AbstractChemicals produced through enzymatic reactions play a key role in the transition from a linear petrol-dependent to a circular bioeconomy. One promising approach is the conversion of single carbon (C1) molecules by biocatalysts to value-added products. Although progress has been made, current biological methods remain less cost-competitive than established chemical processes. Here, we review how single and multi-enzyme transformations, natural C1-trophic microorganisms, and organisms with transplanted synthetic C1 assimilation pathways can synergize to strengthen the competitiveness of C1-based biomanufacturing. To explore the current state-of-the-art and assess the potential of C1 biomanufacturing, we highlight the aforementioned bio-based methodologies and evaluate their industrial applicability through an overview of granted patents.IntroductionAutotrophic carbon fixation converts inorganic carbon into complex organic molecules and is an important bioprocess in nature, providing the building blocks of life1. It also produces organic chemicals and energy that are essential to human society2. The rapid growth of the world’s population over the past century and the depletion of fossil resources have led to a more than fourfold increase in global carbon dioxide (CO2) emissions since the 1960s. Our fossil fuel-based lifestyle, rapidly promoting climate change, already has a deleterious impact on nature and human society. These developments fuel constant efforts to reintegrate the greenhouse gas CO2 into a circular (bio)economy, assisting to provide a sustainable source of energy and feedstock for future generations. One of the potential approaches to advancing Circular Carbon Economy (CCE) is through biotechnological applications that utilize single-carbon (C1) building blocks to upgrade them to more complex chemicals. C1 building blocks, such as CO2, carbon monoxide (CO), methane (CH4), methanol (MeOH), and formic acid/formate (HCOOH/HCO2−), can be derived from various sources, including industrial waste gases, agricultural residues, biomass, and municipal solid waste. In addition, multiple C1 building blocks can be produced through the electrochemical reduction of CO23,4. In addition, these C1 compounds can, in some cases, also serve as both carbon and energy sources4,5, further enhancing their attractiveness as C1 feedstocks. Building blocks like sugars can also be used for the production of chemical compounds, with in vivo approaches via cellular fermentation or in vitro biocatalytic reactions, but they have several disadvantages such as (1) cost and broad availability, (2) land use and potential environmental impact, (3) seasonal dependence, (4) and in some cases competition with food supply which raises ethical concerns and impact global food security6.Ideally, the energy required for CO2 reduction should come from renewable sources such as solar panels and wind turbines7. This, in turn, would also facilitate the storage of excess electricity in stable chemicals such as fuels8,9. In addition, green hydrogen (H2) is considered a promising electron donor in the utilization of C1 compounds, as it can be produced by electrolysis of water using renewable electricity. Formaldehyde (HCHO) is also studied to be obtained from CO2 or being utilized to store H210,11. However, the production of green H2 is not yet cost-competitive with H2 produced from fossil fuels. For example, in 2018, H2 produced by electrolysis using solar electricity was ~25 times more expensive than that derived from hydrocarbons12. Nevertheless, green H2 is likely to play an important role in providing industrial feedstock for cleaner production of ammonia and organic chemicals, as highlighted in the 2022 Intergovernmental Panel on Climate Change report13.Biotechnological processes, such as microbial fermentation and enzyme catalysis, can be employed to convert C1 building blocks into valuable chemicals, polymers, and fuels, thereby contributing to carbon circularity and reducing dependence on fossil-based feedstocks14,15. Currently, microbial cell factories are considered promising biocatalysts for the catalytic upgrading of various C1 feedstocks16,17, but in vitro routes that combine electrochemical and biochemical steps also hold great promise for achieving the necessary high rates and product yields that have been challenging to achieve with fermentative processes due to reduced product yields despite lower substrate costs. To increase the cost competitiveness of microbial C1-based bioprocesses, synthetic biologists and metabolic engineers are currently exploring two routes to increase the efficiency of microbial C1 utilization and conversion: (1) engineering native pathways of autotrophic microorganisms such as microalgae, cyanobacteria, and chemolithoautotrophic bacteria; and (2) designing synthetic C1-utilizing microorganisms and metabolic routes in either autotrophs or biotechnologically relevant heterotrophs. These approaches can create synergies to strengthen the competitiveness of C1-based biomanufacturing, particularly because the synthetic pathways are expected to have several advantages over natural autotrophs, such as improved energetic efficiencies, yields, or minimal overlap with the central carbon metabolism of the host5,18,19,20,21,22,23,24,25,26. Synthetic C1-trophic organisms are still somewhat underutilized, and few of the reported synthetic pathways match the natural pathways of autotrophs after their introduction into heterotrophs25. However, recent achievements in the field—ranging from the first eukaryotic Komagataella phaffii (ex Pichia pastoris)27 and prokaryotic Escherichia coli heterotrophs28 capable of assimilating different C1 molecules29,30,31,32—represent important milestones to further explore the potential of synthetic C1-trophic organisms (Fig. 1)4. For instance, Chen et al. designed the first non-methylotrophic strain (E. coli) that uses MeOH as its sole carbon source32. Luo et al. recently showed the successful design, realization, and optimization of a synthetic CO2 fixation pathway, the reductive tricarboxylic acid (TCA) branch/4-hydroxybutyryl-coenzyme A (CoA)/ethylmalonyl-CoA/acetyl-CoA (THETA) cycle33. These authors demonstrated how this THETA cycle can be modularized and transferred to (heterotrophic) E. coli.Fig. 1: Timeline of scientific milestones regarding biocatalytic applications for the use of C1 building blocks towards a CCE.Milestones are divided into the application of (isolated) enzymes and fermentative approaches operating in (engineered) whole-cells. CETCH Crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA, sMMO Soluble methane monooxygenase, HOPAC Hydroxypropionyl-CoA/acrylyl-CoA, MAP MeOH-alanine pathway, Lcm L-lactyl-CoA mutase, FDH Formate dehydrogenase, FalDH Formaldehyde dehydrogenase.Full size imageConsidering in vitro approaches, several studies focusing on enzymatic carbon fixation strategies are reported in literature. A synthetic pathway—known as the crotonyl- CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle—enables the continuous fixation of CO2 in vitro. This reaction network, involving 17 enzymes, converts CO2 into organic molecules at a rate of 5 nmol CO2 per min per mg of protein34. Regarding the utilization of HCHO for C–C bond formation, researchers have designed a synthetic enzyme cascade for the in vitro fixation of a C1 source like HCHO into the functional C4 sugar erythrulose35. This cascade involves tailoring the enzyme formolase, an optimized variant for fusing HCHO from a three-carbon producer to variants with enhanced two-carbon (glycolaldehyde) or four-carbon (erythrulose) activity. The tailored formolase variants rendered the highest in vitro concentration of erythrulose reported to date. Furthermore, to uncouple CO2 fixation from cellular regulation and growth, cell-free CO2 fixation