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Producing the “molecules of life” from CO2 through hybrid catalytic relay

2021; Elsevier BV; Volume: 7; Issue: 12 Linguagem: Inglês

10.1016/j.chempr.2021.11.018

2451-9308

Peng Zhang, Susie Y. Dai, Joshua S. Yuan,

Catalytic Processes in Materials Science

Carbon dioxide (CO2) is an important feedstock for essential chemicals synthesis, but its transformation into biological macromolecules remains challenging. In a recent issue of Science, Ma et al. integrated chemical and enzymatic catalysis to convert CO2 into starch, a vital source of calories and feedstock in industry, with significantly higher efficiency than plants. Carbon dioxide (CO2) is an important feedstock for essential chemicals synthesis, but its transformation into biological macromolecules remains challenging. In a recent issue of Science, Ma et al. integrated chemical and enzymatic catalysis to convert CO2 into starch, a vital source of calories and feedstock in industry, with significantly higher efficiency than plants. Main textPhotosynthesis is the process by which plants and algae use energy from sunlight to convert carbon dioxide (CO2) and water into carbohydrates. It is fundamental to most life on earth, because both autotrophic and heterotrophic species depend on carbohydrates generated from photosynthesis to sustain them. However, the photosynthetic efficiency for converting light energy to chemical energy is fairly low (<3%), even in some of the fastest-growing phototrophic species. Scientists have been trying to design faster and more efficient routes to produce essential chemicals, fuels, and macromolecules with CO2 and water to sustain the booming human population and global carbon inventory changes caused by economic development.In the past few decades, we have witnessed numerous milestones in "artificial photosynthesis," where chemists and biologists have developed various routes for harnessing the energy in sunlight. However, the primary product of most of these catalytic artificial photosynthesis routes is hydrogen.1Reece S.Y. Hamel J.A. Sung K. Jarvi T.D. Esswein A.J. Pijpers J.J.H. Nocera D.G. Wireless solar water splitting using silicon-based semiconductors and earth-abundant catalysts.Science. 2011; 334: 645-648Google Scholar When CO2 is used as a substrate, some artificial photosynthesis routes can produce one-carbon (C1) compounds like formate2Shan B. Vanka S. Li T.-T. Troian-Gautier L. Brennaman M.K. Mi Z. Meyer T.J. Binary molecular-semiconductor p–n junctions for photoelectrocatalytic CO2 reduction.Nat. Energy. 2019; 4: 290-299Google Scholar and methanol3Wu Y.A. McNulty I. Liu C. Lau K.C. Liu Q. Paulikas A.P. Sun C.-J. Cai Z. Guest J.R. Ren Y. et al.Facet-dependent active sites of a single Cu2O particle photocatalyst for CO2 reduction to methanol.Nat. Energy. 2019; 4: 957-968Google Scholar,4Styring S. Artificial photosynthesis for solar fuels.Faraday Discuss. 2012; 155: 357-376Google Scholar from CO2. Despite all of this progress, these routes don't produce products that are broadly used by heterotrophic species.The integration of chemical catalysis and microbial systems can overcome the barriers facing chemical catalysis alone. In 2016, Yang's group5Sakimoto K.K. Wong A.B. Yang P. Self-photosensitization of nonphotosynthetic bacteria for solar-to-chemical production.Science. 2016; 351: 74-77Google Scholar reported that decorating light-harvesting cadmium sulfide on Moorella thermoacetica enabled this nonphotosynthetic strain to use sunlight and convert CO2 to acetate. This type of photosynthetic biohybrid system (PBS) could transform the heterotrophic species to utilize light. However, microorganisms like M. thermoacetica use the Wood-Ljungdahl pathway, and the current primary product is acetic acid rather than carbohydrate. Furthermore, productivity depends on the addition of cysteine to a certain degree, and long-term cell survival is yet to be achieved.In parallel to using light energy to provide reducing power, electrocatalysis has also been explored to produce either hydrogen or C1 intermediates like formic acid. In 2011, Liao's group6Li H. Opgenorth P.H. Wernick D.G. Rogers S. Wu T.-Y. Higashide W. Malati P. Huo Y.-X. Cho K.M. Liao J.C. Integrated electromicrobial conversion of CO2 to higher alcohols.Science. 2012; 335: 1596Google Scholar demonstrated the conversion of CO2 to higher alcohols by using Ralstonia eutropha H16 in a fermentation system with electricity. CO2 is reduced to formic acid (HCOOH) via electrocatalysis, which can be converted to NADH, providing reducing power and the substrate for the autotrophic strains. In 2016, Silver and Nocera's groups reported a water splitting-biosynthetic system with CO2 fixation efficiencies exceeding photosynthesis.7Liu C. Colón B.C. Ziesack M. Silver P.A. Nocera D.G. Water splitting-biosynthetic system with CO2 reduction efficiencies exceeding photosynthesis.Science. 