Novel carbon immobilization pathway

Abstract

An engineered pathway for carbon immobilization without the use of ribulose diphosphate carboxylase-oxygenase incorporates carbon dioxide into fructose-6-phosphate.

Description

[0001] This invention was made with government support from the Department of Energy (Grant No. DE-FOA-0001540) and the National Science Foundation (Grant Nos. 1437775 and 1442724). The government has certain rights in this invention.

[0002] Sequence Listing

[0003] The sequence listing associated with this application is provided in XML format, not a paper copy, and is therefore incorporated into this specification by reference. The name of the XML file containing the sequence listing is B19-092-2WO.xml. The XML file is approximately 87219KB, was created on April 30, 2023, and was submitted electronically through EFS-Web.

[0004] introduction

[0005] Over 90% of biological carbon fixation is thought to occur via the Calvin Benson cycle, including in all plants, cyanobacteria, and algae. The enzyme ribulose bisphosphate carboxylase-oxygenase, which catalyzes the first step of the Calvin Benson cycle, is the rate-limiting step because it is a slow catalyst compared to the rest of the pathway. Additionally, ribulose bisphosphate carboxylase-oxygenase tends to make costly metabolic errors when using oxygen, which worsens with higher temperatures. Efforts have been made to create synthetic pathways for fixing carbon dioxide. Schwander et al., Science. 2016 Nov 18;354(6314):900-904. SUMMARY OF THE INVENTION

[0007] We have designed new synthetic carbon fixation pathways that use much better CO2 fixing enzymes (technically H2CO3 fixing enzymes, but CO2 is easily interconverted with H2CO3 using carbonic anhydrases) and can therefore increase the yield of carbon currently fixed using the Calvin Benson cycle in any organism, and in particular increase the rate at which plants and other autotrophic organisms can fix carbon. Specific applications include increasing plant yields, reducing water use per unit of yield, and reducing nitrogen fertilizer use per unit of yield.

[0008] The present invention provides methods, systems and compositions for carbon fixation without the use of ribulose bisphosphate carboxylase-oxygenase, including recombinant enzymes / pathways for incorporating carbon dioxide / bicarbonate into fructose-6-phosphate.

[0009] In one aspect, the invention provides a system for carbon fixation without the use of ribulose bisphosphate carboxylase-oxygenase, comprising an engineered pathway configured to incorporate bicarbonate into fructose-6-phosphate, and comprising a recombinant enzyme:

[0010] i) Hps: ketohexose-6-phosphate synthase,

[0011] ii) Phi: ketohexose-6-phosphate isomerase,

[0012] iv) AspC: aspartate aminotransferase,

[0013] v) RpiA: ribose-5-phosphate isomerase,

[0014] vi) YjhH: YjhH 2-keto-3-deoxy-galactonate aldolase (which also performs the 4-hydroxy-2-oxobutyrate aldolase reaction),

[0015] vii) Ppc: phosphoenolpyruvate carboxylase,

[0016] viii) Asd: aspartate-semialdehyde dehydrogenase, and

[0017] ix) ThrA_S345F: fused aspartate kinase / homoserine dehydrogenase.

[0018] In the implementation:

[0019] The enzyme comprises:

[0020] i) bmHps: Bacillus methanolicus ketohexose-6-phosphate synthase,

[0021] ii) bmPhi: Bacillus methanolicus hexulose-6-phosphate isomerase,

[0022] iv) ecAspC: Escherichia coli aspartate aminotransferase,

[0023] v) ecRpiA: Escherichia coli ribose-5-phosphate isomerase,

[0024] vi) ecYjhH: Escherichia coli YjhH 2-keto-3-deoxy-galactonate aldolase (which also performs 4-hydroxy-2-oxobutyrate aldolase reaction),

