When green carbon plants meet synthetic biology

Recycling carbon dioxide (CO2) into chemicals or fuels presents a promising avenue for mitigating carbon emissions and addressing the energy crisis. Plants serve as the ideal platform for the production of materials and chemicals, thanks to their innate capacity to directly use CO2 in the synthesis of various organic compounds. While conventional methods for enhancing plant CO2 fixation may reach their limits, novel technological solutions are imperative. Synthetic biology has illuminated the potential for biosynthesising multiple carbon sources through artificial CO2 fixation pathways in vitro. Recent breakthroughs in photorespiratory bypasses and artificial carboxylation modules offer significant promise for engineering plants to improve carbon fixation, guiding the design and development of plants with more efficient CO2 utilisation. In this context, we begin by summarising recent progress in designing or engineering in vitro CO2 fixation pathways, as well as those solely established in microbes. Subsequently, we delineate strategies employed to enhance CO2 fixation in plants. Finally, we explore potential methods for introducing artificial CO2 fixation pathways into plants. These advancements are critical in advancing synthetic biology's efforts to tackle future challenges related to food and energy scarcity.


INTRODUCTION
Rapid industrial development has led to an increase in natural products and petroleum to meet the economic development and the growing global population. 1 In the past 30 years, the extraction of coal, oil and natural gas has increased from 6.2 billion tons to 15 billion tons per year. 2 With the continuous consumption of fossil fuels, climate and environmental problems are becoming serious.A large amount of carbon dioxide (CO 2 ) is released into the atmosphere as a result of the burning of fossil fuels, which arouses worldwide attention to the greenhouse effect. 3The concentration of atmospheric CO 2 increased from approximately 277 ppm in 1750 4 to 421 ppm in 2022. 5The current increase rate of CO 2 in the atmosphere is at least 10 times faster than at any other time in the past 800,000 years. 6Thus, CO 2 from the atmosphere recycling directly into chemicals or fuels is a potential way to contribute to replacing fossil fuels.
The carbon atoms in CO 2 are in the highest oxidation state, while those in common fuels and chemicals are in the lower oxidation state. 3Thus, the synthesis of fuels and chemicals from CO 2 requires energy input, which is one of the reasons that CO 2 is not currently widely used in chemical industries. 3However, plants and microbes convert CO 2 into organics through six known natural CO 2 fixation pathways, including the Calvin-Benson-Bassham cycle (CBB cycle), 7 the reductive tricarboxylic acid cycle (rTCA cycle), 8 the Wood-Ljungdahl pathway (WL pathway), 9 the 3-hydroxypropionate bicycle (3HP bicycle), 10 the 3-hydroxypropionate/4-hydroxybutyrate cycle (3HP/4HB cycle), 11 and the dicarboxy propionate/4-hydroxybutyrate cycle (Di-4HB cycle). 12lthough CO 2 can be converted into organics in natural pathways, these pathways may not be optimal due to the low enzyme catalytic efficiency and oxygenase sidereaction of Rubisco as well as the remarkable amounts of energy consumption.Therefore, improved artificial pathways remain to be built and implemented.
To explore the potential pathway for artificial CO 2 fixation, Bar-Even et al. 13 first proposed the reductive glycine pathway (rGlyP) by combining computational approaches and systematic analyses.In this pathway, pyruvate is generated with CO 2 and formate.Professor Arren Bar-Even's group has made progress in the production of glycine from CO 2 and formate based on rGlyP in Escherichia coli (E.coli) 14 and yeast, 15 respectively.In addition, a strain of E. coli that grows on CO 2 and formate was obtained by equipping with rGlyP 16 and optimised to produce lactate. 17Recently, Sánchez-Andrea et al. 18 showed that Desulfovibrio desulfuricans assimilates CO 2 by rGlyP, which is a previously proposed yet unconfirmed natural CO 2 fixation pathway.
Compared to natural CO 2 fixation pathways, synthetic biology, as an emerging field, offers a new approach to create designed pathways of CO 2 fixation.It is an engineering approach to design, construct and analyse dynamic molecular devices and pathways aiming for the production of organisms and cells with customised functions from biological components. 19ynthetic biology takes a bottom-up paradigm to create biological systems with desired characteristics or to reconstitute existing natural systems by drawing on the principles of design and construction. 20Artificial CO 2 fixation pathways can be built by freely combining various enzymatic reactions from different biological sources, which are thermodynamically or kinetically preferable to naturally evolved CO 2 fixation routes. 213][24][25][26] However, the microbial chassis remains to be improved due to their restriction of the intracellular membrane, limitation of posttranslational modification, and the nonfunctional nature and negligible activity of target proteins. 27ompared with microbial chassis, plant chassis can be engineered at the level of its cells, tissues or the whole plant. 28Especially, the plant chassis could provide appropriate platform to express plant originated genes.Moreover, the photosynthesis of plants naturally fixes 105 billion tonnes of carbon per year and directly generates 210 billion tonnes of biomass. 29Thus, with the ability to utilise sunlight and CO 2 directly to produce a variety of organic compounds, plants are ideal platforms for the sustainable production of materials and chemicals in the future. 302][33][34] In this review, we initially summarise the new artificial synthetic pathways for direct CO 2 fixation and their respective characteristics.Then, we mainly focus on the methods that were implemented in the plant chassis to improve CO 2 fixation.Finally, we discuss the challenges and directions for future studies of introducing artificial pathways into plants, which will shed light on providing new horizons for the efficient utilisation of CO 2 in the future.