systems represent an alternative approach with independently regulated fixation rates36. Luo et al. recently designed an oxygen-insensitive, self-replenishing CO2 fixation system with opto-sensing37. This approach comprises (1) a synthetic reductive glyoxylate and pyruvate synthesis (rGPS) cycle and (2) the malyl-CoA-glycerate (MCG) pathway to produce acetyl-CoA, pyruvate, and malate from CO2. In general, the exploration of C1 building blocks by biotechnological routes has the potential to complement existing chemical synthetic routes, enabling the production of valuable compounds, and contributing to valorize different wastes in a circular manner (see Fig. 1 for selected examples).This article highlights recent developments featuring isolated enzymes and whole-cell approaches for the production of complex chemicals from C1 building blocks, with a particular focus on patents, reflecting a growing commercial interest and investment in the field. Hybrid and cell-free systems are also discussed. The urgency for sustainable and renewable alternatives to fossil fuels has never been greater, and recent innovations through intellectual property (IP) protection underscore advances in C1-based biomanufacturing as a promising avenue for this transition.Natural and engineered enzymes for C1 platform chemical synthesisUnique sets of natural enzymes and metabolic pathways are responsible for the assimilation of the fully reduced and oxidized C1 compounds CH4 and CO2, respectively, yielding important platform chemicals including MeOH, HCHO, and HCOOH4 (Fig. 2). Therefore, selected enzymes involved in the synthesis and transformation of C1 compounds will be highlighted, together with attempts to customize them by protein engineering towards industrial applications. Of particular interest are methane monooxygenases (MMOs) for the direct assimilation of CH4 to MeOH, methanol dehydrogenases (MDHs) for the conversion of MeOH into HCHO, and formaldehyde dehydrogenase (FalDH). The latter can catalyze the oxidation of HCHO to HCOOH, as discussed below.Fig. 2: Schematic representations of important C1-transforming enzymes.A Methane monooxygenases (MMOs) feature soluble iron-dependent and particulate copper-dependent enzymes (sMMO and pMMO, respectively), catalyzing the oxidation of CH4 to MeOH. B Methanol dehydrogenases MDHs—pyrroloquinoline quinone (PQQ)-dependent MDH, NAD(P)+-dependent MDH, or O2-dependent alcohol oxidase (AOX)—convert MeOH into HCHO. C FalDHs—glutathione independent FalDH and variants with modified NAD(H) binding pockets—oxidize HCHO to HCO2−. D Metal-dependent and—independent FDHs convert HCO2− into CO2.Full size imageIn nature, methanotrophic bacteria can utilize CH4 as the sole carbon and energy source. Consequently, they have been proposed as relevant key-actors to tackle global warming by reducing the amount of atmospheric CH4 emitted through anthropogenic activities38. Recent studies focused on the key enzymes for the conversion of CH4 to MeOH by MMOs39,40,41 and methyl-coenzyme M reductases (MCRs)42 under aerobic and anaerobic conditions, respectively. Methanotrophs can produce two types of MMO—a membrane-associated particulate (pMMO) and a cytoplasmic soluble form (sMMO)41,43 (Fig. 2A). Both MMOs differ in their sequences, overall structure, the requirement of metal cofactors, active site composition, and the catalytic mechanisms44,45. Although naturally predominant, the catalytic mechanism of pMMO remains unresolved due to its air-instability and highly complex structure46. In contrast, the catalytic mechanism of sMMOs is well-elucidated and was recently reviewed by Banerjee and co-workers39. Although sMMOs exhibit higher turnover frequencies than pMMOs and can accept other substrates than CH4, including aromatics47,48,49, difficulties to heterologously produce sMMOs have limited both their broader utilization and the success of engineering campaigns. Recently, the heterologous production of active sMMO from the marine Methylomonas methanica MC09 in E. coli was demonstrated by coexpression of the GroES/EL chaperonin50,51. In addition, Smith and co-workers addressed this issue by constructing vector-based systems for the simplified expression of (mutant) sMMO in a derivative of its native host Methylosinus trichosporium (M. trichosporium) OB3b52,53. In different studies, they targeted residues in the active site (C151, F192, T213, and I217), as well as amino acids close to the entrance of the substrate binding pocket (R98 and L110) by site-directed mutagenesis (SDM). While the binuclear iron center in the active site is coordinated by four glutamate and two histidine residues, which lie in a solvent-accessible cavity lined by hydrophobic residues54, C151 and T213 are the only amino acid residues with protonated side chains not involved in binding the binuclear iron center. Both residues are highly conserved amongst sMMOs52 and homologous monooxygenases with a binuclear iron center55,56, respectively. Although a few conservative amino exchanges, such as C151E and T213S were tolerated, no improved variants, particularly for the oxidation of CH4, could be obtained. Similarly, the engineering of R98 yielded variants, exhibiting up to fivefold increased turnover rates for monoaromatic substrates and biphenyl compared to the wild-type but not CH4. L110 mutants rather influenced the regioselectivity as observed with shifts in the distribution of hydroxylated (aromatic) products49,57.Although only slightly improved, these and related engineering campaigns58 have certainly contributed to a deeper understanding of sMMO enzymes and even inspired the design of both inorganic model complexes mimicking natural diiron clusters39,59,60 and a catalytically active ‘miniature’ sMMO (mini-sMMO)61. Although the oxidation of CH4 has not been achieved yet, these (inorganic) model complexes promise applications in chemical feedstock valorization and fuel conversions in the near future39,60. Importantly, the recent redesign of an apoferritin scaffold yielded a mini-sMMO with moderate turnover frequency (>0.2 s−1). Recombinant expression of the mini-sMMO in E. coli, which lacks a pathway for MeOH oxidation, resulted in an improved MeOH yield and production that is certainly competitive with natural methanotrophic production62,63,64, thereby, representing a scalable platform for the production of MeOH and derivatives (Supplementary Table 1)61.As introduced above, MCRs are crucial in CH4 metabolism in anaerobic environments and have been found in methanogens as well as anaerobic CH4-oxidizing archaea, which are both considered to be key players in the global carbon cycle. Distinct MCRs catalyze the last step in methanogenesis (mMCRs) and the first step in anaerobic CH4 oxidation (aMCRs)46. In direct comparison to MMOs, MCRs are slow due to the multi-step synthesis and regeneration of cofactors47,65, including derivatives of the nickel containing F430 cofactor, and the dependence on posttranslational modifications32,66,67. Although the overall structures of mMCR and aMCR are similar66,68 and the mechanism of mMCR has been studied extensively, the catalytic cycle of aMCR remains unclear46,69. However, to realize the full potential of MCRs and MCR-like enzymes, which have recently been shown to oxidize other alkanes such as ethane or butane70,71, the challenges of (heterologous) expression and the extremely difficult purification of active MCR in its nickel(I) oxidation state have yet to be overcome70,72,73.Methanol is a globally used raw material in the chemical industry74 with a compound annual growth rate of about 5.2%. Its production increased from 85 million metric tons per year in 2016 to over 110 million metric tons in 2021 due to its versatility for different applications. It is an important building block in the synthesis of (bio-based) bulk chemicals such as acetic