[0025] vii) ecPpc: Escherichia coli phosphoenolpyruvate carboxylase,

[0026] viii) ecAsd: Escherichia coli aspartate-semialdehyde dehydrogenase,

[0027] ix) ecThrA_S345F: E. coli fused aspartate kinase / homoserine dehydrogenase with point mutation S345F;

[0028] The system is also configured to convert carbon dioxide to bicarbonate (CO2 to H2CO3), and comprises enzymes: carbonic anhydrase;

[0029] The system is also configured to convert fructose-6-phosphate into mannitol-1-phosphate and comprises the enzymes: iii) MtlD: mannitol-1-phosphate 5-dehydrogenase;

[0030] The system is integrated into the cell;

[0031] The system is integrated into plant cells such as crop plant cells, such as rice, corn, soybean, wheat, rapeseed, sugarcane, cotton, loblolly pine, etc.;

[0032] The system is integrated into microbial cells such as cyanobacteria and algae (microalgae and macroalgae); and / or

[0033] The system is integrated into a cell-free system.

[0034] In various aspects, the invention provides methods of carbon fixation without the use of ribulose bisphosphate carboxylase-oxygenase, comprising providing carbon dioxide or bicarbonate to a system herein, wherein the system incorporates the carbon dioxide or bicarbonate into fructose-6-phosphate.

[0035] In various aspects, the invention provides a method herein comprising further detecting carbon fixation by detecting the resulting decrease in carbon dioxide or bicarbonate, or the resulting increase in fructose-6-phosphate or a reaction product thereof.

[0036] In various aspects, the invention provides a method for producing food (grains, cereals, etc.) or for biomass (fiber, pyrolysis to fuel, etc.), or for sequestering CO2 from air.

[0037] The present invention encompasses all combinations of specific embodiments described herein, as if each combination had been expressly described. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Pathway for incorporation of bicarbonate into mannitol-1-phosphate. bmHps: Bacillus methanolica hexulose-6-phosphate synthase, bmPhi: Bacillus methanolica hexulose-6-phosphate isomerase, ecMtlD: Escherichia coli mannitol-1-phosphate 5-dehydrogenase, ecAspC: Escherichia coli aspartate aminotransferase, ecRpiA: Escherichia coli ribose-5-phosphate isomerase, ecYjhH: Escherichia coli YjhH 2-keto-3-deoxy-galactonate aldolase (which also performs 4-hydroxy-2-oxobutyrate aldolase reaction), ecPpc: Escherichia coli phosphoenolpyruvate carboxylase, ecAsd: Escherichia coli aspartate-semialdehyde dehydrogenase, ecThrA_S345F: Escherichia coli fused aspartate kinase / homoserine dehydrogenase with point mutation S345F.

[0040] Figure 2 . SDS-PAGE gel showing the purity of the purified enzyme.

[0041] Figure 3 . LCMS-TOF data from counted mannitol-1-phosphate accumulation assays of quenched and filtered assay samples are shown. Each condition was run in triplicate and the error bars represent standard deviation.

[0042] Figure 4 .Standard curve of store-bought mannitol-1-phosphate prepared in 50% methanol / 50% ddH2O (vol / vol) and run directly on the LCMS-TOF. The standard curve was not filtered or diluted in quench buffer like the mannitol-1-phosphate accumulation assay samples. These samples were run using the same LCMS-TOF method as the mannitol-1-phosphate accumulation assay. The 0.0390625 μM and 0.078125 μM mannitol-1-phosphate samples are not shown in the graph because they are below the detection limit of the instrument.

[0043] Description of Specific Embodiments of the Invention

[0044] Unless otherwise prohibited or indicated, in these descriptions and throughout the specification, the terms "a" and "an" mean one or more, and the term "or" means and / or. It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and various modifications or changes thereto will be suggested to those skilled in the art, and these modifications or changes will be included in the spirit and scope of the present application and the scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes.