2016; 352: 1210-1213Google Scholar A biocompatible cobalt-based electrocatalyst split water into H2 and O2 at low voltage, and Ralstonia eutropha grown in the system utilized low concentration of CO2 and H2 to produce biomass, fuels, and chemicals. The integration of chemical catalysis with bioconversion has the unique ability to bypass the rate-limiting CO2 fixation steps in the biological systems via converting CO2 to small carbonaceous molecules. However, C1 compounds like formate and methanol have to go through the slow C1 metabolism in cells, and they are often toxic to the cells, making it challenging for microbial bioproduction. For hydrogen-driven CO2 conversion in Ralstonia, the carbon fixation rate is fundamentally limited by gas-to-liquid transfer rate. Therefore, it remains challenging to manufacture bioproducts from these C1 chemical species generated from CO2 reduction, particularly in cellular systems.In an article recently published in Science, Ma and co-workers have reported a cell-free route to synthesize starch from CO2 and hydrogen via a chemical-biochemical hybrid process, called the artificial starch anabolic pathway (ASAP).8Cai T. Sun H. Qiao J. Zhu L. Zhang F. Zhang J. Tang Z. Wei X. Yang J. Yuan Q. et al.Cell-free chemoenzymatic starch synthesis from carbon dioxide.Science. 2021; 373: 1523-1527Google Scholar As in previous studies, the ASAP also exploited the integration of chemical catalysis and biological system, but in an innovative way. Instead of utilizing a cell-based system, the study designed an innovative enzymatic route to convert CO2 hydrogenation-derived formate and methanol into starch. In a sense, the study achieved autotrophic conversion of CO2 to starch via integrating advancements in chemical catalysis for CO2 reduction and synthetic biology design, presenting an artificial route from CO2 to life-essential macromolecules. This system has addressed the aforementioned challenges in previous systems that integrated chemical catalysis with biological systems through at least three separate and innovative advances.First, Ma et al. achieved efficient conversion of CO2 to starch, bypassing the relatively slow photosynthesis system. In nature, starch metabolism begins with carbon anabolism on plant leaves (Figure 1A), in which the chloroplast harnesses the energy from sunlight to split water and produce NADPH and ATP (light reaction), providing reducing power and energy for the CO2 fixation in Calvin-Benson cycle (dark reaction). The slow regeneration of Calvin-Benson cycle intermediates and the low turnover rate of RuBisCo enzyme in the dark reaction limit both the overall photosynthesis efficiency and the productivity of carbohydrate from CO2 in plant or algae cells. In contrast, the researchers built enzymatic reactions to convert C1 chemicals such as formic acid or methanol in the reported cell-free system (Figure 1B). Formic acid and methanol can be efficiently produced by CO2 hydrogenation with inorganic catalysts.9Wang J. Li G. Li Z. Tang C. Feng Z. An H. Liu H. Liu T. Li C. A highly selective and stable ZnO-ZrO2 solid solution catalyst for CO2 hydrogenation to methanol.Sci. Adv. 2017; 3: e1701290Google Scholar,10Shao X. Yang X. Xu J. Liu S. Miao S. Liu X. Su X. Duan H. Huang Y. Zhang T. Iridium Single-Atom Catalyst Performing a Quasi-homogeneous Hydrogenation Transformation of CO2 to Formate.Chem. 2019; 5: 693-705Google Scholar The chemical catalytic CO2 reduction circumvents the rate-limiting CO2 fixation catalyzed by RuBisCo and the intermediate regeneration in the Calvin-Benson cycle, efficiently resolving these limiters of photosynthetic efficiency in natural systems. The new platform significantly advanced the starch synthesis rate from CO2, achieving 22 nmol min−1 mg−1 of total catalyst and proteins, which is calculated to be 8.5-fold higher than normal starch synthesis in maize and 5.7-fold higher than the synthetic CETCH cycle.11Schwander T. Schada von Borzyskowski L. Burgener S. Cortina N.S. Erb T.J. A synthetic pathway for the fixation of carbon dioxide in vitro.Science. 2016; 354: 900-904Google Scholar The rate increase mainly results from the relatively fast rate of CO2 hydrogenation to methanol, highlighting the great potential of hybridizing chemical catalysis and synthetic biology. Furthermore, because H2 could be acquired from green approaches, and the reaction could be driven by renewable energy from sunlight, this chemoenzymatic process could be recognized as another form of artificial photosynthesis. Therefore, the research provides a real case of producing bio-products using CO2 as the sole carbon source in a chemoenzymatic catalytic system, inspiring future efforts to develop more feasible routes toward macromolecule biosynthesis via artificial photosynthesis.Second, the synthetic pathway design is also novel, enabling the highly efficient conversion of C1 compounds into starch (Figure 1C). The cell-free system has an advantage over microbial conversion of C1 compounds, because the C1 pathways have less favorable energy and carbon flux partition in the cell. To design the ASAP pathway, the researchers used bioinformatics analysis to draft possible reaction sets from previous established databases, identifying that starch could be synthesized via nine core reactions from formic acid or methanol. However, unlike the natural pathways—which have evolved and optimized over a long period of time—the artificial pathway faces challenges of inhibition, because the enzymes originate from various organisms and might have disparate kinetics to prevent coordinated substrate utilization. To improve the overall efficiency, the researchers identified critical intermediates in the pathway and deconvoluted this complicated process into several modules, including C1 module for formaldehyde production, C3 module for D-glyceraldehyde 3-phosphate (GAP) production, C6 module for D-glucose-6-phospate (G-6-P) production, and Cn module for glucose assimilation and starch synthesis. In each module, they built multiple candidate reaction sets to address the incompatibility in the module assembly. For instance, when combining C1 and C3 modules, only thermodynamic-favorable methanol oxidation by alcohol oxidase (aox) could provide enough formaldehyde for C3 reactions. Another scenario that could occur during module assembly is that the byproducts or cofactors in former modules could inhibit the enzyme in latter modules. With substitution module reactions to address the incompatibility, the successful assembly of the four modules (C1e+C3a+C6b∗+Cnb) enabled 30 mg/L amylose titer from 20 mM methanol. The system is named ASAP 1.0. Based on this multi-module synthetic pathway, the researchers resolved the main bottlenecks to improve the efficiency in ASAP 2.0. Formolase (fls-M3) activity was improved 4.7-fold with directed evolution, and variants of fructose-bisphosphatase (fbp-AGR) and ADP-glucose pyrophosphorylase (agp-M3) enhanced the enzyme activity as well as ADP/ATP tolerance. The starch productivity of ASAP 2.0 is 7.6-fold of that in the ASAP 1.0. With the integration of CO2 hydrogenation unit, complete CO2 to linear amylose (ASAP 3.0) and branched amylopectin (ASAP 3.1) pathways were established. The keys to the successful assembly of such complex, multi-enzyme reactions are managing the carbon flux and energy consumption and improving the compatibility. This study is representative in this regard and demonstrates how modular design and directed evolution can be used to achieve complicated macromolecule synthesis from C1 compound.Another significant advancement of Ma et al.'s study the improvement of the interface between chemical and biocatalysis. The transformation of CO2 into water-soluble products such as formic acid and methanol is recognized as the critical step to allow biochemists to leverage synthetic biology for macromolecule synthesis. The high water solubility of products facilitates the subsequent enzymatic transformation, representing an advantage over the hydrogen or CO-driven CO2 bioconversion, which is often limited by the gas-to-liquid transfer rate.Starch is regarded as a "molecule of life" because it is a macromolecule commonly consumed by heterotrophic organisms, such as humans, to sustain their growth. The synthesis of starch from CO2 thus represents a significant improvement for artificial photosynthesis, in comparison with the production of smaller molecules like acetate and higher alcohols accomplished in previous studies. Despite considerable progress, there remain several limitations of the study that need to be addressed to achieve broader applications. First, the ASAP 3.0 and 3.1 are stepwise and discontinuous systems that cannot achieve integrated production. Such is a significant disadvantage as compared with the hydrogenotrophic microbial fixation of CO2 or electro-microbial systems for CO2 reduction and conversion.6Li H. Opgenorth P.H. Wernick D.G. Rogers S. Wu T.-Y. Higashide W. Malati P. Huo Y.-X. Cho K.M. Liao J.C. Integrated electromicrobial conversion of CO2 to higher alcohols.Science. 2012; 335: 1596Google Scholar,7Liu C. Colón B.C. Ziesack M. Silver P.A. Nocera D.G. Water splitting-biosynthetic system with CO2 reduction efficiencies exceeding photosynthesis.Science. 2016; 352: 1210-1213Google Scholar Second, the catalytic step for hydrogenation of CO2 requires high temperature (∼320°C) and high pressure (5 MPa), which requires significant energy input and is incompatible with biocatalysis or microbial conversion. Approaches that are amenable to the biological system need to be developed and integrated. Third, the total reaction time tested in the study is approximately 4 h. It is understandable that this study is mainly a proof of concept. However, achieving enzyme stability for a longer reaction time will be challenging. Further study could improve enzyme stability through immobilization and engineering. The microbial system can serve as an alternative, but the compatibility with electro- or photocatalysis still needs to be addressed. Overall, the study by Ma and colleagues is highly inspiring, and future work on system integration will promote broader applications. Main textPhotosynthesis is the process by which plants and algae use energy from sunlight to convert carbon dioxide (CO2) and water into carbohydrates. It is fundamental to most life on earth, because both autotrophic and heterotrophic species depend on carbohydrates generated from photosynthesis to sustain them. However, the photosynthetic efficiency for converting light energy to chemical energy is fairly low (<3%), even in some of the fastest-growing phototrophic species. Scientists have been trying to design faster and more efficient routes to produce essential chemicals, fuels, and macromolecules with CO2 and water to sustain the booming human population and global carbon inventory changes caused by economic development.In the past few decades, we have witnessed numerous milestones in "artificial photosynthesis," where chemists and biologists have developed various routes for harnessing the energy in sunlight. However, the primary product of most of these catalytic artificial photosynthesis routes is hydrogen.1Reece S.Y. Hamel J.A. Sung K. Jarvi T.D. Esswein A.J. Pijpers J.J.H. Nocera D.G. Wireless solar water splitting using silicon-based semiconductors and earth-abundant catalysts.Science. 