[001] This invention was made with government support under the Department of

Energy, grant number DE-FOA-0001540, and the National Science Foundation, grant numbers 1437775 and 1442724. The government has certain rights in the invention.

[002] Sequence Listing

[003] The Sequence Listing associated with this application is provided in XML format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing is B19-092-2WO.xml. The XML file is about 87,219 KB, was created on Apr 30, 2023, and is being submitted electronically via EFS-Web.

[004] Introduction

[005] Over 90% of biological carbon fixation is thought to occur through the Calvin Benson Cycle, including in all plants, cyanobacteria and algae. The enzyme Rubisco, which catalyzes the first step of the Calvin Benson Cycle, is the rate limiting step as it is a slow catalyst compared to the rest of the pathway. In addition, Rubisco tends to make costly metabolic mistakes with oxygen, which worsens with higher temperatures. Efforts have been made to create a synthetic pathway for fixation of carbon dioxide, Schwander et al, Science. 2016 Nov 18; 354(6314): 900-904.

[006] Summary of the Invention

[007] We have designed a novel, synthetic carbon fixation pathway that uses a much better CO2 fixing enzyme (technically, a H2CO3 fixing enzyme, but CO2 is readily inter-converted with H2CO3 with a carbonic anhydrase) that thus can increase the yield of any organism which currently uses the Calvin Benson Cycle to fix carbon, and in particular, increase the speed at which plants and other autotrophs can fix carbon. Particular applications include increased plant yield, reduced water usage per yield, and reduced nitrogen fertilizer use per yield.

[008] The invention provides methods, systems and compositions for carbon fixation without rubisco, comprising recombinant enzymes/pathway to incorporate carbon dioxide/bicarbonate into fructose-6-phosphate.

[009] In an aspect the invention provides a system for carbon fixation without rubisco, comprising an engineered pathway configured to incorporate bicarbonate into fructose-6- phosphate and comprising recombinant enzymes: i) Hps: hexulose-6-phosphate synthase, ii) Phi: hexulose-6-phosphate isomerase, iv) AspC: aspartate transaminase, v) RpiA: ribose-5 -phosphate isomerase, vi) YjhH: 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.

[010] In embodiments:

[Oil] the enzymes comprise: i) bmHps: Bacillus methanolicus hexulose-6-phosphate synthase, ii) bmPhi: Bacillus methanolicus hexulose-6-phosphate isomerase, iv) ecAspC: Escherichia coli aspartate transaminase, v) ecRpiA: Escherichia coli ribose-5-phosphate isomerase, vi) ecYjhH: Escherichia coli YjhH 2-keto-3-deoxy-galactonate aldolase (which also performs the 4-hydroxy-2-oxobutyrate aldolase reaction), vii) ecPpc: Escherichia coli phosphoenolpyruvate carboxylase, viii) ecAsd: Escherichia coli aspartate-semialdehyde dehydrogenase, ix) ecThrA_S345F: Escherichia coli fused aspartate kinase/homoserine dehydrogenase with the point mutation S345F;

[012] the system is further configured to convert carbon dioxide to bicarbonate (CO2 to H2CO3), and comprising enzyme: carbonic anhydrase;

[013] the system is further configured to convert the fructose-6-phosphate to mannitol- 1- phosphate, and comprising enzyme: iii) MtlD: mannitol- 1 -phosphate 5 -dehydrogenase;

[014] the system is integrated into a cell;

[015] the system is integrated into a plant cell, such as a crop plant cell, such as rice, maize, soybean, wheat, canola, sugarcane, cotton, loblolly pine, etc.;

[016] the system is integrated into a microbial cell, such as cyanobacteria and algae (microalgae and macroalgae); and/or

[017] the system is integrated into a cell-free system.