[0045] Example: Incorporation of bicarbonate / carbon dioxide into fructose-6-phosphate and mannitol-1-phosphate

[0046] E. coli cell line BL21DE3* was used for protein expression. E. coli cell line XL1-Blue was used for routine plasmid cloning and storage.

[0047] Carbenicillin disodium salt and kanamycin monosulfate antibiotics were used at final concentrations of 100 μg / mL and 50 μg / mL, respectively, in various growth media.

[0048] LB Lennox medium, also known as LB medium or LB, recipe:

[0049] 10 g tryptone, 5 g sodium chloride, 5 g yeast extract. Dissolve in distilled water to a final volume of 1 L. Autoclave for 45 minutes (121°C, 15 psi) to ensure sterility.

[0050] TB medium, also called TB: 24 g yeast extract, 20 g tryptone, 4 mL glycerol, 9.4 g potassium phosphate dibasic, 2.2 g potassium phosphate monobasic. Dissolve in distilled water to a final volume of 1 L. Autoclave for 45 minutes (121° C., 15 psi) to ensure sterility.

[0051] TSS formulation: 10 g polyethylene glycol 3350, 5 mL dimethyl sulfoxide, 2 mL 1 molar magnesium chloride. Dissolve in LB to a final volume of 100 mL. Sterile filter with a .22 μm filter and store at 4°C.

[0052] 2x KCM composition: 0.06 M potassium chloride, 0.2 M calcium chloride, and 0.1 M magnesium chloride in distilled water. 2x KCM should be sterile filtered through a .22 μm filter prior to use.

[0053] Sodium phosphate lysis buffer composition: 50 mM sodium phosphate buffer at pH 7.2 (approximately 8.925 g of disodium hydrogen phosphate heptahydrate and 2.305 g of sodium dihydrogen phosphate monohydrate dissolved in 1 L of distilled water), 1 mg / mL lysozyme, .1 mg / mL deoxyribonuclease, 20 mM imidazole, 1 mM β-mercaptoethanol (BME), 1 mM magnesium sulfate, 20 μM zinc sulfate, 20 μM manganese sulfate, 20 μM pyridoxal phosphate (PLP). As with all buffers, adjust the pH with sodium hydroxide or hydrochloric acid when necessary.

[0054] Sodium phosphate wash buffer composition: 50 mM sodium phosphate buffer at pH 7.2, 20 mM imidazole, 1 mM BME, 1 mM magnesium sulfate, 20 μM zinc sulfate, 20 μM manganese sulfate, 20 μM PLP.

[0055] Sodium phosphate elution buffer composition: 50 mM sodium phosphate buffer at pH 7.2, 200 mM imidazole, 1 mM BME, 1 mM magnesium sulfate, 20 μM zinc sulfate, 20 μM manganese sulfate, 20 μM PLP.

[0056] The sodium phosphate dialysis buffer composition was: 50 mM sodium phosphate buffer at pH 7.2, 1 mM tris(2-carboxyethyl)phosphine (TCEP), 1 mM magnesium sulfate, 20 μM zinc sulfate, 20 μM manganese sulfate, and 20 μM PLP.

[0057] Chemically competent cell preparation protocol: The desired E. coli strain from a previously stored -80°C glycerol stock is used to inoculate 10 mL LB in a glass culture tube. The culture is then allowed to grow overnight at 37°C with shaking at 200 RPM, approximately 16 hours. 500 μL of culture is then used to inoculate 50 mL of fresh LB medium in a 250 mL baffled flask. The cells are grown at 37°C with shaking at 200 RPM to an OD600 of .35 (approximately 3 hours of growth). The cells are then transferred to two 50 mL conical tubes (25 mL culture each) and incubated on ice for 20 minutes. The culture is then centrifuged at 8000 RCF for 8 minutes at 4°C. The supernatant is discarded from each conical tube. The supernatant is removed by gently vortexing (at 4 °C). Standard Heavy Duty Vortex Mixer ( The first cell pellet was resuspended in 5 ml of 4°C TSS using a Standard Heavy-Duty VortexMixer at about 30% power. The second cell pellet was then resuspended in the same manner using 5 mL of the resuspended cells. 100 μL of the resuspended cells were then aliquoted into .6 mL snap-top tubes, yielding approximately 50 tubes of chemically competent cells. The chemically competent cells were either snap frozen in liquid nitrogen and stored at -80°C until use, or used immediately for plasmid transformation.