2011; 334: 645-648Google Scholar When CO2 is used as a substrate, some artificial photosynthesis routes can produce one-carbon (C1) compounds like formate2Shan B. Vanka S. Li T.-T. Troian-Gautier L. Brennaman M.K. Mi Z. Meyer T.J. Binary molecular-semiconductor p–n junctions for photoelectrocatalytic CO2 reduction.Nat. Energy. 2019; 4: 290-299Google Scholar and methanol3Wu Y.A. McNulty I. Liu C. Lau K.C. Liu Q. Paulikas A.P. Sun C.-J. Cai Z. Guest J.R. Ren Y. et al.Facet-dependent active sites of a single Cu2O particle photocatalyst for CO2 reduction to methanol.Nat. Energy. 2019; 4: 957-968Google Scholar,4Styring S. Artificial photosynthesis for solar fuels.Faraday Discuss. 2012; 155: 357-376Google Scholar from CO2. Despite all of this progress, these routes don't produce products that are broadly used by heterotrophic species.The integration of chemical catalysis and microbial systems can overcome the barriers facing chemical catalysis alone. In 2016, Yang's group5Sakimoto K.K. Wong A.B. Yang P. Self-photosensitization of nonphotosynthetic bacteria for solar-to-chemical production.Science. 2016; 351: 74-77Google Scholar reported that decorating light-harvesting cadmium sulfide on Moorella thermoacetica enabled this nonphotosynthetic strain to use sunlight and convert CO2 to acetate. This type of photosynthetic biohybrid system (PBS) could transform the heterotrophic species to utilize light. However, microorganisms like M. thermoacetica use the Wood-Ljungdahl pathway, and the current primary product is acetic acid rather than carbohydrate. Furthermore, productivity depends on the addition of cysteine to a certain degree, and long-term cell survival is yet to be achieved.In parallel to using light energy to provide reducing power, electrocatalysis has also been explored to produce either hydrogen or C1 intermediates like formic acid. In 2011, Liao's group6Li H. Opgenorth P.H. Wernick D.G. Rogers S. Wu T.-Y. Higashide W. Malati P. Huo Y.-X. Cho K.M. Liao J.C. Integrated electromicrobial conversion of CO2 to higher alcohols.Science. 2012; 335: 1596Google Scholar demonstrated the conversion of CO2 to higher alcohols by using Ralstonia eutropha H16 in a fermentation system with electricity. CO2 is reduced to formic acid (HCOOH) via electrocatalysis, which can be converted to NADH, providing reducing power and the substrate for the autotrophic strains. In 2016, Silver and Nocera's groups reported a water splitting-biosynthetic system with CO2 fixation efficiencies exceeding photosynthesis.7Liu C. Colón B.C. Ziesack M. Silver P.A. Nocera D.G. Water splitting-biosynthetic system with CO2 reduction efficiencies exceeding photosynthesis.Science. 2016; 352: 1210-1213Google Scholar A biocompatible cobalt-based electrocatalyst split water into H2 and O2 at low voltage, and Ralstonia eutropha grown in the system utilized low concentration of CO2 and H2 to produce biomass, fuels, and chemicals. The integration of chemical catalysis with bioconversion has the unique ability to bypass the rate-limiting CO2 fixation steps in the biological systems via converting CO2 to small carbonaceous molecules. However, C1 compounds like formate and methanol have to go through the slow C1 metabolism in cells, and they are often toxic to the cells, making it challenging for microbial bioproduction. For hydrogen-driven CO2 conversion in Ralstonia, the carbon fixation rate is fundamentally limited by gas-to-liquid transfer rate. Therefore, it remains challenging to manufacture bioproducts from these C1 chemical species generated from CO2 reduction, particularly in cellular systems.In an article recently published in Science, Ma and co-workers have reported a cell-free route to synthesize starch from CO2 and hydrogen via a chemical-biochemical hybrid process, called the artificial starch anabolic pathway (ASAP).8Cai T. Sun H. Qiao J. Zhu L. Zhang F. Zhang J. Tang Z. Wei X. Yang J. Yuan Q. et al.Cell-free chemoenzymatic starch synthesis from carbon dioxide.Science. 2021; 373: 1523-1527Google Scholar As in previous studies, the ASAP also exploited the integration of chemical catalysis and biological system, but in an innovative way. Instead of utilizing a cell-based system, the study designed an innovative enzymatic route to convert CO2 hydrogenation-derived formate and methanol into starch. In a sense, the study achieved autotrophic conversion of CO2 to starch via integrating advancements in chemical catalysis for CO2 reduction and synthetic biology design, presenting an artificial route from CO2 to life-essential macromolecules. This system has addressed the aforementioned challenges in previous systems that integrated chemical catalysis with biological systems through at least three separate and innovative advances.First, Ma et al. achieved efficient conversion of CO2 to starch, bypassing the relatively slow photosynthesis system. In nature, starch metabolism begins with carbon anabolism on plant leaves (Figure 1A), in which the chloroplast harnesses the energy from sunlight to split water and produce NADPH and ATP (light reaction), providing reducing power and energy for the CO2 fixation in Calvin-Benson cycle (dark reaction). The slow regeneration of Calvin-Benson cycle intermediates and the low turnover rate of RuBisCo enzyme in the dark reaction limit