ARTIFICIAL CO 2 FIXATION PATHWAYS DESIGNED BY SYNTHETIC BIOLOGY IN VITRO OR IN MICROBES THAT COULD BE IMPLEMENTED IN PLANTS
CO 2 has already been converted into ethanol, 35 acetone, 36 lactic acid, 37 propanediol, 38 2-methyl-1butanol 39 and other chemicals by synthetic biology in recent decades.However, the productivity of these biological processes remains to be improved due to the limitation of natural carbon fixation.Thus, intensive studies have focused on the design of novel unnatural pathways to reduce CO 2 loss and improve fixation efficiency.
The developed novel pathways of CO 2 assimilation include cyclic and linear pathways.In the cyclic pathways, a continuous regenerative process is developed using the resulting product to restart the reaction.In contrast, a series of intermediates are generated to produce the final materials in the linear pathways.Thus, the artificial CO 2 fixation pathways will be presented in cyclic and linear pathways.

Cyclic pathways of CO 2 fixation pathways
Crotonyl-coenzyme A/ethylmalonyl-coenzyme A/hydroxybutyryl-coenzyme A (CETCH) cycle Schwander et al. 21designed and established the CETCH cycle of 17 enzymes to convert CO 2 into organic products in vitro, which was more efficient than photosynthesis (Figure 1a, version 5.4).However, the direct product glyoxylate of this pathway is a less active metabolite that is not well connected to other metabolic MODERN AGRICULTURE pathways.Thus, the CETCH cycle could be coupled with the β-hydroxyaspartate cycle (BHAC) and part of the serine cycle in vitro to fix CO 2 and generate acetyl-CoA. 40Both monoterpenes and sesquiterpenes were produced from CO 2 by combining terpene biosynthetic modules with CETCH-BHAC modules.Recently, 6deoxyerythronolide B (6-DEB) was produced with coupled CETCH-BHAC modules plus either the rTCA cycle, glyoxylate cycle, 3HP bicycle or ethylmalonyl-CoA (EMC) pathway in vitro. 41With the best combination of designed modules, 6-DEB was directly synthesised from CO 2 with an effective carbon conversion rate of 172% (carbon percentage yield of 6-DEB over starting substrate). 41he in vitro CO 2 fixation rate of the CETCH cycle was increased up to 5-fold than that reported for the CBB cycle in cell extracts per total amount of enzyme protein in the reaction mixture. 21This cycle can be further extended by combining with other pathways to broaden the product spectrum. 40,41Thus, the CETCH cycle could be a high-efficiency alternative to the CBB cycle.
However, because the CETCH cycle is a network of 17 enzymes and 13 core reactions, it is a challenge to balance the expression of 17 separate enzymes in the complex environment of the heterologous system.The expression level and localisation of proteins, enzyme activity and stability, and silencing of transgenes are the main challenges. 42After the introduction of the CETCH pathway, a great deal of optimisation should be carried out in CO 2 fixation efficiency and exogenous gene regulation to regulate the metabolic capacity of plants. 42ruvate carboxylase, oxaloacetate acetylhydrolase, acetate-CoA ligase, and pyruvate synthase (POAP) cycle The POAP cycle, as a minimised synthetic CO 2 fixation cycle that yields oxalate in vitro, was designed with pyruvate carboxylase, oxaloacetate acetylhydrolase, acetate-CoA ligase, and pyruvate synthase (Figure 1b). 33The CO 2 fixation rate of the POAP cycle could achieve 8.0 nmol CO 2 min −1 mg −1 enzymes. 33ince only four steps are involved in this cycle, the POAP cycle showed great advantages in the construction of plant expression vectors and genetic transformation.However, this cycle has the higher activity only under anaerobic conditions at 50°C because of the catalytic characteristics of pyruvate synthase.Thus, the POAP cycle is difficult to operate in plants if the catalytic properties of this enzyme cannot be overcome.In addition, the significant disadvantage of this cycle is that its product, oxalate, is highly oxidised and difficult to be converted to other metabolite.