acid, methyl tert-butyl ether, and gasoline75. As mentioned before, MeOH can be further oxidized to HCHO by MDHs (Fig. 2B). Noteworthy, the utilization of MeOH is particularly interesting since it can be directly sourced from CO2, which can be captured from high-emission industries75,76. This is beneficial from an economic and an environmental point of view and will also be revisited below.As of today, three different types of MDH enzymes have been identified: (1) pyrroloquinoline quinone (PQQ)-dependent MDH from Gram-negative bacteria77, (2) NAD(P)+-dependent MDH from Gram-positive bacteria78, and (3) O2-dependent AOX from yeast79. Various recent reviews not only have covered the unique features of different MDHs and their limitations, as well as the native pathways for the assimilation of MeOH and HCHO as described below80. Among the three MDH classes, the NAD+-dependent enzyme variants are probably the most suitable for their implementation in heterologous hosts. Firstly, these MDHs can operate under both aerobic and anaerobic conditions81. Furthermore, the use of NAD+ as cofactor is advantageous as it is more stable and cheaper than NADP+, two important considerations not only for in vitro but also for industrial applications82. One key-feature of dehydrogenase-catalyzed reactions, including MDHs here and the formate dehydrogenases (FDHs) discussed below, is the reversibility of the reaction. Both can drive the NAD(P)+-dependent oxidation and the NAD(P)H-dependent reduction reactions. Hence, the direction of the desired reaction has to be considered when choosing (enzyme-coupled) systems for the recycling of cofactors82,83. One potential disadvantage of PQQ-dependent MDHs and AOXs is the requirement of molecular oxygen (O2) for PQQ biosynthesis84 and as a co-substrate, respectively. AOX also depends on flavin cofactors such as flavine adenine dinucleotide and the production of hydrogen peroxide (H2O2) is cytotoxic at elevated levels82. Alternatively, other alcohol dehydrogenases (ADHs) have been suggested for the oxidation of MeOH to HCHO and might also be valuable engineering targets85,86,87.Besides the considerable effect of metal ions, which facilitate cofactor binding in NAD+-dependent MDHs, high activities are usually observed at high temperatures (>50 °C) and pH (9–10)88,89. To improve the activity of MDHs, for example, at ambient temperatures (20–37 °C) and at neutral cytosolic pH in well-established hosts like E. coli, SDM90,91, site-saturation mutagenesis (SSM)87, and random mutagenesis89 have been used to construct MDH mutants.A representative study targeted the MDH-2 from Cupriavidus necator N-1 by SSM. Subsequently, promising variants were subjected to error-prone polymerase chain reaction87. Ultimately, the Nash assay-based high-throughput screening (HTS) of generated libraries yielded a triple mutant (A169V, A26V, and A31V), exhibiting a low KM (∼22 mM) and an unchanged kcat (0.2 s−1) compared to the wild-type enzyme (KM 132 mM). Similarly, the group of Lee created different mutants of a MDH from Lysinibacillus xylanilyticus by SDM of active site residues92 as well as random mutagenesis89. Both studies yielded beneficial mutations, resulting in improved affinities (S101V: KM 10.35 mM; T141S: KM 51.24 mM; A164F: KM 36.83 mM) and specific activities towards MeOH compared to the wild-type MDH. Importantly, the implementation of a genetically encoded biosensor for the detection of HCHO93 facilitated the identification of several mutants, exhibiting almost 80-fold enhanced catalytic efficiency over the wild-type enzyme and a low KM value of 0.01 mM89. This example not only points towards the transfer of improved MDH variants into (synthetic) strains capable of HCHO assimilation as described below11,94; the use of genetically encoded biosensor systems, which have been recognized as versatile tools for the detection and quantification of a broad range of small molecules in living cells, might be particularly interesting for the optimization of both C1-converting enzymes and (artificial) carbon assimilation pathways95.Since HCHO is a toxic metabolite that may cause the crosslinking of DNA and proteins and induce oxidative stress, it is rapidly converted in methanotrophs and other (micro)organisms46. HCHO can be metabolized into CO2 by FalDH (Fig. 2C) and FDH (Fig. 2D) directly or through pathways mediated by tetrahydrofolate (H4F), tetrahydromethanopterin, or various thiols such as glutathione or bacillithiol11,96,97. As discussed above for MDHs, the requirement of different redox cofactors must be considered. While whole-cells regenerate cofactors through different metabolic pathways, efficient recycling systems have to be implemented in vitro82,98,99.Particularly interesting for biotechnological applications is the FalDH from Pseudomonas putida. It catalyzes the glutathione-independent oxidation of HCHO into HCOOH and has industrial potential because of its strong catalytic specificity and high transformation efficiency100. Recently, Wang et al. constructed FalDH variants by structure-guided modification of the NAD(H) binding pocket to favor the non-native nicotinamide cytosine dinucleotide (NCD) cofactor. The best mutant, FalDH-9B2, showed >150-fold higher preference for NCD than NAD, which offers opportunities to assemble orthogonal pathways for the biological conversion of C1 compounds101. Alternatively, HCO2− can be produced (enzymatically) from multiple (renewable) feedstocks including CO2. Like MeOH and HCHO, HCOOH can be further converted into various platform chemicals, hence, is of interest for different industries1,102.FDHs catalyze the decarboxylation of cheap HCO2− and were first discovered in pea seeds more than 60 years ago. Today, metal-dependent (Mo- or W-containing) and metal-independent FDHs have been characterized. The latter are widely applied for the regeneration of NAD(P)H over a broad range of reaction conditions (e.g., pH 6–9). The release of gaseous CO2 is essentially irreversible, which has been utilized to shift the reaction equilibrium of dehydrogenase-catalyzed reactions99. However, recent reports suggested that CO2 reduction might be possible, although with very low efficiency since HCO2− oxidation is highly favored (Supplementary Table 1)103,104,105. One drawback of FDHs is their somewhat limited operational stability82,99,106. The latter is due to the presence of cysteine residues that are susceptible to chemical modification and oxidation by air, leading to the inactivation of the enzyme. These and other hurdles have been addressed by protein engineering, providing variants with improved kinetic properties as well as increased thermal stability107, or switched cofactor specificity108,109,110. Tishkov and Popov highlighted these achievements106, together with FDH mutants for the enhanced soluble expression in heterologous hosts like E. coli106,111,112.The direct capture of CO2 using H2 to convert it directly to HCO2- or HCOOH is attractive as a strategy for transporting and storing H2113. Since a major disadvantage of FDHs is that they require stoichiometric amounts of NAD(P)H, a H2-dependent carbon dioxide reductase (HDCR) could be instead used for the direct conversion of H2 and CO2 to HCO2−. Schuchmann and Müller studied this enzyme in detail and elucidated its biochemical properties114. The HDCR complex consists of four subunits: two large subunits formed by the (presumably Se- and Mo-containing) FDH and an [FeFe]-hydrogenase, complemented by two small electron transfer subunits. This complex was shown to hydrogenate CO2 with a turnover frequency (TOF) of 28.2 s−1 (Supplementary Table 1), which is comparable to the performance of chemical catalysts113. In addition to HDCR, formate hydrogenlyase (FHL) from E. coli, which normally oxidizes HCO2− to CO2 by coupling the reaction to the reduction of protons