[0058] Heat shock protocol: Thaw chemically competent cells if necessary. Add 1 μL of the desired plasmid at a concentration of about 50 ng / μL to 100 μL of chemically competent cells. Then add 100 μL of 2x KCM to the cells. Allow the cells to incubate on ice for 20 minutes. Then heat shock the cells at 42°C for 90 seconds and then incubate on ice for 1 minute. Then allow the cells to recover at 37°C (without shaking) for one hour. Then spread 200 μL of cells on LB agar plates with appropriate antibiotics to select the plasmid to be transformed. The plates are incubated at 37°C overnight (about 16 hours) until colonies have formed.

[0059] Enzyme expression in E. coli BL21DE3*: A single E. coli BL21DE3* colony containing the desired plasmid was picked into 10 mL LB medium with appropriate antibiotics in a glass culture tube and grown overnight (about 16 hours) at 37°C with shaking at 200RPM. Then 500 μL of the culture was used to inoculate 50 mL TB medium in a 250 mL baffled flask. The culture was then grown at 37°C at 200RPM until an OD600 of .8 was reached. The culture was then induced by adding IPTG (isopropyl β-D-1-thiogalactoside) at a final concentration of 1 mM. The cells were then incubated at 18°C ​​with shaking at 200RPM for 24 hours. Then 40 mL of the culture was transferred to a 50 mL conical tube and centrifuged at 8000RCF for 10 minutes at 4°C. The supernatant was discarded. The cell pellet was either stored at -20°C until use or used immediately for enzyme purification.

[0060] Enzyme purification by immobilized metal affinity chromatography: All enzyme purification steps were performed in a cold room at 4°C. If necessary, the cell pellet was thawed on ice from -20°C to 4°C. The cell pellet was then resuspended in 5 mL of sodium phosphate lysis buffer pre-cooled to 4°C by vortexing. The 50 mL conical tube containing the resuspended cells was then placed in an ice bucket at 4°C and sonicated using a QSonica Q500 sonicator. The sonication program cycled between 25% maximum amplitude for 5 seconds and 0% maximum amplitude for 10 seconds, with a total cycle time of 3 minutes (1 minute "working time"). The lysed culture was then centrifuged at 10,000 RCF for 20 minutes at 4°C. While waiting for the centrifugation to be completed, a gravity column containing 1 mL of Ni-NTA agarose beads was equilibrated with 20 column volumes of sodium phosphate wash buffer pre-cooled to 4°C, making it possible to perform immobilized metal affinity chromatography on proteins with polyhistidine tags. After the centrifugation had been completed, the clarified supernatant was applied to the Ni-NTA column. 40 column volumes of sodium phosphate wash buffer pre-cooled to 4° C. were then applied to each column. The enzyme was eluted into a 10K MWCO spin concentrator using 15 column volumes of sodium phosphate elution buffer also pre-cooled to 4° C.