Malonyl-CoA-glycerate (MCG) cycle
The MCG cycle was engineered to combine a threecarbon substrate and HCO 3 − to generate acetyl-CoA to increase the intracellular CoA concentration and established in vitro and in E. coli and cyanobacteria (Figure 1c). 43Both the intracellular acetyl-CoA pool and HCO 3 − assimilation are enhanced by approximately 2fold through this cycle in Synechococcus elongates. 43oreover, Luo et al. 34 designed a self-replenishing system by combining the reductive glyoxylate synthesis pathway, reductive pyruvate synthesis and MCG pathway, developing the rGPS-MCG cycle in which HCO 3 − is converted into acetyl-CoA, pyruvate and malate.The rGPS-MCG cycle showed the highest steady-state CO 2 fixation rate compared with fully established complex biocatalytic in vitro pathways. 34he MCG cycle can couple with the CBB cycle by using phosphoglycerate mutase, which converts 3phosphoglycerate to 2-phosphoglycerate and compensates for the deficiency of the CBB cycle in the efficient synthesis of acetyl-CoA. 43Glyoxylate, as an intermediate of photorespiration in plants, could be assimilated into acetyl-CoA by the MCG cycle to reduce photorespiration while increasing CO 2 fixation.In addition, two C4 pathway-related enzymes, phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase (MDH) are involved in the MCG cycle.Thus, it is possible to introduce part of the MCG cycle into C4 plants or synthetic C4 plants to increase CO 2 fixation while generating acetyl-CoA.

Gnd-Enter-Doudoroff (GED) cycle
The GED cycle was designed and established in E. coli to assimilate CO 2 and ribulose-5-phosphate to produce 6-phosphogluconate using endogenous E. coli enzymes (Figure 1d). 446-phosphogluconate is further converted into glyceraldehyde-3-phosphate and pyruvate by the Enter-Doudoroff pathway.Pyruvate is then converted to glyceraldehyde-3-phosphate in the process of gluconeogenesis.Glyceraldehyde-3-phosphate is recycled by the pentose phosphate pathway to regenerate ribulose-5-phosphate.The GED cycle is more ATP efficient than the CBB cycle.Therefore, the GED pathway could be used as an alternative pathway to the natural CBB cycle with relatively modest adjustments to the endogenous metabolic structure of carbon fixation.However, only developing seeds and roots sometimes require gluconeogenesis. 45Therefore, it is necessary to coordinate the localisation and expression of multiple enzymes in this cycle to introduce it into plants as an alternative CBB cycle.

Modified serine cycle
A modified serine cycle was constructed in E. coli to convert HCO 3 − to acetyl-CoA (Figure 1e). 46Compared to the natural serine cycle, this cycle consumes one more ATP in producing acetyl-CoA from HCO 3 − and methanol. 46However, some of the natural reactions were replaced by simplified steps to better fit the endogenous metabolism in the host. 46Similar to the MCG cycle, PEPC and MDH in the C4 pathway are also shared in this cycle.Thus, part of the modified serine cycle may be introduced into C4 plants to increase CO 2 fixation.

Linear pathways of CO 2 fixation pathways
Formolase (FLS) pathway and half-Wood-Ljungdahl-formolase (HWLS) pathway FLS, a computationally designed enzyme, catalyses the conversion of formaldehyde into dihydroxyacetone. 47FLS was used to design a carbon fixation pathway in vitro to convert CO 2 into dihydroxyacetone phosphate in five steps (Figure 1h). 47Hu et al. 48then designed the HWLS pathway to generate dihydroxyacetone phosphate from CO 2 and obtained an FLS* mutant to replace FLS in the HWLS pathway (Figure 1f).The production rate of dihydroxyacetone phosphate was increased 2.3-fold, and it was introduced into E. coli to increase the yield of L-malate and butyrate.The FLS pathway showed obvious advantages in reaction conditions, chemical driving force and biomass yield. 49Furthermore, the product of FLS and HWLS, dihydroxyacetone phosphate, is directly linked to glycolysis. 47,48Therefore, these pathways have become more feasible for application in vivo.