to H2, has been studied for the reverse reaction115,116. Interestingly, FHL can operate as an HDCR when pressurized CO2 and H2 are used117. Roger et al. demonstrated that intact whole E. coli cells rapidly converted 100% of gaseous CO2 to HCO2− in a pressurized system, accumulating >500 mM HCO2− in solution (Supplementary Table 1)115.Lastly, CO2 can be fixed by the natural Calvin-Benson-Bassham (CBB) cycle, which is mainly operated by autotrophs (e.g., plants, algae, cyanobacteria)1. Enzymes well-known for the fixation of CO2 are ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), phosphoenolpyruvate carboxylase, propionyl-CoA carboxylase, pyruvate carboxylase, acetyl-CoA carboxylase, and carbonic anhydrases, which have been identified in methanotrophs like M. trichosporium OB3b through whole-genome screenings, for example118. Selected examples of these carboxylating enzymes, as well as other natural and synthetic routes to assimilate HCHO, HCO2−, and CO2 will be discussed below. One of the most intriguing but challenging engineering targets has been RuBisCO. Due to numerous works summarizing recent advances in that area, the engineering of RuBisCO will not be included herein119,120,121,122.Remaining challenges, such as the inhibition of microorganisms and enzymes by (cytotoxic) pathway intermediates, the low activity of biocatalysts, or difficulties in recombinant expression, have been (partly) addressed by protein design and engineering as highlighted by the selected examples above. Although value-added compounds such as fuels or vitamins have been produced from CH4 and derived C1 building blocks in vivo123,124,125,126, improved genetic tools—not only for the genetic manipulation of native and synthetic methanotrophs—are highly demanded46,127,128,129,130. Complementary, advanced reactor designs aim at improving the low efficiency of gas-liquid fermentations as well as their productivities to meet industrial performance metrics47,67. Furthermore, recent trends in biocatalysis and biotechnology feature, for example, genetically encoded biosensors for the HTS of enzyme libraries95, the photocatalytic reduction of CO2 in combination with CO2-binding enzymes131, or emerging bacterial model strains132. The latter includes Clostridium autoethanogenum, which grows under high CO concentrations and converts waste gases into biofuels133, or genetically engineered C. necator, which oxidizes CO to CO2 by displaying an enzyme complex on the cell surface, ultimately, producing bioplastic134.This section introduced C1-based biomanufacturing enzymes and provided some performance metrics for representative examples (Supplementary Table 1), which are further discussed in the final section on industrial applicability. The bio-based oxidation and transformation of CH4 involved highly specialized enzymes: MMOs and MCRs from methanotrophs for the conversion of CH4 into MeOH in the presence and absence of O2, respectively, as well as MDHs to produce HCHO. The latter can be efficiently metabolized by FalDHs into HCO2−. FDHs, heavily utilized for the regeneration of NAD(P)H, release CO2 upon the oxidation of HCO2−.Since natural C1-transforming enzymes usually work in tandem, the next section will highlight their metabolic pathways and contexts, as well as de novo routes for the assimilation of C1 building blocks.Exploiting natural and engineered carbon metabolismThe ability of microbes to assimilate C1 molecules has been uncovered in several organisms. Currently known native pathways for the assimilation of CO2 or other C1 compounds are the following: (1) CBB cycle, (2) Wood-Ljungdahl pathway, (3) reductive glycine pathway (rGlyP), (4) serine pathway, (5) reductive TCA cycle, (6) xylulose monophosphate cycle, (7) ribulose monophosphate cycle, (8) 3-hydroxypropionate (3-HPate) bicycle, (9) 3-HPate/4-hydroxybutyrate (3-HPate/4-HB) cycle, and (10) dicarboxylate/4-hydroxybutyrate (DC/4-HB) cycle. These pathways have been thoroughly discussed already in different reviews and will not be revisited in detail here1,135,136,137,138. However, it is important to note that each of these pathways involves a range of different starting substrates and key intermediates, which are interesting for further utilization by microbes for energy generation or the production of valuable compounds (Fig. 3). Importantly, several of the highlighted natural pathways utilize enzymes that were introduced in the previous chapter.Fig. 3: Native pathways in microorganisms for the assimilation of C1 molecules.The C1 substrates were ranked based on the oxidation state of the carbon atom from fully reduced (CH4) to fully oxidized (CO2). rGlyP Reductive glycine pathway, CBB Calvin-Benson-Bassham, TCA Tricarboxylic acid, 3-HPate 3-Hydroxypropionate, 3-HPate/4-HB 3-Hydroxypropionate/4-hydroxybutyrate, DC/4-HB Dicarboxylate/4-hydroxybutyrate.Full size imageNative pathways have evolved and adapted based on the needs of the microorganism to survive in a particular niche, hence, do not perform at a level of efficiency needed for biotechnological and industrial applications. Additionally, some of the metabolic reactions introduced above only function under strictly anaerobic conditions, for example, CH4-assimilating pathways employing MCRs42. Alternative anaerobic pathways starting from CO2 are the Wood-Ljungdahl pathway and the reverse TCA cycle. Furthermore, microbes that can naturally use CO2 and C1 compounds often live in extreme environments, like different Beggiatoa, Chlorobium limicola, Clostridium ljungdahlii, Methylococcus capsulatus, and some Thiobacillus species to name a few139,140. Harnessing these microorganisms for our benefit can be difficult, as it is necessary to mimic their energy requirements and habitat, and the lack of available genetic tools to improve them often represents a major bottleneck141 and has been discussed above for methanotrophs and related microorganisms.The advent of modern synthetic biology allows us to redesign and alter natural pathways, particularly in the context of domesticated microorganisms such as E. coli or yeast142,143. Here, we discuss some of the progress regarding the engineering of natural C1 metabolism and its utilization for synthetic applications (Fig. 4, left part).Fig. 4: Selected artificial pathways for C1 utilization.Examples for the utilization of a variety of C1 building blocks can operate in vivo (left), in vitro, or as hybrid systems (right). CODH/ACS Carbon monoxide dehydrogenase/acetyl-CoA synthase, sMMO soluble methane monooxygenase.Full size imageE. coli, a bacterium that does not have an endogenous ADH able to convert MeOH to HCHO, has now been engineered towards synthetic methylotrophy (Fig. 5)32,86,144,145. The first major success in turning E. coli into an efficient methylotroph was reported by Chen et al.32. In this study, the authors identified several bottlenecks related to DNA-protein crosslinking that prevented E. coli from growing efficiently on MeOH as the sole carbon source. Through genome editing, copy number variations, and laboratory evolution, a synthetic methylotrophic E. coli strain was developed that grows to high optical densities (ODs) with a doubling time of 8 h.Fig. 5: Utilization of methylotrophic E. coli for the bioproduction of organic acids.To enable MeOH assimilation, three genes were expressed heterologously in E. coli: a MDH (encoded by mdh), a 3-hexulose 6-phosphate synthase (hps), and a 6-phospho 3-hexuloisomerase (phi). This synthetic chassis was utilized for the biosynthesis of lactic acid, itaconic acid, p-aminobenzoic acid, and polyhydroxybutyrate, exploiting different metabolites from various endogenous pathways (pyruvate or acetyl-CoA, TCA or chorismate pathway, respectively)145.Full size imageComplementary, the yeast K. phaffii is being improved for MeOH-based manufacturing146,147,148. The success of engineering