both the overall photosynthesis efficiency and the productivity of carbohydrate from CO2 in plant or algae cells. In contrast, the researchers built enzymatic reactions to convert C1 chemicals such as formic acid or methanol in the reported cell-free system (Figure 1B). Formic acid and methanol can be efficiently produced by CO2 hydrogenation with inorganic catalysts.9Wang J. Li G. Li Z. Tang C. Feng Z. An H. Liu H. Liu T. Li C. A highly selective and stable ZnO-ZrO2 solid solution catalyst for CO2 hydrogenation to methanol.Sci. Adv. 2017; 3: e1701290Google Scholar,10Shao X. Yang X. Xu J. Liu S. Miao S. Liu X. Su X. Duan H. Huang Y. Zhang T. Iridium Single-Atom Catalyst Performing a Quasi-homogeneous Hydrogenation Transformation of CO2 to Formate.Chem. 2019; 5: 693-705Google Scholar The chemical catalytic CO2 reduction circumvents the rate-limiting CO2 fixation catalyzed by RuBisCo and the intermediate regeneration in the Calvin-Benson cycle, efficiently resolving these limiters of photosynthetic efficiency in natural systems. The new platform significantly advanced the starch synthesis rate from CO2, achieving 22 nmol min−1 mg−1 of total catalyst and proteins, which is calculated to be 8.5-fold higher than normal starch synthesis in maize and 5.7-fold higher than the synthetic CETCH cycle.11Schwander T. Schada von Borzyskowski L. Burgener S. Cortina N.S. Erb T.J. A synthetic pathway for the fixation of carbon dioxide in vitro.Science. 2016; 354: 900-904Google Scholar The rate increase mainly results from the relatively fast rate of CO2 hydrogenation to methanol, highlighting the great potential of hybridizing chemical catalysis and synthetic biology. Furthermore, because H2 could be acquired from green approaches, and the reaction could be driven by renewable energy from sunlight, this chemoenzymatic process could be recognized as another form of artificial photosynthesis. Therefore, the research provides a real case of producing bio-products using CO2 as the sole carbon source in a chemoenzymatic catalytic system, inspiring future efforts to develop more feasible routes toward macromolecule biosynthesis via artificial photosynthesis.Second, the synthetic pathway design is also novel, enabling the highly efficient conversion of C1 compounds into starch (Figure 1C). The cell-free system has an advantage over microbial conversion of C1 compounds, because the C1 pathways have less favorable energy and carbon flux partition in the cell. To design the ASAP pathway, the researchers used bioinformatics analysis to draft possible reaction sets from previous established databases, identifying that starch could be synthesized via nine core reactions from formic acid or methanol. However, unlike the natural pathways—which have evolved and optimized over a long period of time—the artificial pathway faces challenges of inhibition, because the enzymes originate from various organisms and might have disparate kinetics to prevent coordinated substrate utilization. To improve the overall efficiency, the researchers identified critical intermediates in the pathway and deconvoluted this complicated process into several modules, including C1 module for formaldehyde production, C3 module for D-glyceraldehyde 3-phosphate (GAP) production, C6 module for D-glucose-6-phospate (G-6-P) production, and Cn module for glucose assimilation and starch synthesis. In each module, they built multiple candidate reaction sets to address the incompatibility in the module assembly. For instance, when combining C1 and C3 modules, only thermodynamic-favorable methanol oxidation by alcohol oxidase (aox) could provide enough formaldehyde for C3 reactions. Another scenario that could occur during module assembly is that the byproducts or cofactors in former modules could inhibit the enzyme in latter modules. With substitution module reactions to address the incompatibility, the successful assembly of the four modules (C1e+C3a+C6b∗+Cnb) enabled 30 mg/L amylose titer from 20 mM methanol. The system is named ASAP 1.0. Based on this multi-module synthetic pathway, the researchers resolved the main bottlenecks to improve the efficiency in ASAP 2.0. Formolase (fls-M3) activity was improved 4.7-fold with directed evolution, and variants of fructose-bisphosphatase (fbp-AGR) and ADP-glucose pyrophosphorylase (agp-M3) enhanced the enzyme activity as well as ADP/ATP tolerance. The starch productivity of ASAP 2.0 is 7.6-fold of that in the ASAP 1.0. With the integration of CO2 hydrogenation unit, complete CO2 to linear amylose (ASAP 3.0) and branched amylopectin (ASAP 3.1) pathways were established. The keys to the successful assembly of such complex, multi-enzyme reactions are managing the carbon flux and energy consumption and improving the compatibility. This study is representative in this regard and demonstrates how modular design and directed evolution can be used to achieve complicated macromolecule synthesis from C1 compound.Another significant advancement of Ma et al.'s study the improvement of the interface between chemical and biocatalysis. The transformation of CO2 into water-soluble products such as formic acid and methanol is recognized as the critical step to allow biochemists to leverage synthetic biology for macromolecule synthesis. The high water solubility of products facilitates the subsequent enzymatic transformation, representing an advantage over the hydrogen or