Artificial starch anabolic pathway (ASAP)
The ASAP, a hybrid chemical-biochemical pathway from CO 2 to starch in vitro, has been established in a cell-free system by computational pathway design, modular assembly and protein engineering with a rate 8.5-fold higher than that in maize (Figure 1g). 32With the resulting C6 products, commercial compounds such as starch could be synthesised by extending the reaction pathway.If this pathway is introduced into plants, the glyceraldehyde-3-phosphate produced through the CBB cycle 50 could be used as the substrate for starch synthesis in ASAP.Thus, the steps from CO 2 to glyceraldehyde-3-phosphate in ASAP could be introduced into plants to simplify the CBB cycle.Therefore, ASAP manifests as an efficient synthetic system for MODERN AGRICULTURE -101 CO 2 fixation and sheds light on simplifying and improving starch synthesis in plants.

Other linear pathways
In addition to the linear pathways starting with CO 2 , there are some CO 2 -added linear pathways.For example, the tartronyl-CoA (TaCo) pathway was designed to convert glycolate into glycerate with a novel carbon fixation step through the carboxylation of HCO 3 − with glycolyl-CoA. 31This pathway could act as a photorespiratory bypass to increase CO 2 fixation or be combined with biotechnological and agricultural processes for downstream reactions. 31

IMPROVED CO 2 FIXATION PATHWAY DESIGNED BY SYNTHETIC BIOLOGY AND IMPLEMENTED IN PLANTS IN VIVO
In the above section, we described the artificial pathways that are designed to assimilate CO 2 directly in vitro or in microbes, based on which potential methods used for improving plant CO 2 fixation in vivo are summarised and discussed in the present section.
The CBB cycle, as the universal pathway for CO 2 fixation in natural plants, has been intensively studied and characterised.Rubisco, as the first and key enzyme in the CBB cycle, was initially designed to increase CO 2 fixation by increasing CO 2 specificity 51 and catalytic rates. 52However, only limited success has been made for the imbalance between the activity and specificity of Rubisco, 53 and the related studies have been well reviewed by Sharwood, 54 Andralojc et al. 55 and Jin et al. 56 Moreover, Schulz et al. 57 found that small subunits of Rubisco are responsible for the high specificity of CO 2 .The gain of this accessory subunit could be more productive rather than changing amino acids in Rubisco.Thus, the traditional approaches of improving Rubisco by solely modifying its amino acids may need to be corrected.Here, we mainly discuss the improved CO 2 fixation pathway designed by synthetic biology, implemented in plants in vivo and evaluated by experiments, including alternative pathways to the photorespiration/C2 pathway and the implementation of carbon concentrating mechanisms (CCMs) in plants.