these model systems also advanced the genetic modification of Saccharomyces cerevisiae, different Bacillus strains, and Corynebacterium glutamicum, facilitating their utilization to produce complex (bio)chemicals149,150,151,152. Alternatively, cell-free approaches have also been shown as an effective way to perform proof-of-concept experiments, which can be integrated into in vivo systems34,153.As mentioned above, the main limiting reaction for C1 assimilation in E. coli is the lack of an ADH that can oxidize MeOH to HCHO. Interestingly, E. coli has a detoxification mechanism for HCHO. Müller et al. showed that by introducing three genes—an MDH, a hexulose-6-phosphate synthase, and a 6-phospho-3-hexulo-isomerase—the ribulose monophosphate (RuMP) cycle could be established in this host (Supplementary Table 2)86. The success of synthetic methylotrophy in E. coli was followed by other routes such as the establishment of erythrulose monophosphate cycle154 and the rGlyP (Supplementary Table 2)31,155. The rGlyP was introduced through the introduction of four core modules to allow growth on MeOH and HCO2−. The first module converts HCO2− to 5,10-methylenetetrahydrofolate (methylene-TH4F), while the second module condenses methylene-TH4F with CO2 and ammonia to produce glycine. Meanwhile, the third module combines glycine and methylene-TH4F to produce serine and pyruvate. The fourth module is an energy module that contains a FDH to generate reducing power and energy from HCO2−.Adapted laboratory evolution has also been a key driver to the success of synthetic methylotrophy in E. coli as there are improved strains. It was shown that the methylotrophic growth of E. coli can be further optimized to achieve doubling times of approximately 4 h, comparable to many natural methylotrophs145,154,156,157. These examples highlight that the availability of numerous genetic tools for E. coli enable its development and use as a platform chassis for the methylotrophic production of valuable compounds. Bioproducts derived from MeOH like lactic acid, polyhydroxybutyrate, itaconic acid, and p-aminobenzoic acid, and other industrially relevant compounds were recently produced in E. coli145. These molecules are important precursors for a variety of compounds, ranging from biopolymers to fine chemicals, preservatives, and pharmaceuticals.Complementary, the design of orthogonal pathways has also been investigated in E. coli, such as the establishment of formyl-CoA elongation (FORCE) pathway158. C1 substrates, such as HCO2− can be converted to formyl-CoA by CoA transferases or CoA ligases. Ultimately, formyl-CoA can be further processed to yield aldehydes, which can be transformed into acids, diols, polyols, and alcohols82,93,95.As briefly mentioned above, another target for improved synthetic methylotrophs is the yeast K. phaffii, which can assimilate MeOH—as the sole carbon source or in combination with a co-carbon source—due to an endogenous AOX that converts MeOH and O2 to HCHO and H2O2159. HCHO can be metabolized through the xylulose monophosphate pathway, which yields glyceraldehyde-3-phosphate, and is further converted to fructose 1,6-bisphosphate. The latter can be reused for driving the pathway again148. MeOH-based chemicals produced in K. phaffii include various organic acids like fatty acids or polyketides160. Recently, K. phaffii was used to host a synthetic β-alanine pathway through the expression of panD, yhxA, and ydfG to produce 3-hydroxypropionic acid (3-HP) directly from MeOH as the only carbon source160. This pathway transforms L-aspartate by the activity of an aspartate-1-decarboxylase into ß-alanine, which, in turn, is converted into malonyl-semialdehyde by a β-alanine-pyruvate aminotransferase. 3-HP is finally obtained by the action of 3-HP dehydrogenase. Further strain engineering by overexpressing a NADP+-dependent FDH variant led to a final 3-HP titer of >20 g L−1. 3-HP is listed as one of the most important chemicals that could be produced from renewable resources and can be used to produce acrylic acid, as well as other commodities and specialty chemicals such as acrylamide and methyl acrylate161.Although the engineering of native pathways has already been realized and artificial metabolic modules could be transferred to biotechnological hosts like E. coli and K. phaffii (Supplementary Table 2), the optimization of metabolic fluxes is strictly required but might be challenging to achieve in vivo—despite the availability of (bioinformatic) tools such as metabolic flux analysis82. A promising solution is offered by easy-to-adjust cell-free systems or in vivo/in vitro hybrid systems as discussed in the following section.Hybrid and cell-free approachesMetabolic fluxes and the overall thermodynamics of intricate reactions not only affect the physiology of microorganisms but can be hard to predict and even harder to control by design162,163,164. Therefore, cell-free and in vitro/in vivo hybrid approaches have been utilized to optimize C1 assimilation and to produce valuable intermediates and products (e.g., acetyl-CoA, amino acids, or starch) (Fig. 4, right part; Supplementary Table 3).The possibility of synthesizing starch from cellulose is a prominent example and was first demonstrated by You et al. 165. It was achieved by concocting a non-natural synthetic enzymatic pathway, consisting of endoglucanase, cellobiohydrolyase, cellobiose phosphorylases, and α-glucan phosphorylase. Through this cascade, cellulose was partially converted to starch, while the unconverted residue was hydrolyzed to glucose for ethanol production166. This study also inspired an artificial starch anabolic pathway (ASAP), in which starch was produced by converting CO2 to MeOH, followed by a series of enzymatic reactions (Supplementary Table 3)167. CO2 was converted into starch at a rate of 22 nmol CO2 min−1 mgcat−1. Overall, Cai et al. claimed an 8.5-fold higher productivity than the natural starch biosynthesis in maize (a C4 plant), demonstrating that human-designed and laboratory-evolved cell-free synthetic routes can outcompete naturally occurring organisms168. In addition, the product titer (>1 g L−1) and high productivity (>300 mg L−1 h−1) significantly outperform those achieved with the synthetic CETCH cycle (Supplementary Table 3)34. Previously, formolase, the key enzyme used in ASAP, was computationally designed by the Baker group to convert HCHO (C1) to dihydroxyacetone (C3)169. In this initial study, the FLS was combined with several naturally occurring enzymes to create the formolase pathway, but the enzyme activities were low, which likely contributed to the lack of detectable growth on HCO2−. However, this artificially designed carboligase attracted much attention, paving the way to its application in various artificial C1 compound conversion pathways35,170,171,172. One example is the synthetic acetyl-CoA (SACA) pathway reported by Lu et al. that represents a linear C1 assimilation pathway realized by combining protein design and pathway construction in vitro and in vivo, employing E. coli as a host (Supplementary Table 2)170. Using this thermodynamically favorable pathway, the biosynthesis of acetyl-CoA from HCHO has been achieved. While this pathway has significant potential in synthetic enzyme cascades and in vivo metabolism, several limitations related to enzyme inhibition by HCHO or its toxicity to cells must still be addressed to make it practical for applications in the context of living cells.In this regard, the engineering of formolase is crucial to develop variants with improved activity at low HCHO concentrations. A recent study shows that a channel-modulating helix, which forms a zipper structure with its neighboring helix, regulates the activity of the enzyme at low HCHO concentrations165. Based on this, the authors engineered this helix, resulting in a 24-fold