[0061] Purified enzyme concentration and dialysis: The enzyme in the 10K MWCO spin concentrator was centrifuged at 4°C at the maximum speed recommended by the manufacturer until the volume of the enzyme was concentrated to less than 800 μL. The concentrated enzyme was transferred to a dialysis cassette with a 3.5K MWCO membrane and placed in a sodium phosphate dialysis buffer prepared at 4°C and allowed to dialyze overnight (at least 8 hours) in a cold room at 4°C. The stirring bar ensured constant but slow mixing of the dialysis buffer at about 60RPM. Up to 8 enzyme dialysis cassettes were dialyzed using 1L sodium phosphate dialysis buffer. The enzyme cassette was then dialyzed a second time in fresh sodium phosphate dialysis buffer, as described above, but only for 4 hours. The enzyme was then transferred to a 1.7mL microcentrifuge tube on ice and checked for any precipitation. Any enzyme with precipitation in the sample was spun at 14000RCF for 2 minutes at 4°C to precipitate any insoluble material. The clarified supernatant was then transferred to a new microcentrifuge tube. At this point, we believe that the enzymes are completely purified. The concentration of each enzyme was measured by measuring the absorbance at 280 nm using a NanoDrop 1000 spectrophotometer. Enzymes were either used immediately or glycerol was added to a final concentration of 20% (v / v) before the enzymes were snap frozen in liquid nitrogen and stored at -80°C until use.

[0062] Enzyme purity is also checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A brief gel electrophoresis scheme is as follows. 10 μL of purified and dialyzed enzymes are mixed with 2 μL of SDS-PAGE sample loading buffer 6X of Cepham Life Sciences in a PCR tube. The enzyme and loading buffer mixture are then boiled at 95°C for 10 minutes in a PCR block. After cooling to room temperature, 10 μL of boiled enzyme and loading buffer mixture are applied to a 4-20% small protein TGX precast protein gel, which has been properly inserted into a small protein Tetra cell. The gel is run at a constant 200V until the running buffer dye leaves the bottom of the gel (approximately 25 minutes). Then by placing the gel in a tray, adding approximately 100ml ddH2O completely submerges the gel, allowing it to be gently shaken on a gel shaker for 5 minutes, using ddH2O water to wash the gel. The used ddH2O is then discarded. This washing step is repeated twice, for a total of three washes. The gel was then stained in 100 mL GelCode (manufactured by ThermoScientific) for 1 hour while gently shaking. Thereafter, the GelCode was removed and the gel was briefly washed three times with 100 mL ddH2O (without shaking, only ddH2O was added and then removed). The gel was then destained in 100 mL ddH2O with gentle shaking overnight (about 16 hours) and a Kimwipe was added. The gel was then imaged.

[0063] Incorporation of bicarbonate into mannitol-1-phosphate: Reaction solution A consists of: 3 mM reduced nicotinamide adenine dinucleotide phosphate (NADPH), 3 mM reduced nicotinamide adenine dinucleotide (NADH), 4 mM phosphoenolpyruvate (PEP), 1 mM sodium bicarbonate, 2 mM adenosine triphosphate (ATP), 2 mM ribose-5-phosphate, 2 mM α-ketoglutarate, 2 mM glutamate, 1 mM acetyl coenzyme A (acetyl-CoA), 2 mM guanosine triphosphate (GTP), 50 mM sodium phosphate buffer at pH 7.2 (about 8.925 g of disodium hydrogen phosphate heptahydrate and 2.305 g of sodium dihydrogen phosphate monohydrate dissolved in 1 L of distilled water), 1 mM TCEP, 1 mM magnesium sulfate, 20 μM zinc sulfate, 20 μM manganese sulfate, 20 μM PLP. The pH value is adjusted with sodium hydroxide or hydrochloric acid as needed.

[0064] Reaction solution B consisted of: 0.8215 mg / mL bmHps, .2495 mg / mL bmPhi, .18 mg / mL ecMtlD, .5695 mg / mL ecAspC, .214 mg / mL ecRpiA, .1155 mg / mL ecYjhH, .1615 mg / mL ecPpc, .2825 mg / mL ecAsd, .1345 mg / mL ecThrA_S345F, 50 mM sodium phosphate buffer at pH 7.2 (approximately 8.925 g of disodium hydrogen phosphate heptahydrate and 2.305 g of sodium dihydrogen phosphate monohydrate dissolved in 1 L of distilled water), 1 mM TCEP, 1 mM magnesium sulfate, 20 μM zinc sulfate, 20 μM manganese sulfate, 20 μM PLP. The pH was adjusted with sodium hydroxide or hydrochloric acid as needed.