Alternative pathways to photorespiration/ C2 pathway
The photorespiration/C2 pathway originates from the oxygenase activity of Rubisco, starting from the oxidation of RuBP and generating 3-phosphoglycerate and phosphoglycolate. 58Phosphoglycolate is toxic in chloroplasts because of its strong inhibition of several key enzymes in the CBB cycle. 59,60To detoxify phosphoglycolate, two molecules of phosphoglycolate are converted into one molecule of 3-phosphoglycerate, during which one carbon atom is lost as CO 2 and results in 25% carbon loss. 61Therefore, there is great potential for increasing CO 2 fixation by designing alternative pathways to photorespiratory bypasses and implementing them in plants to decrease CO 2 loss.To date, five photorespiration bypasses have been assembled and implemented in plants, including three targeted to chloroplasts and two located in peroxisomes (Table 1). 62Detailed information on the alternative pathways to the photorespiration/C2 cycle is as follows.
][65][66][67][68][69] Glycolate is oxidised to glyoxylate by glycolate dehydrogenase (GDH) or glycolate oxidase (GLO).The byproduct H 2 O 2 of GLO-mediated catalysis is scavenged by catalase (CAT). 67The glyoxylate is then catalysed by glyoxylate carboligase (GCL) to generate tartronic semialdehyde and CO 2 , and the resulting tartronic semialdehyde is converted to glycerate by tartronic semialdehyde reductase.CO 2 is released into the chloroplast because all the catalytic steps are established in the chloroplast, where it can be recycled into the CBB cycle.Thus, the CO 2 concentration is enhanced by the glycolate catabolic pathway, which facilitates the carboxylation of Rubisco while decreasing CO 2 loss in the photorespiration pathway.
Compared to the partial decarboxylated glycolate cycle mentioned above, a complete decarboxylated version has also been introduced into the chloroplast. 65,70,71Glycolate is converted to glyoxylate by GDH or GLO, followed by the generation of malate from glyoxylate and acetyl-CoA via malate synthase.Malate is then decarboxylated into pyruvate and CO 2 by malic enzyme (ME).Pyruvate is then converted into acetyl-CoA and CO 2 by pyruvate dehydrogenase.Finally, acetyl-CoA is combined with glyoxylate to re-enter the bypass.In this pathway, CO 2 is released into the chloroplast to be recycled into the CBB cycle.Therefore, the byproducts of photorespiration are totally recycled back into the CBB cycle by generating CO 2 completely.
In addition, glycolate has also been totally converted into CO 2 in a series of catalysis reactions located in the chloroplast. 72In this pathway, glycolate is converted to oxalate and H 2 O 2 by GLO, and then oxalate is oxidised to H 2 O 2 and CO 2 by oxalate oxidase.H 2 O 2 is further decomposed by CAT.Similar to the bypass described above, CO 2 is released into the chloroplast but in a total ratio.
In the above three pathways, two different enzymes are used to catalyse the conversion of glycolate to glyoxylate.In higher plants, GLO in peroxisomes is responsible for this conversion.However, this enzyme wastes reducing power because it uses O 2 as an electron acceptor to form H 2 O 2 .Compared to oxygendependent GLO, GDH uses cofactors as the H acceptor to preserve reducing power. 73In the study of South et al., 65 three enzymes from different sources were used to catalyse this step.AP1 is a glycolate catabolic pathway that uses GDH from E. coli, and AP2 and AP3 are the complete decarboxylated pathways that use GLO from Arabidopsis and GDH from Chlamydomonas reinhardtii, respectively.However, only AP1 and AP3 plants that used GDH showed significant increases in biomass compared with WT, and AP3 lines exhibited the greatest growth accumulation.It appears that the introduction of GDH is a valuable strategy for improving plant growth.Furthermore, GDH from E. coli contains three subunits, which is difficult in vector construction and plant genetic transformation.Therefore, GDH from Chlamydomonas reinhardtii may be the best alternative enzyme at present.
Instead of the strategies that increase the concentration of CO 2 in chloroplasts, the pathways in peroxisomes focus on avoiding CO 2 release in mitochondria.In the first design of peroxisomes, Carvalho et al. 74 converted glyoxylate into tartronic semialdehyde by GCL, and the resulting tartronic semialdehyde is catalysed into hydroxypyruvate by hydroxypyruvate isomerase (HYI) to feed back into the photorespiration pathway. 74Unfortunately, this pathway is not completely established because HYI is not expressed in all transgenic plants.The second photorespiration bypass in peroxisomes is the BHAC cycle. 75Glyoxylate and aspartate are converted into oxaloacetate and glycine by aspartate:glyoxylate aminotransferase, and then glyoxylate and glycine are condensed into β-hydroxyaspartate in the presence of β-hydroxyaspartate aldolase.The resulting β-hydroxyaspartate is converted to iminosuccinate by β-hydroxyaspartate dehydratase.Finally, iminosuccinate is reduced to aspartate via iminosuccinate reductase to regenerate the amino group donor that participates in this bypass.This work showed no carbon loss and suggested that alternative C2 pathways can produce other products involved in the biological cycle in plants rather than just returning to the CBB cycle.
In addition, when comparing pathway introduction in these two organelles, chemical energy (ATP), reducing power (NADPH) and primary and secondary metabolites are produced by photosynthesis in chloroplasts, 76 making this organelle attractive for synthetic biology.However, chloroplasts are also the place of various metabolic reactions, including the production of amino acids, lipids and hormones. 77Therefore, the metabolic pathways involved in chloroplasts are complex and difficult to regulate.It is necessary to adapt the regulation of natural metabolic pathways when synthetic pathways are introduced.In contrast, peroxisomes are small cell organelles that are mainly involved in photorespiration and fatty acid β-oxidation. 78It may be simpler to manipulate in peroxisomes than in more complex organelles.Moreover, targeting the synthetic pathways to plant peroxisomes may also minimise the plant growth defects caused by targeting chloroplasts. 79However, although great progress has been made in understanding peroxisomal metabolism, much remains to be discovered.Thus, the introduction of novel synthetic pathways may have negative effects on substrate homoeostasis and whole cellular metabolism.