increase in catalytic efficiency. In particular, the activity of the best variant was increased 27-fold at 20 mM HCHO and 86-fold at 40 mM HCHO. This promising improvement in enzyme performance not only opens the door for further engineering efforts in the future, but may also allow for increased efficiency when formolase is used in multi-enzyme systems or in vivo pathways.Noteworthy, the reversal of known catabolic pathways has also been carried out to produce amino acids. The Ehrlich pathway breaks down amino acids into alcohols through a series of enzymatic reactions involving deamination, decarboxylation, and reduction. Martin et al. demonstrated that the reversal of this catabolic pathway can yield L-methionine from the abundant industrial intermediate methional by direct incorporation of CO2173. Despite the unfavorable chemical equilibrium of the decarboxylase, the enzyme displayed half-maximal activity even at low CO2 pressure for its reverse reaction, yielding 4-methylthio-2-oxobutanoate (an α-keto acid). This intermediate was then further converted to L-methionine using an aminotransferase or an amino acid dehydrogenase.Similarly, another cell-free approach to amino acid production uses the MAP (MeOH-alanine pathway) as a cell-free enzymatic cascade to produce L-alanine directly from MeOH174. A one-pot cascade consisting of a total of nine enzymes, including an intrinsic cofactor recycling system, produces L-alanine with a maximum of 90% theoretical yield (about 3.9 g L−1 L-alanine, Supplementary Table 3).Even more complex systems are the reported new-to-nature pathways for the fixation of CO2, such as the CETCH pathway. Developed by Schwander and co-workers, it involves a total of 17 enzymes from different organisms to enhance the conversion of CO2 to organic molecules (Supplementary Table 3)34. As this pathway was realized in vitro, the question of whether it can be implemented in vivo is still open. As a follow-up to the CETCH pathway, the HOPAC cycle was designed through metabolic retrosynthesis (Fig. 6A; see also Fig. 1)175. The HOPAC cycle focuses on the reductive carboxylation of acrylyl-CoA, which has been implemented in a stepwise fashion and further optimized using rational engineering and machine learning. The final version of this cycle consists of a total of 11 enzymes and achieves a CO2 fixation rate of 2.4 nmol CO2 min−1 mg−1 protein. Although the HOPAC cycle is nature-inspired and based on the 3-HPate bi-cycle of Chloroflexus aurantiacus, which converts acetyl-CoA to pyruvate from three molecules of bicarbonate fixed in two separate rounds, other alternatives with potentially higher feasibility for in vivo application have been developed. One of these is the THETA cycle, which has recently been developed to enable the implementation of synthetic CO2 pathways in vivo (Fig. 6B)33. Here, a modularized CO2 fixation pathway was developed and divided into three in vivo modules, paving the way for hybrid modules. The THETA cycle yielded a CO2 fixation rate of 2.7 nmol min−1 mg−1. Although this cycle is not as efficient as the CETCH pathway, it provides a stepping stone for complex synthetic pathways to be realized in vivo, and may hold future promising prognoses for industrial implementation.Fig. 6: New-to-nature pathways.A Overall scheme of the HOPAC cycle, consisting of a reductive (yellow) and an oxidative (red) part for the conversion of two molecules of HCO3− and/or CO2 into one molecule of glyoxylate. The reductive pathway consists of five and the oxidative pathway of six enzymatic steps. Variant pathways have been designed for the reductive and oxidative parts, as shown inside of the circle. B Design and application of the THETA cycle for CO2 fixation. The THETA cycle is designed in three modules and constructed in vitro. Modules 1 and 2 consist of five enzymatic steps each, whereas module 3 consists of seven enzymatic transformations. The inner circle represents a simplified scheme of the THETA cycle, while the outer circle highlights each reaction step of the pathway in detail.Full size imageLastly, Schulz-Mirbach et al. developed a CO2-dependent acetyl-CoA assimilation pathway called the Lcm module142. The Lcm module is based on the novel activity of a coenzyme B12-dependent mutase. At the heart of the pathway, the mutase converts 3-hydroxypropionyl-CoA to lactyl-CoA. Lactyl-CoA is further converted to lactate and finally to pyruvate for biomass production through the endogenous central carbon metabolism. The Lcm module fixes CO2 rather than releasing it, unlike other available aerobic pathways. Interestingly, the pathway works both in vitro and in vivo, although application in a cell-free context will require further optimization regarding the stability and oxygen-sensitivity of the Lcm. However, the application of the Lcm module in vivo allows the co-expression of chaperones, the rescue of damaged coenzyme B12, and offers improved protection of the coenzyme in the reductive intracellular environment. Thus, in vivo rather than in vitro applications are more promising for future implementations.Assessment of industrial applicabilityThe need for a transition to greener energy systems puts pressure on developing more robust enzymatic systems that can be of utility (G20 Saudi Arabia. Guide to the CCE (2020); https://www.cceguide.org/). This may pose various challenges across disciplines and legal fields, including those pertaining to intellectual property rights (IPRs), particularly within the framework of the European Green Deal. The latter aims at a resource-efficient and competitive economy with net-zero greenhouse gas emissions by 2050176. IPRs are being adapted to support activities like re-using, repairing, refurbishing, remanufacturing, and recycling (collectively, the “R activities”). These activities are essential for reducing waste and conserving resources. IPRs may play a pivotal role at both the operational and the infrastructural levels of a future circular economy176. However, the need to foster innovation through IP protection should not obstruct from the broader objectives of a CCE177,178.Today, IPRs need to protect several aspects of biotechnological processes, such as—but not limited to—the type of reaction, enzyme variants (e.g., generated by protein engineering), and the application scope that surpasses the state-of-the-art (Chemicals Knowledge Hub (2020); https://www.chemicalsknowledgehub.com/). Regarding biobased processes involving enzymes (FDHs, carboxylase, decarboxylase, MMOs, FalDH, and MDHs), broad patent activities were found, related to many different applications, and being FDHs and carboxylases/decarboxylases the most prominent ones. This result is not surprising as FDHs have shown broad applications in biocatalysis, in particular for cofactor regeneration as discussed above (Fig. 2D)99,179. More specifically, when the patent search is narrowed to enzyme and specific molecules for C1 valorization (CO2, CH4, HCHO, and MeOH), also large activities are retrieved, with overall >130 granted patent families as of April 2025 (retrieved by LENS.org). Among these, formaldehyde and MDHs result the most protected ones (Fig. 7). Generally, the low enzymatic activity observed for CO2 fixation has been traditionally a bottleneck for its implementation at industrial level180,181. However, they are still worth patenting because of their potential for significant advances in biotechnological applications. These enzymes offer unique capabilities for carbon capture and utilization, which are crucial for the development of sustainable industrial processes. In addition, continued research and development can lead to improved efficiencies and novel methods of enzyme engineering, making (de)carboxylases valuable assets in the ongoing efforts to mitigate climate change and reduce industrial carbon footprints. IP could