[018] In aspects the invention provides a method of carbon fixation without rubisco, comprising providing carbon dioxide or bicarbonate to a system herein, wherein the system incorporates the carbon dioxide or bicarbonate into fructose-6-phosphate. [019] In aspects the invention provides a method herein, comprising further detecting carbon fixation by detecting a resultant decrease in carbon dioxide or bicarbonate, or a resultant increase in fructose- 6-phosphate or reaction product thereof.

[020] In aspects the invention provides a method herein used for production of food (grains, cereals, etc.) or for biomass (fiber, pyrolysis into fuels, etc.), or to sequester CO2 from air. [021] The invention encompasses all combinations of the particular embodiments recited herein, as if each combination had been laboriously recited.

[022] Brief Description of the Drawings

[023] Fig. 1. Pathway to incorporate bicarbonate into mannitol- 1 -phosphate. bmHps: Bacillus methanolicus hexulose-6-phosphate synthase, bmPhi: Bacillus methanolicus hexulose-6- phosphat isomerase, ecMtlD: Escherichia coli mannitol- 1 -phosphate 5 -dehydrogenase, ecAspC: Escherichia coli aspartate transaminase, ecRpiA: Escherichia coli ribose-5 -phosphate isomerase, ecYjhH: Escherichia coli YjhH 2-keto-3-deoxy-galactonate aldolase (which also performs the 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 the point mutation S345F

[024] Fig. 2. SDS-PAGE gel showing enzyme purity after purification.

[025] Fig. 3. LCMS-TOF data from the mannitol- 1 -phosphate accumulation assay showing the counts from the quenched and filtered assay samples. Each condition was run in triplicate, and error bars represent standard deviation.

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

[027] Description of Particular Embodiments of the Invention

[028] Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, the term “or” means and/or. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and 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. [029] Examples: Incorporation of bicarbonate/carbon dioxide into fructose- 6-phosphate and mannitol- 1 -phosphate

[030] The Escherichia coli cell line BL21DE3* was used for protein expressions. E. coli cell line XL 1 -Blue was used for routine plasmid cloning and storage.

[031] Carbenicillin disodium salt and kanamycin monosulfate antibiotics were used at a final concentration of 100 pg/mL and 50 g/mL respectively in various growth media.

[032] LB Lennox media, also referred to as LB media or LB, recipe:

10 grams tryptone, 5 grams sodium chloride, 5 grams yeast extract. Dissolve in distilled water so that the final volume is 1 L. Autoclave for 45 min (121 °C, 15 psi) to ensure sterility.

[033] TB media, also referred to as TB: 24 grams yeast extract, 20 grams tryptone, 4 mL glycerol, 9.4 grams potassium phosphate dibasic, 2.2 grams potassium phosphate monobasic. Dissolve in distilled water so that the final volume is 1 L. Autoclave for 45 min (121°C, 15 psi) to ensure sterility.

[034] TSS recipe: 10 grams polyethylene glycol 3350, 5 mL of dimethyl sulfoxide, 2 mL of 1 molar magnesium chloride. Dissolve in LB so that the final volume is 100 mL. Sterile filter with a .22 pm filter, and store at 4°C.

[035] 2x KCM composition: 0.06 molar potassium chloride, 0.2 molar calcium chloride, and 0.1 molar magnesium chloride in distilled water. 2x KCM should be sterile filtered with a .22 pm filter before use.

[036] Sodium phosphate lysis buffer composition: 50 rnM sodium phosphate buffer pH 7.2 (approximately 8.925 grams sodium phosphate dibasic heptahydrate and 2.305 grams sodium phosphate monobasic monohydrate dissolved in 1 L of distilled water), 1 mg/mL lysozyme, .1 mg/mL deoxyribonuclease, 20 mM imidazole, 1 rnM P-mercaptoethanol (BME), 1 mM magnesium sulfate, 20 pM zinc sulfate, 20 pM manganese sulfate, 20 pM pyridoxal phosphate (PLP). As with all the buffers, pH was adjusted as necessary with sodium hydroxide or hydrochloric acid.