[0065] Reaction solution C consisted of: 0.8215 mg / mL bmHps, .2495 mg / mL bmPhi, .18 mg / mL ecMtlD, .5695 mg / mL ecAspC, .214 mg / mL ecRpiA, .1155 mg / mL ecYjhH, 0.1615 mg / mL ecPpc previously boiled at 95°C for 10 minutes, .2825 mg / mL ecAsd, .1345 mg / mL ecThrA_S345F, 50 mM sodium phosphate buffer at pH 7.2 (approximately 8.925 g of disodium hydrogen phosphate heptahydrate and 2.305 g of sodium dihydrogen phosphate monohydrate dissolved in 1 L of distilled water), 1 mM TCEP, 1 mM magnesium sulfate, 20 μM zinc sulfate, 20 μM manganese sulfate, 20 μM PLP. The pH was adjusted with sodium hydroxide or hydrochloric acid as needed.

[0066] Quenching buffer: 50% methanol, 50% deionized distilled water (ddH2O) (vol / vol)

[0067] Mannitol-1-phosphate accumulation assay procedure: 50 μL reaction buffer A was combined with 50 μL reaction solution B to start the test condition reaction. 50 μL reaction buffer A was combined with 50 μL reaction solution C to start the negative control reaction. Each of these reactions was performed three times. The reaction was stopped by taking 6.6 μL of the desired reaction mixture and mixing it with 144 μL quenching buffer, taking time points at 0 minutes (immediately after combining reaction solution A and reaction solution B, or reaction solution A and reaction solution C), 20 minutes, 40 minutes, 1 hour, 2 hours, 4 hours and 6 hours. The quenched samples were stored at -20°C until all time points were collected. The quenched reaction samples were then centrifuged through a 96-well PallLaboratory AcroPrep Omega Membrane 10K MWCO filter plate to remove the enzyme at 4°C with the maximum recommended relative centrifugal force (RCF) of the manufacture of the swing bucket rotor. Centrifugation was stopped periodically to check and see if the flow through was sufficient to meet the minimum volume required for the LCMS vial to properly facilitate LCMS-TOF injection. Once all samples had a volume of at least twice the minimum volume, the flow through was transferred to an LCMS vial and stored at -80°C until analysis by LCMS-TOF.

[0068] Analytical methods: We used liquid chromatography-time of flight mass spectrometry (LCMS-TOF) as our analytical method on the Agilent 6545. We used the method of Baidoo, Edward EK et al. "Liquid chromatography and mass spectrometry analysis ofisoprenoid intermediates in Escherichia coli." Microbial Metabolomics. Humana Press, New York, NY, 2019. 209-224, following the variants listed in Note 6 of that publication. The LCMS-TOF was set to detect m / z from 70 to 1100. Mannitol-1-phosphate with an m / z of 261.0381 could be detected.

[0069] Store purchased mannitol-1-phosphate was used as an analytical standard to create a standard curve to verify the retention time and mass-to-charge ratio (m / z) of mannitol-1-phosphate and to verify the detection limit of our analytical instrument, method, and target compound. The standard curve was constructed in 50% methanol, 50% water (vol / vol) buffer. The standard curve concentrations of mannitol-1-phosphate were 0.0390625 μM, 0.078125 μM, 0.15625 μM, 0.3125 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, 20 μM. 0.0390625 μM and 0.078125 μM were below the detection limit.