ensure that innovation in this field is protected, thereby encouraging further investment and development.Fig. 7: Overview of the granted patents related to C1-associated enzymes.LENS.org was used to sort out the granted patents by sectors and families. To narrow the search, the single enzyme type was considered and combined with the expected substrate or product (CO2, CH4, HCHO, and MeOH): Granted patents were searched for that included an enzyme type (formate dehydrogenase, carboxylase, decarboxylase, methane monooxygenase, formaldehyde dehydrogenase, and methanol dehydrogenase) in the title, abstract, or claims, together with the expected substrate to be fixed, also in the claims (e.g., CO2). From the total number of granted patents on enzymes using HCO2−, CO2, CH4, HCHO, or MeOH, which has been steadily increasing since 2000, the respective proportions were calculated. A representative structure of the enzymes FDH, carboxylase, decarboxylase, MMO, FalDH, and MDH is shown in the pie chart. Source: LENS.org, accessed on the 14th of April 2025.Full size imageA more detailed assessment of IPRs yielded 60 granted patents that fall within the most important aspects discussed in this article. Novel applications cover the synthesis of different chemicals such as biofuels (e.g., butanol and ethanol), aldehydes, fatty acids, as well as diamines and related derivatives by (engineered) microorganisms and enzymes. The innovation strategy observed in the patents mostly focuses on the development of novel microorganisms and enzymes that can lead to improved systems, in line with the need of establishing intensified processes to reach industrial standards. The 7 shortlisted cases of these granted patents are summarized in Table 1. No hits were found in the search for IP on hybrid and cell-free systems.Table 1 Representative granted patents focused on inventions for C1 compound utilization based on enzymatic reactionsFull size tableIn analogous areas, recent advancements have paved the way for innovative applications of autotrophic microorganisms in industrial processes. For example, LanzaTech has successfully commercialized a fermentation process that converts industrial waste gases such as CO2 and H2 into valuable products like ethanol and n-octanol, demonstrating a high Technology Readiness Level (TRL) of 9. Most notably, its first commercial plant in Caofeidian (China), built with its joint venture partner, the Shougang Group, a leading Chinese iron and steel producer, operates with a production capacity of 46,000 tons of ethanol per year182. Although the genetic engineering of the acetogenic bacteria used by LanzaTech has been used to deliver other products such as acetone183, 2-propanol184, butanol (pending patent), the production of more complex molecules appears more challenging due to the metabolic limitations of these organisms182,185. Nonetheless, this technology not only reduces CO2 emissions but also valorizes waste carbon into useful materials, contributing to a CCE186. The industrial implementation of autotrophic microorganisms in a commercial production plant, as demonstrated by LanzaTech, has spurred further commercialization of these technologies.In particular, it has contributed to additional investments in such and similar gas fermentation technologies (see the following patents: AU2024204385A1, pending; AU2024203354A1, pending; US8809015B2, active). Emerging interest is further exemplified by start-ups that are building their businesses around this area. For instance, Solar Foods uses H2-oxidizing bacteria to produce Solein®, a protein-rich powder directly produced from CO2 and H2 that provides a sustainable alternative to traditional agricultural methods. Other start-up companies, such as Econutri and Arkeon, are also exploring the potential of autotrophic microorganisms to produce amino acids (see the following patent applications: WO2023247741A1, published; WO2023247740A1, published) and proteins, respectively, further demonstrating the commercial applicability of these biological systems for highly demanded target products.As highlighted above, the current developments are encouraging for the industrial implementation of microbes assimilating C1 molecules with native pathways (e.g., CBB cycle or Wood-Ljungdahl pathway) for bioproduction, but the TRL of engineered in vivo/in vitro systems still remains low. This is underlined by the fact that the search for IP did not yield hits for hybrid and cell-free systems, suggesting that they might be currently the interest in academic research rather than being ready for industrial purposes. In fact, the hybrid systems highlighted above have only been demonstrated at low TRLs (Supplementary Table 3), but have not yet been scaled up to increase product titers and STYs using process engineering techniques, for example. Similar observations have been made for the productivity of microbes with engineered carbon metabolism (mainly E. coli and K. phaffii) used to furnish value-added products from MeOH (Supplementary Table 2). However, several of these examples demonstrate fed-batch fermentations in liter-scale bioreactors, achieving promising product titers (up to 1 g L−1 itaconic acid in E. coli145 and 21 g L−1 3-HP in K. phaffii160) and already showing a slightly higher TRL of 4.However, even at low TRL, these technologies provide key parameters for yield, titers, and productivity to make a meaningful assessment for future improvements. For example, the cell-free enzymatic synthesis of L-alanine from MeOH reported by Willers et al. achieves up to 3.9 g L−1 L-alanine in nine reaction steps (Supplementary Table 3)174. In contrast, traditional L-alanine production based on the synthesis of petroleum-based L-aspartic acid using immobilized cells or cell suspensions or the fermentation of glucose achieves high titers of up to 121 g L−1187. It should be noted that a direct and straight-forward comparison of these processes is difficult as they are completely different in nature, but it still highlights obvious productivity gaps. In addition to overall productivity, other parameters such as cost, development time, process implementation, and market needs must be balanced too, as recently summarized for biocatalytic processes98,188. In particular, the energy and mass flows within up- and downstream units are important. The ease (or difficulty) of downstream operations (in terms of cost and yields) is usually crucial to be addressed for industrial implementation. Moreover, water consumption and handling of wastewater are mandatory elements that need to be considered for the successful practical use of biotechnological systems. In the case of C1-based bioprocesses (whether in vivo or in vitro), another important consideration is the source and type of C1 starting material. For liquid C1 feedstocks (e.g., MeOH, HCOOH), a hybrid or cell-free reaction system has the advantage that no special equipment is required, and reactors commonly used for chemical synthesis can be employed98. If a gaseous C1 compound is the substrate, the most appropriate solution is to establish the bioprocess where the gaseous substrate is released (e.g., syngas generated from municipal waste, industrial or agricultural waste, reformed biogas), ultimately, to avoid difficulties and costs of transporting such gases. This, in turn, creates the need for large investments, which might contribute to why these examples are currently rare. On the other hand, the use of gaseous feedstocks also requires more advanced reactor setups for gas fermentations. This suggests that synthetic biology, metabolic engineering, and process engineering need to be integrated to achieve an economically viable process from the outset. A representative example is the production of the bulk chemical isopropanol in C. necator. First, a metabolic engineering strategy was