The efficient capture of CO2 by carboxylases is crucial for sustainable biocatalysis and a carbon-neutral bioeconomy. However, the number of naturally occurring enzymes capable of this is limited. The conversion of C2 compounds, such as glycolate and glyoxylate, into C3 metabolites is vital in various carbon metabolic processes, including photorespiration, fatty acid assimilation, formato-, and methylotrophy. Glyoxylate also serves as a key metabolite in several synthetic CO2 fixation pathways. Existing natural pathways for C2 to C3 conversion are inefficient, involving carbon loss. Photorespiration, for example, is estimated to reduce agricultural crop yields by up to 50% in hot and dry climates due to CO2 loss. The tartronyl-CoA (TaCo) pathway was proposed as a hypothetical, more efficient pathway, but its realization required the engineering of glycolyl-CoA carboxylase (GCC), its key enzyme, which was the main challenge addressed in this study. This research focuses on the successful reconstitution and in vitro implementation of the complete TaCo pathway using rational design and high-throughput enzyme evolution, and it demonstrates the pathway's application in relevant biotechnological and agricultural processes.

The literature review highlights the limitations of existing natural pathways for converting C2 compounds (glycolate and glyoxylate) to C3 metabolites. These pathways, including the glyoxylate cycle, the β-hydroxyaspartate cycle, and photorespiration, all result in the net loss of carbon, significantly reducing their carbon efficiency. This inefficiency is especially pronounced in photorespiration, leading to considerable yield losses in agriculture. The study references previous work on synthetic CO2 fixation pathways and the proposed TaCo pathway as a potential solution to improve glycolate assimilation. Existing research on engineering biotin-dependent acyl-CoA carboxylases is also discussed, highlighting the challenge of altering substrate preference.

The study employed a multi-faceted approach combining rational design and high-throughput evolution. Initially, the researchers investigated two strategies for synthesizing the non-native carboxylation substrate glycolyl-CoA: CoA transfer from another acyl-CoA donor and direct ligation of glycolate to CoA. Screening of various enzymes revealed *Clostridium aminobutyricum* AbfT as the best CoA transferase and an acetyl-CoA synthetase (ACS) homologue from *Erythrobacter* sp. NAP1 (EryACS1) as the best CoA synthetase. EryACS1 was further engineered to enhance its catalytic efficiency. The researchers created a lysine acetylase knockout strain to prevent post-translational inactivation. Further directed mutagenesis targeted Val379, resulting in the variant Val379Ala (GCS) with improved kinetic parameters. For the carboxylation of glycolyl-CoA to tartronyl-CoA, the researchers screened different biotin-dependent propionyl-CoA carboxylases (PCCs). MePCC from *Methylorubrum extorquens* exhibited the highest activity, though low. Structure-guided rational design, using a 3.48-Å cryo-EM structure of MePCC, identified key residues for mutagenesis. Variants were created to accommodate the hydroxyl group of glycolyl-CoA. Subsequently, directed evolution using high-throughput microfluidics and microplate screens further optimized the enzyme, resulting in GCC M5 with a catalytic efficiency three orders of magnitude higher than the wild type. The 1.96-Å resolution cryo-EM structure of GCC M5 validated the design strategy. The TaCo pathway was reconstituted in vitro, and its thermodynamic profile was calculated. To optimize continuous pathway operation, it was coupled with different ATP regeneration modules. The TaCo pathway was then interfaced with photorespiration, ethylene glycol conversion, and the CETCH cycle to test its functionality in various applications. Detailed methods for library generation, screening, pathway reconstruction, and cryo-EM are provided in the Supplementary Information.

The key findings include the successful engineering of GCC M5, a new-to-nature enzyme with a catalytic efficiency comparable to natural CO2-fixing enzymes. The 1.96 Å cryo-EM structure of GCC M5 validated the structural modifications enhancing substrate specificity. The complete TaCo pathway was reconstituted in vitro and shown to efficiently convert glycolate to glycerate. The TaCo pathway was successfully interfaced with photorespiration, significantly increasing the carbon efficiency (from 75% to 150%) and reducing ATP and reducing equivalent requirements compared to natural photorespiration. It also successfully improved the carbon efficiency of ethylene glycol conversion and the synthetic CETCH cycle, illustrating its versatility in various applications. Stoichiometric calculations showed the potential for up to a 150% increase in carbon efficiency and decreased energy demand in these processes.

The successful engineering of GCC and the reconstitution of the TaCo pathway demonstrate the feasibility of creating new-to-nature metabolic pathways with improved carbon and energy efficiency. The integration of the TaCo pathway with various processes significantly enhances their overall performance, particularly in addressing the inefficiencies of natural photorespiration. The findings highlight the potential of synthetic biology to improve agricultural productivity and create sustainable biotechnological solutions. The observed improvement in carbon and energy efficiency suggests that similar approaches could be applied to other metabolic pathways. The limitations in the wild-type enzyme and the need for ATP regeneration are addressed.

This study successfully engineered a new-to-nature CO2-fixing enzyme, GCC, and integrated it into a functional TaCo pathway. The TaCo pathway significantly improved the efficiency of natural and synthetic CO2 fixation processes. Further research could focus on improving enzyme activities through directed evolution, enhancing pathway flux, and minimizing futile ATP hydrolysis. The approach taken here opens new avenues for expanding the solution space of natural metabolism and creating superior biotechnological and agricultural applications.

While the study demonstrated significant improvements in carbon and energy efficiency, further optimization of the TaCo pathway is needed. The futile ATP hydrolysis by GCC, although significantly reduced in GCC M5, could be further minimized. The in vitro studies might not fully reflect the complexities of in vivo conditions. The long-term stability and scalability of the engineered system remain to be investigated.