[0070] Construction of recombinant pathways in cyanobacteria. A disclosed system for carbon fixation was introduced into cyanobacteria (Synezocystis sp. PCC 6803) using natural transformation to provide stable expression of recombinant enzymes, including an engineered pathway configured to incorporate bicarbonate into fructose-6-phosphate: (i) Hps: hexulose-6-phosphate synthase, (ii) Phi: hexulose-6-phosphate isomerase, (iv) AspC: aspartate aminotransferase, (v) RpiA: ribose-5-phosphate isomerase, (vi) YjhH: 2-keto-3-deoxy-galactonate aldolase, (vii) Ppc: phosphoenolpyruvate carboxylase, (viii) Asd: aspartate-semialdehyde dehydrogenase, and (ix) ThrA_S345F: fused aspartate kinase / homoserine dehydrogenase. These cells similarly demonstrated bicarbonate / carbon dioxide incorporation into fructose-6-phosphate and mannitol-1-phosphate without the use of ribulose bisphosphate carboxylase-oxygenase.

Claims

1. A system for carbon fixation without the use of ribulose bisphosphate carboxylase-oxygenase, comprising an engineered pathway configured to incorporate bicarbonate into fructose-6-phosphate, and comprising a recombinant enzyme: i) Hps: ketohexose-6-phosphate synthase, ii) Phi: ketohexose-6-phosphate isomerase, iv) AspC: aspartate aminotransferase, v) RpiA: ribose-5-phosphate isomerase, vi) YjhH: 2-keto-3-deoxy-galactonate aldolase (which also performs the 4-hydroxy-2-oxobutyrate aldolase reaction), vii) Ppc: phosphoenolpyruvate carboxylase, viii) Asd: aspartate-semialdehyde dehydrogenase, and ix) ThrA_S345F: fused aspartate kinase / homoserine dehydrogenase.

2. The system of claim 1, wherein the enzyme comprises: i) bmHps: Bacillus methanolicus hexulose-6-phosphate synthase, ii) bmPhi: Bacillus methanolicus hexose-6-phosphate isomerase, iv) ecAspC: Escherichia coli aspartate aminotransferase, v) ecRpiA: Escherichia coli ribose-5-phosphate isomerase, vi) ecYjhH: Escherichia coli 2-keto-3-deoxy-galactonate aldolase (which also performs 4-hydroxy-2-oxobutyrate aldolase reaction), vii) ecPpc: Escherichia coli phosphoenolpyruvate carboxylase, viii) ecAsd: Escherichia coli aspartate-semialdehyde dehydrogenase, ix) ecThrA_S345F: E. coli fused aspartate kinase / homoserine dehydrogenase with point mutation S345F.

3. The system of claim 1, further configured to convert carbon dioxide to bicarbonate (CO2 to H2CO3) and comprising the enzyme: carbonic anhydrase.

4. The system of claim 1, further configured to convert fructose-6-phosphate to mannitol-1-phosphate, and comprising the enzyme: iii) MtlD: Mannitol-1-phosphate 5-dehydrogenase.

5. The system of claim 1, which is integrated into a cell.

6. The system of claim 1, which is integrated into engineered plant cells such as crop plant cells, such as rice, corn, soybean, wheat, rapeseed, sugarcane, cotton, loblolly pine, etc.

7. The system of claim 1, which is integrated into engineered microbial cells such as cyanobacteria and algae (microalgae and macroalgae).

8. The system of claim 1, which is in a cell-free system.

9. An engineered cell comprising the system of claim 1.

10. A method of carbon fixation without using ribulose bisphosphate carboxylase-oxygenase, comprising providing carbon dioxide or bicarbonate to the system of claim 1, wherein the system incorporates the carbon dioxide or bicarbonate into fructose-6-phosphate.

11. The method of claim 10, comprising further detecting carbon fixation by detecting the resulting decrease in carbon dioxide or bicarbonate, or the resulting increase in fructose-6-phosphate or a reaction product thereof.

12. The method as claimed in claim 11 for use in the production of food (cereals, grains, etc.) or for biomass (fibers, pyrolysis to fuel, etc.), or for sequestering CO2 from air.

Images