CO-driven CO2 bioconversion, which is often limited by the gas-to-liquid transfer rate.Starch is regarded as a "molecule of life" because it is a macromolecule commonly consumed by heterotrophic organisms, such as humans, to sustain their growth. The synthesis of starch from CO2 thus represents a significant improvement for artificial photosynthesis, in comparison with the production of smaller molecules like acetate and higher alcohols accomplished in previous studies. Despite considerable progress, there remain several limitations of the study that need to be addressed to achieve broader applications. First, the ASAP 3.0 and 3.1 are stepwise and discontinuous systems that cannot achieve integrated production. Such is a significant disadvantage as compared with the hydrogenotrophic microbial fixation of CO2 or electro-microbial systems for CO2 reduction and conversion.6Li H. Opgenorth P.H. Wernick D.G. Rogers S. Wu T.-Y. Higashide W. Malati P. Huo Y.-X. Cho K.M. Liao J.C. Integrated electromicrobial conversion of CO2 to higher alcohols.Science. 2012; 335: 1596Google Scholar,7Liu C. Colón B.C. Ziesack M. Silver P.A. Nocera D.G. Water splitting-biosynthetic system with CO2 reduction efficiencies exceeding photosynthesis.Science. 2016; 352: 1210-1213Google Scholar Second, the catalytic step for hydrogenation of CO2 requires high temperature (∼320°C) and high pressure (5 MPa), which requires significant energy input and is incompatible with biocatalysis or microbial conversion. Approaches that are amenable to the biological system need to be developed and integrated. Third, the total reaction time tested in the study is approximately 4 h. It is understandable that this study is mainly a proof of concept. However, achieving enzyme stability for a longer reaction time will be challenging. Further study could improve enzyme stability through immobilization and engineering. The microbial system can serve as an alternative, but the compatibility with electro- or photocatalysis still needs to be addressed. Overall, the study by Ma and colleagues is highly inspiring, and future work on system integration will promote broader applications. Photosynthesis is the process by which plants and algae use energy from sunlight to convert carbon dioxide (CO2) and water into carbohydrates. It is fundamental to most life on earth, because both autotrophic and heterotrophic species depend on carbohydrates generated from photosynthesis to sustain them. However, the photosynthetic efficiency for converting light energy to chemical energy is fairly low (<3%), even in some of the fastest-growing phototrophic species. Scientists have been trying to design faster and more efficient routes to produce essential chemicals, fuels, and macromolecules with CO2 and water to sustain the booming human population and global carbon inventory changes caused by economic development. In the past few decades, we have witnessed numerous milestones in "artificial photosynthesis," where chemists and biologists have developed various routes for harnessing the energy in sunlight. However, the primary product of most of these catalytic artificial photosynthesis routes is hydrogen.1Reece S.Y. Hamel J.A. Sung K. Jarvi T.D. Esswein A.J. Pijpers J.J.H. Nocera D.G. Wireless solar water splitting using silicon-based semiconductors and earth-abundant catalysts.Science. 2011; 334: 645-648Google Scholar When CO2 is used as a substrate, some artificial photosynthesis routes can produce one-carbon (C1) compounds like formate2Shan B. Vanka S. Li T.-T. Troian-Gautier L. Brennaman M.K. Mi Z. Meyer T.J. Binary molecular-semiconductor p–n junctions for photoelectrocatalytic CO2 reduction.Nat. Energy. 2019; 4: 290-299Google Scholar and methanol3Wu Y.A. McNulty I. Liu C. Lau K.C. Liu Q. Paulikas A.P. Sun C.-J. Cai Z. Guest J.R. Ren Y. et al.Facet-dependent active sites of a single Cu2O particle photocatalyst for CO2 reduction to methanol.Nat. Energy. 2019; 4: 957-968Google Scholar,4Styring S. Artificial photosynthesis for solar fuels.Faraday Discuss. 2012; 155: 357-376Google Scholar from CO2. Despite all of this progress, these routes don't produce products that are broadly used by heterotrophic species. The integration of chemical catalysis and microbial systems can overcome the barriers facing chemical catalysis alone. In 2016, Yang's group5Sakimoto K.K. Wong A.B. Yang P. Self-photosensitization of nonphotosynthetic bacteria for solar-to-chemical production.Science. 2016; 351: 74-77Google Scholar reported that decorating light-harvesting cadmium sulfide on Moorella thermoacetica enabled this nonphotosynthetic strain to use sunlight and convert CO2 to acetate. This type of photosynthetic biohybrid system (PBS) could transform the heterotrophic species to utilize light. However, microorganisms like M. thermoacetica use the Wood-Ljungdahl pathway, and the current primary product is acetic acid rather than carbohydrate. Furthermore, productivity depends on the addition of cysteine to a certain degree, and long-term cell survival is yet to be achieved. In parallel to using light energy to provide reducing power, electrocatalysis has also been explored to produce either hydrogen or C1 intermediates like formic acid. In 2011, Liao's group6Li H. Opgenorth P.H. Wernick D.G. Rogers S. Wu T.-Y. Higashide W. Malati P. Huo Y.-X. Cho K.M. Liao J.C. Integrated electromicrobial conversion of CO2 to higher alcohols.Science. 