supplemented with 10 mM sodium hexametaphosphate, 50 μg / mL polyphosphokinase, and 10 μg / mL each of adenosine kinase and pyrophosphate hydrolase. Sample 3 system: Based on Sample 2, supplemented with 20 mM sodium formate and 10 μg / mL formate dehydrogenase. Sample 4 system: Based on Sample 2, supplemented with 20 mM glucose, and 1 μg / mL each of glucokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconide lactonease, and 6-phosphogluconide dehydrogenase. Sample 5 system: Based on Sample 2, supplemented with 10 mM dextrin, 50 μg / mL α-glucan phosphorylase, and 1 μg / mL phosphogluconomutase.

[0070] Control group 1: Compared to sample 1, it lacked only polyhydroxybutyrate synthase. Control group 2: Compared to sample 1, it lacked only glycolyl-CoA carboxylase. Results are as follows... Figure 5 As shown in Figure B, no poly-3-hydroxybutyric acid (3HB) was detected in the treated control group samples, while the concentration of 3HB products from the sample combinations ranged from 0.2 to 0.6 mM. Specifically, Sample 2, using sodium hexametaphosphate as the ATP regeneration method, saw its product concentration increase from 0.21 mM in Sample 1 to 0.37 mM, an increase of 76%. In Samples 3 and 5, which used formic acid and dextrin to regenerate NADPH, the 3HB product concentrations were 0.43 mM and 0.48 mM, respectively, representing increases of 16% and 30% compared to Sample 2. In contrast, Sample 4, using glucose as the NADPH regeneration strategy, had the highest product concentration at 0.56 mM, an increase of 51% compared to Sample 2.

[0071] Example 6

[0072] Enolase, phosphoenolpyruvate carboxylase, and malate dehydrogenase are endogenous genes of the strain and do not require integration. Heterologous genes involved in the LATCH pathway, including glycerol kinase (NCBI accession number AFK19321), mayl-CoA ligase (NCBI accession numbers AAU91976 and AAU91975 for its two subunits), mayl-CoA lyase (NCBI accession number ABY30201), glyoxylate reductase (NCBI accession number AQS85295), and glycolyl-CoA synthase (whose amino acid sequence contains V379A in the wild-type amino acid sequence), were integrated. Mutations were integrated into the genome of *Escherichia coli* K-12 MG1655, including wild-type amino acid sequence accession number AKQ42064, glycolyl-CoA carboxylase (with an L100S-Y143H-D407I-I450V-W502R mutation relative to wild-type subunit I amino acid sequence; wild-type subunit I amino acid sequence NCBI accession number ABY28706; subunit II amino acid sequence NCBI accession number ABY31385), and tartrateyl-CoA reductase (NCBI accession number ABY35820). All genes used strong Trc promoters. Simultaneously, the endogenous carbonic anhydrase *Can* (NCBI accession number NP_414668) was overexpressed using the pBBR1k plasmid carrying the kanamycin resistance gene. Control strain 1 was the wild-type MG1655 strain, transformed with the empty pBBR1k plasmid, designated MG1655_pBBR1k; Sample 1 strain was the wild-type MG1655 strain, transformed with the pBBR1k plasmid integrating the endogenous carbonic anhydrase gene, designated MG1655_Can. Sample 2 strain was a strain integrating the LATCH pathway, transformed with the empty pBBR1k plasmid, designated MG1655_LATCH; Sample 3 strain was a strain integrating the LATCH pathway, transformed with the pBBR1k plasmid carrying the endogenous carbonic anhydrase, designated MG1655_LATCH_Can.

[0073] The above-mentioned strains were seed cultured in LB medium. After 12 hours, they were transferred at a 0.5% inoculum to M9 medium (without glucose) supplemented with 0.2% yeast extract for further culture and testing. The kanamycin concentration in both LB and M9 media was 50 μg / mL, pH 7.5. Tests were performed using 48-well plates, with 1 mL of solution per well, in triplicate. Culture and OD... 600 Real-time monitoring was conducted in a Jeling growth tester with an oscillation rate of 800 rpm and a temperature of 30°C.

[0074] The results are as follows Figure 5As shown in Figure C, none of the four strains exhibited a distinct delayed phase, and the slopes of their logarithmic growth curves were similar, indicating little difference in doubling time. This suggests that integration of the LATCH pathway and overexpression of carbonic anhydrase did not significantly affect the growth rate of the strains. The maximum biomass at the stationary phase (in OD0.05) was [data missing]. 600 From a characterization perspective, strains that overexpressed carbonic anhydrase alone and strains that integrated the LATCH pathway gene alone were superior to the wild-type control strain, while strains that overexpressed both carbonic anhydrase and integrated the LATCH pathway gene performed best. This indicates that both overexpression of carbonic anhydrase and integration of the LATCH pathway gene can increase the biomass of the bacteria during the stationary phase, and the biomass is further increased when the two factors are combined.

[0075] like Figure 5 As shown in Figure D, biomass peak data (22 hours) during the stationary phase of four strains were selected for comparison. The OD values ​​were: control strain MG1655_pBBR1k (a control strain carrying the pBBRk empty plasmid) and three sample strains: MG1655_Can (a strain overexpressing the carbonic anhydrase gene using the pBBR1k plasmid), MG1655_LATCH (a strain with a heterologous gene integrated into its genome for the LATCH pathway), and MG1655_LATCH_Can (a strain with a heterologous gene integrated into its genome for the LATCH pathway and overexpressing the carbonic anhydrase gene using the pBBR1k plasmid). 600 The values ​​were 0.38, 0.45, 0.48, and 0.57, respectively. The maximum biomass during the stationary phase of the three sample strains was increased by 18%, 26%, and 50% compared to the control strain, respectively.