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

[038] Sodium phosphate elution buffer composition: 50 mM sodium phosphate buffer pH 7.2, 200 mM imidazole, 1 mM BME, 1 mM magnesium sulfate, 20 pM zinc sulfate, 20 pM manganese sulfate, 20 pM PLP. [039] Sodium phosphate dialysis buffer composition: 50 mM sodium phosphate buffer pH 7.2, f mM tris(2-carboxyethyl)phosphine (TCEP), f mM magnesium sulfate, 20 M zinc sulfate, 20 pM manganese sulfate, 20 pM PLP.

[040] Chemical competent cell preparation protocol: The desired E. coll strain from a previously stored -80 °C glycerol stock, was used to inoculate 10 mL of LB in a glass culture tube. The culture was then allowed to grow overnight, approximately 16 hours, at 37 °C shaking at 200 RPM. 500 pL of culture was then used to inoculate 50 mL fresh LB media in a 250 mL baffled flask. Cells were grown at 37°C shaking at 200 RPM to an OD600 of .35 (approximately 3 hours of growth). Cells were then transferred to two 50 mL conical tubes (25 mL of culture each) and incubated on ice for 20 min. The cultures were then centrifuged at 8,000 RCF for 8 minutes at 4 °C. The supernatant was discarded from each conical tube. The first cell pellet was resuspended in 5 mL of 4 °C TSS by light vortexing (approximately 30% power on a VWR® Standard Heavy-Duty Vortex Mixer). The 5 mL of resuspended cells were then used to resuspend the second cell pellet in the same manner. 100 pL of resuspended cells were then aliquoted into .6 mL snap cap tubes, yielding approximately 50 tubes worth of chemical competent cells. Chemical competent cells were either flash frozen in liquid nitrogen and stored at -80 °C until use, or used immediately for plasmid transformations.

[041] Heat shock protocol: Chemical competent cells were thawed if necessary, f pL of desired plasmid, at approximately 50 ng/pL concentration, was added into 100 pL of chemical competent cells. 100 pL of 2x KCM was then added to the cells. Cells were allowed to incubate on ice for 20 min. Cells were then heat shocked at 42 °C for 90 seconds, then incubated on ice for 1 min. Cells were then allowed to recover at 37 °C (not shaking) for one hour. Then 200 pL of cells were plated on LB agar plates with the appropriate antibiotics to select for the plasmid being transformed. Plates were incubated at 37 °C overnight (approximately 16 hours), until colonies had formed.

[042] Enzyme expression in E. coli BL21DE3*: Single E. coli BL21DE3* colonies containing the desired plasmid were picked into 10 mL of LB media with the appropriate antibiotic in glass culture tubes and grown overnight (approximately 16 hours) at 37 °C shaking at 200 RPM. 500 pL of culture was then used to inoculate 50 mL of TB media in 250 mL baffled flasks. Cultures were then grown at 37 °C at 200 RPM until reaching an OD600 of .8. Then the cultures were induced by adding IPTG (Isopropyl P-D-l-thiogalactopyranoside) at a final concentration of 1 mM. Cells were then incubated at 18 °C for 24 hours shaking at 200 RPM. 40 mL of culture was then transferred to 50 mL conical tubes, and centrifuged at 8,000 RCF for 10 min at 4 °C. Supernatant was discarded. Cell pellets were either stored at -20 °C until use, or used for enzyme purifications immediately. [043] Enzyme purification by immobilized metal affinity chromatography: All enzyme purification steps were conducted in a cold room at 4 °C. If necessary, cell pellets were thawed from -20 °C to 4 °C on ice. Cell pellets were then resuspended in 5 mL of sodium phosphate lysis buffer, pre chilled to 4 °C, by vortexing. 50 mL conical tubes containing resuspended cells were 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, for a total cycle time of 3 min (1 minute of “on time”). Lysed culture was then centrifuged at 10,000 RCF for 20 min at 4 °C. While waiting for the centrifugation to complete, gravity columns containing 1 mL of Ni-NTA agarose beads, enabling immobilized metal affinity chromatography of proteins with a polyhistidine-tag, were equilibrated with 20 column volumes of sodium phosphate wash buffer prechilled to 4 °C. After the centrifugation had finished, clarified supernatant was applied to the Ni-NTA columns. 40 column volumes of sodium phosphate wash buffer, prechilled to 4 °C, were then applied to each column. 15 column volumes of sodium phosphate elution buffer, also pre chilled to 4 °C, were used to elute the enzymes into 10K MWCO spin concentrators.