2012; 335: 1596Google Scholar demonstrated the conversion of CO2 to higher alcohols by using Ralstonia eutropha H16 in a fermentation system with electricity. CO2 is reduced to formic acid (HCOOH) via electrocatalysis, which can be converted to NADH, providing reducing power and the substrate for the autotrophic strains. In 2016, Silver and Nocera's groups reported a water splitting-biosynthetic system with CO2 fixation efficiencies exceeding photosynthesis.7Liu C. Colón B.C. Ziesack M. Silver P.A. Nocera D.G. Water splitting-biosynthetic system with CO2 reduction efficiencies exceeding photosynthesis.Science. 2016; 352: 1210-1213Google Scholar A biocompatible cobalt-based electrocatalyst split water into H2 and O2 at low voltage, and Ralstonia eutropha grown in the system utilized low concentration of CO2 and H2 to produce biomass, fuels, and chemicals. The integration of chemical catalysis with bioconversion has the unique ability to bypass the rate-limiting CO2 fixation steps in the biological systems via converting CO2 to small carbonaceous molecules. However, C1 compounds like formate and methanol have to go through the slow C1 metabolism in cells, and they are often toxic to the cells, making it challenging for microbial bioproduction. For hydrogen-driven CO2 conversion in Ralstonia, the carbon fixation rate is fundamentally limited by gas-to-liquid transfer rate. Therefore, it remains challenging to manufacture bioproducts from these C1 chemical species generated from CO2 reduction, particularly in cellular systems. In an article recently published in Science, Ma and co-workers have reported a cell-free route to synthesize starch from CO2 and hydrogen via a chemical-biochemical hybrid process, called the artificial starch anabolic pathway (ASAP).8Cai T. Sun H. Qiao J. Zhu L. Zhang F. Zhang J. Tang Z. Wei X. Yang J. Yuan Q. et al.Cell-free chemoenzymatic starch synthesis from carbon dioxide.Science. 2021; 373: 1523-1527Google Scholar As in previous studies, the ASAP also exploited the integration of chemical catalysis and biological system, but in an innovative way. Instead of utilizing a cell-based system, the study designed an innovative enzymatic route to convert CO2 hydrogenation-derived formate and methanol into starch. In a sense, the study achieved autotrophic conversion of CO2 to starch via integrating advancements in chemical catalysis for CO2 reduction and synthetic biology design, presenting an artificial route from CO2 to life-essential macromolecules. This system has addressed the aforementioned challenges in previous systems that integrated chemical catalysis with biological systems through at least three separate and innovative advances. First, Ma et al. achieved efficient conversion of CO2 to starch, bypassing the relatively slow photosynthesis system. In nature, starch metabolism begins with carbon anabolism on plant leaves (Figure 1A), in which the chloroplast harnesses the energy from sunlight to split water and produce NADPH and ATP (light reaction), providing reducing power and energy for the CO2 fixation in Calvin-Benson cycle (dark reaction). The slow regeneration of Calvin-Benson cycle intermediates and the low turnover rate of RuBisCo enzyme in the dark reaction limit both the overall photosynthesis efficiency and the productivity of carbohydrate from CO2 in plant or algae cells. In contrast, the researchers built enzymatic reactions to convert C1 chemicals such as formic acid or methanol in the reported cell-free system (Figure 1B). Formic acid and methanol can be efficiently produced by CO2 hydrogenation with inorganic catalysts.9Wang J. Li G. Li Z. Tang C. Feng Z. An H. Liu H. Liu T. Li C. A highly selective and stable ZnO-ZrO2 solid solution catalyst for CO2 hydrogenation to methanol.Sci. Adv. 2017; 3: e1701290Google Scholar,10Shao X. Yang X. Xu J. Liu S. Miao S. Liu X. Su X. Duan H. Huang Y. Zhang T. Iridium Single-Atom Catalyst Performing a Quasi-homogeneous Hydrogenation Transformation of CO2 to Formate.Chem. 2019; 5: 693-705Google Scholar The chemical catalytic CO2 reduction circumvents the rate-limiting CO2 fixation catalyzed by RuBisCo and the intermediate regeneration in the Calvin-Benson cycle, efficiently resolving these limiters of photosynthetic efficiency in natural systems. The new platform significantly advanced the starch synthesis rate from CO2, achieving 22 nmol min−1 mg−1 of total catalyst and proteins, which is calculated to be 8.5-fold higher than normal starch synthesis in maize and 5.7-fold higher than the synthetic CETCH cycle.11Schwander T. Schada von Borzyskowski L. Burgener S. Cortina N.S. Erb T.J. A synthetic pathway for the fixation of carbon dioxide in vitro.Science. 