used to develop a C. necator strain that produced 3.4 g L−1 isopropanol using fructose as the carbon source189. This strain was then further optimized for gas fermentation using CO2 as the sole carbon source, resulting in isopropanol production of 250 mg L−1 in 12 h190. A bioreactor was then designed to provide high gas mass transfer to achieve higher product titers. The adapted bioreactor configuration and cultivation strategy enabled the production of 3.5 g L−1 isopropanol191. This example illustrates that the integration of different engineering strategies is essential to achieve higher yields and industrially relevant productivities, which is particularly critical for bulk chemicals such as isopropanol, where low market prices and large volumes are expected.While gaseous substrates place specific demands on the process setup, a promising solution paving the way to industrial applicability is the combination of electrochemical reduction of CO2 to a liquid C1 compound (e.g., HCOOH, MeOH), which can be coupled to a subsequent bioprocess using formatotrophic or methylotrophic microbes. For example, b.fab GmbH (Germany) uses an engineered C. necator strain employing the rGlyP for growth on HCOOH5 to produce value-added chemicals, single-cell proteins, amino acids, and biopolymers192.With these current developments, it is expected that more applications of (engineered) (autotrophic) microorganisms will reach industrial maturity, given productivities and related metrics being competitive with economically established processes.Future perspectives and challengesThis article has highlighted the potential of more sustainable biotechnological applications in advancing a CCE. The transformation of various C1 building blocks (e.g., CO2, CH4, and CO) into complex chemicals can address the pressing issues of greenhouse gas emissions while providing valuable compounds for human applications from renewable resources. Furthermore, the highlighted processes have the potential to convert waste gases into valuable products, thereby closing the carbon loop and helping to mitigate climate change. However, despite significant progress in this area, several challenges remain, which have been addressed by several examples: (1) The improvement of biocatalysts (by protein engineering and synthetic pathway design), (2) the optimization of reaction and process conditions (by the recycling of cofactors or advanced reactor designs), which, together, facilitated the (3) upscaling of the technologies investigated towards industrial applications. These challenges may also reflect the inherent difficulties of biological systems to compensate for the thermodynamic constraints that determine the structure of carbon fixation pathways. For instance, natural evolution took a billion years, and yet RuBisCO is a slow-evolving, notoriously constrained enzyme that does not discriminate properly between CO2 and O2, for example. Nature helps itself by making large amounts of RuBisCO to compensate for the low CO2 fixation capacity. Several research groups have attempted to improve the catalytic properties of RuBisCO through protein engineering, but with limited success121,122. However, recent work by the Erb group not only sheds light on how RuBisCO has evolved in response to rising atmospheric O2 levels, but also provides intriguing insights into the function of interactions within the enzyme that may guide further engineering efforts in the future18,120. Besides the thermodynamically challenging reduction of CO2, several enzymes that can catalyze this reaction are extremely difficult to handle due to their oxygen sensitivity or the demand of complex cofactors for catalysis114. Thus, the challenge for the scientific community is to discover alternative biocatalysts that can become applicable soon, for example, through a combination of advanced techniques, including but certainly not limited to bioinformatic- and machine learning-assisted protein design and tailoring as well as metabolic and strain engineering15,193. While emerging enzyme classes like carboxylases/decarboxylases have already been applied towards their CO2 fixation capabilities, which is also reflected in the number of patents filed (Fig. 7 and Supplementary Table 1), this activity has yet to be investigated and engineered thoroughly for FDHs, which are well-established as cofactor recycling enzymes (Figs. 2D and 7 and Supplementary Table 1). Considering industrial developments, driven by LanzaTech, for example, who use wild-type and engineered Clostridia strains for the production of bulk chemicals194. Along these lines, the increasing number of start-up companies in this and related fields are quickly adapting novel technologies and testing them in different markets today.Overall, the future of sustainable biotechnological applications offers promising research avenues. The combined efforts by scientific communities, political organizations, and societies drive innovation and hold the potential to transform our petrol-dependent economy into a CCE that preserves natural resources, protects the environment, and turns waste into wealth for future generations to come.ReferencesSantos Correa, S., Schultz, J., Lauersen, K. J. & Soares Rosado, A. Natural carbon fixation and advances in synthetic engineering for redesigning and creating new fixation pathways. J. Adv. Res. 47, 75–92 (2023).CAS Google Scholar Levi, P. G. & Cullen, J. M. Mapping global flows of chemicals: from fossil fuel feedstocks to chemical products. Environ. Sci. 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A.C.R.N. and D.T. were supported by the DFG grant 536337083 in the frame of priority program SPP2240 eBiotech.Author informationAuthors and AffiliationsInstitute of Molecular Biosciences, University of Graz, Graz, AustriaGiovanni Davide BaroneDepartment of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, Greifswald, GermanyIna Somvilla, Thomas Bayer & Uwe T. BornscheuerDepartment of Chemical and Pharmaceutical Biology, University of Groningen, Groningen, The NetherlandsHannah Pia Franziska Meier & Sandy SchmidtMicrobial Biotechnology, Faculty of Biology and Biotechnology, Ruhr University Bochum, Bochum, GermanyAnna Christina R. Ngo & Dirk TischlerDepartment of Chemistry, Materials and Chemical Engineering “Giulio Natta”, Polytechnic of Milan, Milan, ItalyFabio ParmeggianiInstitute of Chemistry, University of Graz, Graz, AustriaViktoria Rehbein & Johann A. HlinaSustainable Momentum SL, Las Palmas de Gran Canaria, Canary Islands, SpainPablo Domínguez de MaríaAuthorsGiovanni Davide BaroneView author publicationsSearch author on:PubMed Google ScholarIna SomvillaView author publicationsSearch author on:PubMed Google ScholarHannah Pia Franziska MeierView author publicationsSearch author on:PubMed Google ScholarAnna Christina R. NgoView author publicationsSearch author on:PubMed Google ScholarThomas BayerView author publicationsSearch author on:PubMed Google ScholarFabio ParmeggianiView author publicationsSearch author on:PubMed Google ScholarViktoria RehbeinView author publicationsSearch author on:PubMed Google ScholarJohann A. HlinaView author publicationsSearch author on:PubMed Google ScholarPablo Domínguez de MaríaView author publicationsSearch author on:PubMed Google ScholarUwe T. BornscheuerView author publicationsSearch author on:PubMed Google ScholarDirk TischlerView author publicationsSearch author on:PubMed Google ScholarSandy SchmidtView author publicationsSearch author on:PubMed Google ScholarContributionsG.D.B. and S.S. conceived the manuscript. G.D.B., S.S., I.S., A.C.R.N., T.B., V.R., J.A.H., H.P.F.M., and P.D.M. co-wrote the paragraphs. H.P.F.M., G.D.B., A.C.R.N., P.D.M., and S.S. designed the figures. T.B., H.P.F.M., F.P., P.D.M., D.T., U.T.B., G.D.B., and S.S. jointly edited the manuscript. 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