Establishment of CCMs in plants
To alleviate the CO 2 loss in photorespiration induced by Rubisco oxygenation or CO 2 leakage, CCMs are well developed during evolution among microalgal species, which results in a CO 2 -enriched environment around Rubisco.Therefore, scientists have endeavoured to introduce CCMs to increase carbon fixation (Figures 2a  and 2b) by implementing either biophysical CCMs or C4 photosynthesis into plants.

Implementation of biophysical CCMs into plants
Biophysical CCMs are widely identified in green algae 80 and cyanobacteria, 81 and they are mainly composed of HCO 3 − transporters, carbonic anhydrase (CA), and pyrenoids or carboxysomes. 82,83 HCO 3  − is transported into the cytosol by HCO 3 − transporters and further transported into pyrenoids or carboxysomes packed with Rubisco, where HCO 3 − is dehydrated to CO 2 by CA. 84 Pyrenoids and carboxysomes present a diffusion barrier for keeping CO 2 in and O 2 out to enhance Rubisco carboxylation. 85Thus, CCMs provide an ideal environment for CO 2 fixation, which would enforce Rubisco carboxylation when implemented in higher plants.People have made great progress in understanding the underlying mechanisms of CCMs, which makes the introduction of CCMs into plants possible.
In testing the possibility of pyrenoid assembly in higher plants, McCormick's group conducted a series of studies on appropriate subcellular localisations of CCM components in tobacco or Arabidopsis and the assembly of Rubisco from Arabidopsis and algae. 86,87However, Rubisco condensation was not observed in Arabidopsis-algal hybrid Rubisco, although the full coding sequence of essential pyrenoid component 1 (EPYC1) was expressed. 88Interestingly, the expression MODERN AGRICULTURE of mature EPYC1 resulted in the spontaneous condensation of Rubisco into a phase separation in Arabidopsis chloroplasts with a plant-algal hybrid Rubisco background, which was similar to pyrenoid substrates. 89herefore, the interaction between EPYC1 and hybrid/ native Rubisco remains to be explored to optimise the results.
In evaluating carboxysome assembly in plants, Lin et al. reported that β-carboxysome shell proteins were able to assemble in tobacco chloroplasts, 90 and the Rubisco large and small subunits from cyanobacteria could replace the natural Rubisco large subunit in tobacco transplastomic lines to form macromolecular complexes in the chloroplast stroma by expressing along with an assembly chaperone or an internal carboxysomal protein. 91With cyanobacterial Form-1A genes encoding Rubisco large and small subunits, Long et al. 92 replaced endogenous genes in tobacco, and along with two key genes of α-carboxysome structural proteins introduced, simplified carboxysomes were produced that enabled plants to grow autotrophically under high CO 2 conditions.These results indicate that the carboxysome could be simply assembled in the chloroplasts of plants.
Both strategies provide ideal microenvironmental conditions for CCMs by forming compartments.The structural specificity, functional stability, and localisation accuracy of pyrenoid and carboxysomes introduced into chloroplasts are important for CO 2 concentration.In addition, the issues of the function of bicarbonate channels and the activity of CA need to be addressed to transport inorganic carbon precisely and decarboxylate HCO 3 − efficiently.Admittedly, the functional characterisation of pyrenoids and carboxysomes requires many studies, which hinders their implementation in plants.The successful implementation of biophysical CCMs in plants will pave the way for improving CO 2 fixation in the future.

Rebuilding of CO 2 fixation in plants based on the C4 pathway
The rebuilding of the CO 2 fixation system of C4 photosynthesis in C3 plants provides an alternative pathway to biophysical CCMs.In the C4 pathway, CO 2 is converted to HCO 3 −, which is then assimilated with phosphoenolpyruvate (PEP) to oxaloacetate by PEPC. 93Oxaloacetate is reduced to malate, which is transported to the bundle sheath cells and decarboxylated to pyruvate and CO 2 to enter the CBB cycle. 93yruvate is converted to PEP to restart the C4 pathway. 93CO 2 absorption and fixation by the CBB cycle occur in two separate cells, which increase the CO 2 concentration around Rubisco and reduce the photorespiration process. 94Lin et al. 95 introduced four key enzymes of the C4 pathway from maize to rice, but there was no carbon flux elevation into the CBB cycle based on a nonsignificant difference in the labelling of 3-phosphoglycerate.Ermakova et al. 96 assembled a single construct containing five core C4 photosynthetic genes of maize and transformed it into rice, but the flux from malate through pyruvate to PEP remained low despite the increased flux of PEPC.The lack of proper compartmentalisation of the expression and activity of these genes may prevent the function of C4 photosynthesis.Thus, Kranz two cell-type anatomy modifications need to be installed in these plants to achieve the full function of the C4 pathway.However, the functional characterisation of Kranz anatomyassociated genes is considered a challenging project. 97Therefore, it is necessary to understand the structural and functional differentiation of C4 plants.This will provide a milestone towards rebuilding a functional C4 module in C3 plants aiming to improve CO 2 fixation.