[044] Purified enzyme concentration and dialysis: Enzymes in 10K MWCO spin concentrators were centrifuged at the manufactures recommended maximum speed at 4 °C until the volume of enzymes was concentrated to less than 800 pL. Concentrated enzymes were transferred to dialysis cassettes with 3.5K MWCO membranes and placed in sodium phosphate dialysis buffer prepared at 4 °C, and allowed to dialyze overnight (at least 8 hours) in a cold room also at 4 °C. A stir bar ensured constant, but slow, approximately 60 RPM, mixing of the dialysis buffer. 1 L of sodium phosphate dialysis buffer was used to dialyze up to 8 enzyme dialysis cassettes. The enzyme cassettes were then dialyzed a second time in fresh sodium phosphate dialysis buffer, as before, but only for 4 hours. Enzymes were then transferred to 1.7 mL microcentrifuge tubes on ice and checked for any precipitation. Any enzyme with precipitation in the sample was spun at 14,000 RCF for 2 minutes at 4 °C to pellet any insoluble material. The clarified supernatant was then transferred to a new microcentrifuge tube. At this point, we consider these enzymes fully purified. A NanoDrop 1000 spectrophotometer was used to measure the concentration of each enzyme by measuring absorbance at 280 nm. Enzymes were either used immediately, or glycerol was added to a final concentration of 20% (v/v) before enzymes were flash frozen in liquid nitrogen and stored at -80 °C until use.

[045] Enzyme purity was also checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The brief gel electrophoresis protocol is as follows. 10 pL of purified and dialyzed enzyme was mixed with 2 pL of SDS-PAGE Sample Loading Buffer 6X by Cepham Life Sciences in PCR tubes. The enzyme and loading buffer mix was then boiled for 10 minutes at 95 °C in a PCR block. After cooling to room temperature, 10 pL of the boiled enzyme and loading buffer mix was then applied to a 4-20% mini-PROTEAN TGX precast protein gel which was already inserted appropriately in a Mini-PROTEAN Tetra Cell. The gel was run at a constant 200 V until the running buffer dye exited the bottom of the gel (approximately 25 minutes). The gel was then washed in ddH2O water by placing it in a tray, adding approximately 100 ml of ddH2O completely submerging the gel, and allowing it to rock gently for 5 min on a gel rocker. The used ddH2O was then discarded. This wash step was repeated twice, for a total of three washes. The gel was then allowed to stain in 100 mL GelCode (by Thermo Scientific) for 1 hour, also gently rocking. After this, the GelCode was removed, and the gel washed briefly with 100 mL of ddH2O three times (no rocking, just addition of ddH2O, then ddH2O removal). Then the gel was the de-stained overnight (approximately 16 hours) by rocking gently in 100 mL of ddH2O, with a Kimwipe added. The gel was then imaged after.

[046] Incorporation of bicarbonate into mannitol- 1 -phosphate: Reaction solution A composition: 3 mM nicotinamide adenine dinucleotide phosphate reduced (NADPH), 3 mM nicotinamide adenine dinucleotide reduced (NADH), 4 mM phosphoenolpyruvate (PEP), 1 mM sodium bicarbonate, 2 mM adenosine triphosphate (ATP), 2 mM ribose-5-phosphate, 2 mM alpha-ketoglutarate, 2 mM glutamate, 1 mM acetyl coenzyme A (acetyl-CoA), 2 mM guanosine triphosphate (GTP), 50 mM sodium phosphate buffer pH 7.2 (approximately 8.925 grams sodium phosphate dibasic heptahydrate and 2.305 grams sodium phosphate monobasic monohydrate dissolved in 1 L of distilled water), 1 mM TCEP, 1 mM magnesium sulfate, 20 pM zinc sulfate, 20 M manganese sulfate, 20 pM PLP. Adjusting pH was done with either sodium hydroxide or hydrochloric acid as needed.