[0076] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for artificial carbon dioxide fixation, characterized in that, An artificial carbon dioxide fixation metabolic pathway, LATCH, was constructed. The LATCH pathway is an autocatalytic cycling pathway consisting of 10 enzymatic steps. It integrates the serine cycle and the TaCo module through glyoxylate reductase. Each cycle can convert carbon dioxide or bicarbonate into acetyl-CoA.

2. The method according to claim 1, characterized in that, The enzymes involved in the LATCH pathway include glycerol kinase, enolase, phosphoenolpyruvate carboxylase, malate dehydrogenase, mayl-CoA ligase, mayl-CoA lyase, glyoxylate reductase, alcoholyl-CoA synthase, alcoholyl-CoA carboxylase, and tartrate-CoA reductase.

3. The method according to claim 2, characterized in that, The glycerokinase is derived from denitrifying filamentous microbes or thermophilic acidophilic bacteria, and its amino acid sequences have NCBI accession numbers AFK19321 and AAK40970, respectively. The enolase, phosphoenolpyruvate carboxylase, and malate dehydrogenase are derived from Escherichia coli, and their amino acid sequences have NCBI accession numbers NP_417259, NP_418391, and NP_417703, respectively. The malico-CoA ligase is derived from *Tricholoma materans* or *Methanococcus capsulatum*. The malico-CoA ligase is a two-subunit enzyme. When derived from *Tricholoma materans*, the NCBI accession numbers for the amino acid sequences of the two subunits are ABY30198 and ABY30199, respectively. When derived from *Methanococcus capsulatum*, the NCBI accession numbers for the amino acid sequences of the two subunits are AAU91976 and AAU91975, respectively. The NCBI accession number for the amino acid sequence of the malic acyl-CoA lyase is ABY30201. The glyoxylate reductase is derived from Acetic Acid Bacillus, and its amino acid sequence has the NCBI accession number AQS85295. The glycolyl-CoA synthase is derived from a strain of Rhodobulbus. The amino acid sequence of the glycolyl-CoA synthase has a mutation at amino acid position 379 compared to the wild-type amino acid sequence. Amino acid position 379 is mutated to A. The NCBI accession number of the wild-type amino acid sequence of the glycolyl-CoA synthase is AKQ42064. The glycolyl-CoA carboxylase is derived from *Thunb.*, and is a bisubunit enzyme consisting of glycolyl-CoA carboxylase subunit I and glycolyl-CoA carboxylase subunit II. The glycolyl-CoA carboxylase exhibits mutations at the following sites relative to wild-type glycolyl-CoA carboxylase: positions 100, 143, 407, 450, and / or 50. Preferably, the 100th position is mutated to S, the 143rd position is mutated to H, the 407th position is mutated to I, the 450th position is mutated to V, and the 502nd position is mutated to R. The INCBI accession number for the two subunits of the wild-type glycolyl-CoA carboxylase is ABY28706, and the INCBI accession number for the amino acid sequence of glycolyl-CoA carboxylase subunit II is ABY31385. The tartrate-CoA reductase is derived from *Hyperhygrophytes thermophila*, and its amino acid sequence has the NCBI accession number ABY35820.

4. The method according to claim 1, characterized in that, Each cycle converts 2 molecules of carbon dioxide or bicarbonate into 1 molecule of acetyl-CoA.

5. The method according to claim 1, characterized in that, The LATCH pathway relies on ATP and / or NADPH for energy and reducing power. The substance that provides ATP is polyphosphate, and the substance that provides NADPH is selected from formic acid, glucose, or dextrin.

6. The method according to claim 5, characterized in that, The polyphosphate is sodium hexametaphosphate, with an addition concentration of 2-80 mM; the formic acid is sodium formate, with an addition concentration of 8-25 mM; the glucose addition concentration is 10-70 mM; and the dextrin addition amount is 5-35 mM. Preferably, the sodium hexametaphosphate addition concentration is 10-60 mM; the sodium formate addition concentration is 10-20 mM; the glucose addition concentration is 15-60 mM; and the dextrin addition amount is 7.5-15 mM.

7. A recombinant microorganism, characterized in that, The LATCH pathway is constructed using the method described in any one of claims 1-6, and the gene encoded by the enzyme involved in the LATCH pathway is integrated into a host cell. Preferably, the host cell is Escherichia coli, Bacillus, yeast, or plant cell.

8. The recombinant microorganism according to claim 7, characterized in that, It also contains a plasmid overexpressing a carbonic anhydrase encoding gene, the amino acid sequence of which has the NCBI accession number NP_414668.

9. The application of the method according to any one of claims 1-6 in the synthesis of acetyl-CoA and its derivatives, wherein the derivatives are selected from polyhydroxyalkanoates, biofuels, sugars, amino acids, organic acids and biomass, preferably, the derivatives are polyhydroxybutyric acid.

10. The application according to claim 9, characterized in that, The application can be synthesized in vitro or in vivo by microorganisms.

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