INTRODUCTION OF ARTIFICIAL CO 2 FIXATION PATHWAYS INTO PLANTS
The establishment of photorespiration bypasses and CCMs manifested promising results in enhancing CO 2 fixation in vivo; nonetheless, the reported pathways are designed based on grafting natural pathways into plants.To overcome this natural limitation, the introduction of the artificial CO 2 fixation pathways described above into plants provides potential methods to greatly increase CO 2 fixation.Here, we proposed possible solutions for artificial CO 2 fixation pathway introduction in plants based on the reported pathway.

Determining the cellular localisations of the introduced proteins and enzymes
In introducing artificial CO 2 fixation pathways into plants, it is essential to consider potential subcellular localisation.Since plants have evolved differentiated cells and tissues, the complex structure and gene regulation could affect the functions of the introduced genes. 28Thus, adding, removing or modifying the targeting peptides could alter the subcellular location of heterologous proteins in the artificial pathway and affect the resulting CO 2 fixation. 98he targeting peptides are short chains of 3-70 amino acids that direct peptide and protein transport to specific regions in cells. 999][110] Two types of peroxisomal targeting signals have been identified within the C-terminus (PTS1) and N-terminus (PTS2) of peroxisomal-destined proteins. 111The overall sequence of PTS1 contains three amino acids following the consensus of [SA][RK] [LM] in plants, 112 and PTS2 is a nonapeptide with the consensus R[ILQ]X 5 HL. 113,114he difficulty in heterologous protein targeting is that the same targeting peptides may have different effects and targeting efficiencies on various proteins and may even be unable to target proteins to specific organelles. 111Therefore, protein structure analysis, transient expression and western blot analysis are essential to determine the localisation of heterologous proteins before genetic transformation.

Optimising the expression of introduced genes
In addition to the target peptides, heterologous protein expression is another challenge when introducing artificial CO 2 fixation pathways into plants owing to a series of factors, such as codon usage, 115 and promotor and terminator combinations. 116,117Codon optimisation, a process of altering the codons in the gene sequence without altering the protein sequence to improve the expression of recombinant proteins, has been widely considered in studies. 118oreover, promoters, an important module in the regulation of transgenic expression, have received much attention in recent years. 119The cauliflower mosaic virus (CaMV)-originated 35S promoter and maize ubiquitin-1 (Ubi-1) promoter are widely used in dicot and monocot plants, respectively. 119Mandadi's group developed a combinatorial stacking gene-promoter expression system that coexpressed recombinant proteins under a triple promoter and quadruple promoter to significantly increase Galanthus nivalis L. (snowdrop) agglutinin and bovine lysozyme, respectively. 120,121eanwhile, terminator effective polyadenylation has a great influence on gene transcription and mRNA translation from the nucleus to the cytoplasm. 122The nopaline synthase (NOS) terminator is commonly used in plant expression constructs. 123Rosenthal et al. 124 found that the tobacco extensin gene terminator without its native intron could enhance the production of recombinant proteins in plants compared with NOS, CaMV35S terminator and soybean vegetative storage protein terminator.
Thus, similar to determining the localisation of heterologous proteins, transient expression systems are also used to confirm that the gene expression cassettes could achieve appropriate levels of protein expression.In addition, multiple designs of promoter and terminator combinations need to be tested to optimise protein expression.