2016; 354: 900-904Google Scholar The rate increase mainly results from the relatively fast rate of CO2 hydrogenation to methanol, highlighting the great potential of hybridizing chemical catalysis and synthetic biology. Furthermore, because H2 could be acquired from green approaches, and the reaction could be driven by renewable energy from sunlight, this chemoenzymatic process could be recognized as another form of artificial photosynthesis. Therefore, the research provides a real case of producing bio-products using CO2 as the sole carbon source in a chemoenzymatic catalytic system, inspiring future efforts to develop more feasible routes toward macromolecule biosynthesis via artificial photosynthesis. Second, the synthetic pathway design is also novel, enabling the highly efficient conversion of C1 compounds into starch (Figure 1C). The cell-free system has an advantage over microbial conversion of C1 compounds, because the C1 pathways have less favorable energy and carbon flux partition in the cell. To design the ASAP pathway, the researchers used bioinformatics analysis to draft possible reaction sets from previous established databases, identifying that starch could be synthesized via nine core reactions from formic acid or methanol. However, unlike the natural pathways—which have evolved and optimized over a long period of time—the artificial pathway faces challenges of inhibition, because the enzymes originate from various organisms and might have disparate kinetics to prevent coordinated substrate utilization. To improve the overall efficiency, the researchers identified critical intermediates in the pathway and deconvoluted this complicated process into several modules, including C1 module for formaldehyde production, C3 module for D-glyceraldehyde 3-phosphate (GAP) production, C6 module for D-glucose-6-phospate (G-6-P) production, and Cn module for glucose assimilation and starch synthesis. In each module, they built multiple candidate reaction sets to address the incompatibility in the module assembly. For instance, when combining C1 and C3 modules, only thermodynamic-favorable methanol oxidation by alcohol oxidase (aox) could provide enough formaldehyde for C3 reactions. Another scenario that could occur during module assembly is that the byproducts or cofactors in former modules could inhibit the enzyme in latter modules. With substitution module reactions to address the incompatibility, the successful assembly of the four modules (C1e+C3a+C6b∗+Cnb) enabled 30 mg/L amylose titer from 20 mM methanol. The system is named ASAP 1.0. Based on this multi-module synthetic pathway, the researchers resolved the main bottlenecks to improve the efficiency in ASAP 2.0. Formolase (fls-M3) activity was improved 4.7-fold with directed evolution, and variants of fructose-bisphosphatase (fbp-AGR) and ADP-glucose pyrophosphorylase (agp-M3) enhanced the enzyme activity as well as ADP/ATP tolerance. The starch productivity of ASAP 2.0 is 7.6-fold of that in the ASAP 1.0. With the integration of CO2 hydrogenation unit, complete CO2 to linear amylose (ASAP 3.0) and branched amylopectin (ASAP 3.1) pathways were established. The keys to the successful assembly of such complex, multi-enzyme reactions are managing the carbon flux and energy consumption and improving the compatibility. This study is representative in this regard and demonstrates how modular design and directed evolution can be used to achieve complicated macromolecule synthesis from C1 compound. Another significant advancement of Ma et al.'s study the improvement of the interface between chemical and biocatalysis. The transformation of CO2 into water-soluble products such as formic acid and methanol is recognized as the critical step to allow biochemists to leverage synthetic biology for macromolecule synthesis. The high water solubility of products facilitates the subsequent enzymatic transformation, representing an advantage over the hydrogen or CO-driven CO2 bioconversion, which is often limited by the gas-to-liquid transfer rate. Starch is regarded as a "molecule of life" because it is a macromolecule commonly consumed by heterotrophic organisms, such as humans, to sustain their growth. The synthesis of starch from CO2 thus represents a significant improvement for artificial photosynthesis, in comparison with the production of smaller molecules like acetate and higher alcohols accomplished in previous studies. Despite considerable progress, there remain several limitations of the study that need to be addressed to achieve broader applications. First, the ASAP 3.0 and 3.1 are stepwise and discontinuous systems that cannot achieve integrated production. Such is a significant disadvantage as compared with the hydrogenotrophic microbial fixation of CO2 or electro-microbial systems for CO2 reduction and conversion.6Li H. Opgenorth P.H. Wernick D.G. Rogers S. Wu T.-Y. Higashide W. Malati P. Huo Y.-X. Cho K.M. Liao J.C. Integrated electromicrobial conversion of CO2 to higher alcohols.Science. 2012; 335: 1596Google Scholar,7Liu C. Colón B.C. Ziesack M. Silver P.A. Nocera D.G. Water splitting-biosynthetic system with CO2 reduction efficiencies exceeding photosynthesis.Science. 2016; 352: 1210-1213Google Scholar Second, the catalytic step for hydrogenation of CO2 requires high temperature (∼320°C) and high pressure (5 MPa), which requires significant energy input and is incompatible with biocatalysis or microbial conversion. Approaches that are amenable to the biological system need to be developed and integrated. Third, the total reaction time tested in the study is approximately 4 h. It is understandable that this study is mainly a proof of concept. However, achieving enzyme stability for a longer reaction time will be challenging. Further study could improve enzyme stability through immobilization and engineering. The microbial system can serve as an alternative, but the compatibility with electro- or photocatalysis still needs to be addressed. Overall, the study by Ma and colleagues is highly inspiring, and future work on system integration will promote broader applications.