[047] Reaction solution B composition: 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 pH 7.2 (approximately 8.925 grams sodium phosphate dibasic heptahydrate and 2.305 grams sodium phosphate monobasic monohydrate dissolved in 1 L of distilled water), 1 mM TCEP, 1 mM magnesium sulfate, 20 pM zinc sulfate, 20 pM manganese sulfate, 20 pM PLP. Adjusting pH was done with either sodium hydroxide or hydrochloric acid as needed.

[048] Reaction solution C composition: 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 previously boiled at 95 °C for 10 minutes, .2825 mg/mL ecAsd, .1345 mg/mL ecThrA_S345F, 50 mM sodium phosphate buffer pH 7.2 (approximately 8.925 grams sodium phosphate dibasic heptahydrate and 2.305 grams sodium phosphate monobasic monohydrate dissolved in 1 L of distilled water), 1 mM TCEP, 1 mM magnesium sulfate, 20 pM zinc sulfate, 20 |iM manganese sulfate, 20 |iM PLP. Adjusting pH was done with either sodium hydroxide or hydrochloric acid as needed.

[049] Quench buffer: 50% methanol, 50% deionized distilled water (ddH2O) (v/v)

[050] Mannitol- 1 -phosphate accumulation assay procedure: 50 pL of reaction buffer A was combined with 50 pL reaction B to initiate the test condition reaction. 50 pL of reaction buffer A was combined with 50 pL reaction C to initiate the negative control reaction. Each of these reactions was performed in triplicate. Time points were taken 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 by taking 6.6 pl of the desired reaction mixture, and mixing it with 144 pL of quench buffer to stop the reaction. Quenched samples were stored at -20 °C until all the time points were collected. Quenched reaction samples were then centrifuged through a 96 well Pall Laboratory AcroPrep Omega Membrane 10K MWCO filter plate to remove enzymes at the manufactured maximum recommended relative centrifugal force (RCF) for a swinging bucket rotor at 4 °C. The centrifugation was periodically stopped to check and see if the amount of flow through was sufficient to satisfy the minimum volume required for LCMS vials to properly facilitate LCMS- TOF injections. Once all the samples had at least twice the minimum volume, the flowthrough was transferred to LCMS vials and stored at -80 °C until analysis via LCMS-TOF.

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

[052] Store bought mannitol- 1 -phosphate served as the analytical standard, and was used to create a standard curve, to validate retention time and mass to charge ratio (m/z) of mannitol- 1- phosphate, and to validate the limit of detection given our analytical instrument, method, and compound of interest. The standard curve was constructed in 50% methanol, 50% water (v/v) buffer. Standard curve concentrations for mannitol- 1 -phosphate were 0.0390625 pM, 0.078125 pM, 0.15625 pM, 0.3125 pM, 0.625 pM, 1.25 pM, 2.5 pM, 5 pM, 10 pM, 20 pM. 0.0390625 pM and 0.078125 pM were below the limit of detection.

[053] Recombinant pathway constitution in cyanobacteria. The disclosed system for carbon fixation comprising an engineered pathway configured to incorporate bicarbonate into fructose- 6-phosphate was introduced in cyantobacteria (Synechocystis sp. PCC 6803) using natural transformation, providing stable expression of recombinant enzymes: (i) Hps: hexulose-6- phosphate synthase, (ii) Phi: hexulose-6-phosphate isomerase, (iv) AspC: aspartate transaminase, (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 demonstrate incorporation of bicarbonate/carbon dioxide into fructose-6- phosphate and mannitol- 1 -phosphate without rubisco.