Decrease the interference and increase the product of pathway introduction into plants
In addition to the localisation and expression of individual proteins or enzymes, systematic regulation also needs to be considered.The complicated CO 2 fixation in plant systems is notoriously difficult to design because even small changes can have significant effects that are hard to predict. 84Furthermore, the engineering of targeted pathways will interfere due to the strict metabolic regulation and complex feedback regulation mechanism in plants. 28Although mathematical models can help manage the complexity of plant CO 2 fixation, a lack of knowledge of the relevant components commonly compromises accurate predictions. 84The transcriptome and metabolome of transgenic plants with different pathways could be determined to explain the transcriptional basis and signal transduction and help elucidate metabolic crosstalk.In addition, metabolite synthesis and accumulation in specific tissues by the development and application of transgene expression could avoid negative effects on plant growth. 125For example, Guiziou et al. 126 innovated an integrase-mediated DNA switch in Arabidopsis under the control of a lateral root development promoter to label all lateral root descendants, making it possible to restrict gene expression at specific times and spaces during plant development.Apart from internal events, external stimuli can activate synthetic regulatory elements.Kallam et al. 127 developed a copper-inducible expression system in Nicotiana benthamiana to control the timing and levels of insect sex pheromone production to reduce the adverse effect on plants.Therefore, the utilisation of advanced regulatory elements could help regulate the spatial and temporal expression of the pathway during the whole development of plants to decrease interference with plant growth.
Another consideration is that production in a heterogeneous host may reduce the yield of the product. 128Kim et al. 129 increased the production of etoposide aglycone (EA) by coexpressing AtMYB85 with EA pathway genes in Nicotiana benthamiana to improve precursor availability.These results suggested that transcription factors play an important role in promoting specialised metabolite biosynthesis in a plant heterologous host.In addition, Barnum et al. 130 identified auxiliary genes by screening a combination of uncharacterised coexpressed genes and selecting genes alongside glucoraphanin biosynthetic pathways and finally achieved high glucoraphanin production in Nicotiana benthamiana.These results indicated that the optimal combination of heterologous expression with the metabolism of the host could potentially increase bioproduction. 128

CONCLUSIONS
Carbon is an important element in life and ecological processes.At present, scientists are mainly focusing on studies of artificial CO 2 fixation pathways in vitro or in microbes, while plant systems remain to be explored.It is estimated that approximately one-fifth of the carbon in the atmosphere is fixed by plants per year, 131 which demonstrates the great potential and efficiency of plant chassis for CO 2 fixation.Considering that the improvement and utilisation of plant carbon fixation are still limited to the engineering of natural pathways, we will have to conduct intensive studies on plant synthetic biology to uncover novel carbon fixation pathways that may provide clues to overcome natural limitations (Figure 3).Although the in vivo or in vitro pathways of CO 2 fixation in plants and microbes have been described individually, these pathways could be recombined and built for metabolic engineering.For example, the defects of hydroxypyruvate reductase results in an increase in glycolate, 132 which allows us to implement the artificial synthetic pathway into the mutant to enhance the carbon flow to targeted highvalue products.Additionally, the amino acid metabolic profile could be improved in accordance with the amino acid composition of the target product.Furthermore, the creation of novel aspect of gene-mRNA-proteinmetabolite regulatory networks may aid in the development of novel strategies for engineering plants to assimilate CO 2 while producing valuable products.Therefore, rebuilding of metabolism and reshaping of core biochemical processes are essential for the implementation of biosynthetic/artificial pathways in plants, and these will unleash the potential of plant

F I G U R E 1
Synthetic cyclic pathways and linear pathways for CO 2 fixation.(a) The crotonyl-coenzyme A/ethylmalonyl-coenzyme A/ hydroxybutyryl-coenzyme A (CETCH) cycle.(b) Pyruvate carboxylase, oxaloacetate acetylhydrolase, acetate-CoA ligase, and pyruvate synthase (POAP) cycle.(c) Malonyl-coenzyme A-glycerate (MCG) cycle.(d) Gnd-Enter-Doudoroff (GED) cycle.(e) The modified serine cycle.(f) The half-Wood-Ljungdahl-formolase (HWLS) pathway.(g) The artificial starch anabolic pathway (ASAP).(h) The formolase (FLS) pathway.The green square represents the consumption of one molecule NAD(P)H.The yellow square represents the consumption of one molecule of ATP.The red colour indicates that these pathways are conducted in vitro.The green colour indicates that these pathways are conducted in E.scherichia coli.The yellow colour indicates that this pathway is conducted in vitro and in E. coli and cyanobacteria.

F I G U R E 2
Establishment of carbon concentrating mechanisms (CCMs) in plants to improve the CO 2 fixation.(a) Transferring green algae and cyanobacteria CCMs into chloroplasts.The blue hexagon represents the installation of pyrenoid or carboxysome structures.(b) Introducing the C4 mechanism into C3 plants.CA, carbonic anhydrase; PEPC, phosphoenolpyruvate carboxylase; MDH, malate dehydrogenase; ME, malic enzyme; PPDK, pyruvate phosphate dikinase; OAA, oxaloacetate.

F I G U R E 3
Improvement of CO 2 fixation in plants by synthetic biology approaches.
meet the requirements of humans in the future.