CO2 Fixation Pathways Research Articles

The transition from 2G to 3G-feedstocks enabled efficient production of fuels and chemicals

For decades micoorganisms have been engineered for the utilization of lignocellulose-based second-generation (2G) feedstocks, but with the concerns of increased levels of atmospheric CO2 causing global warming there is an emergent need to transition from the utilization of 2G feedstocks to third-generation (3G) feedstocks such as CO2 and its derivatives. Here, we established a yeast platform that is capable of simultaneously converting 2G and 3G feedstocks into bulk and value-added chemicals. We demonstrated that by adopting 3G substrates such as CO2 and formate, the conversion of 2G feedstocks could be substantially improved. Specifically, formate could provide reducing power and energy for xylose conversion into valuable chemicals. Simultaneously, it can form a concentrated CO2 pool inside the cell, providing thermodynamically and kinetically favoured amounts of precursors for CO2 fixation pathways, e.g. the Calvin–Benson–Bassham (CBB) cycle. Furthermore, we demonstrated that formate could directly be utilized as a carbon source by yeast to synthesize endogenous amino acids. The engineered strain achieved a one-carbon (C1) assimilation efficiency of 9.2 %, which was the highest efficiency observed in the co-utilization of 2G and 3G feedstocks. We applied this strategy for productions of both bulk and value-added chemicals, including ethanol, free fatty acids (FFAs), and longifolene, resulting in yield enhancements of 18.4 %, 49.0 %, and ∼100 %, respectively. The strategy demonstrated here for co-utilization of 2G and 3G feedstocks sheds lights on both basic and applied research for the up-coming establishment of 3G biorefineries.

From Rags to Riches: Exploiting the Calvin‐Benson‐Bassham Cycle for Biomanufacturing

AbstractIndustrial chemical production largely relies on fossil fuels, resulting in the unavoidable release of carbon dioxide (CO2) into the atmosphere. The concept of a circular carbon bioeconomy has been proposed to address this issue, wherein CO2 is captured and used as a raw material for manufacturing new chemicals. Microbial cell factories and, in particular, autotrophic microorganisms capable of utilizing CO2 as the sole carbon source, emerged as potential catalysts for upcycling CO2 to valuable products. The Calvin‐Benson‐Bassham cycle (CBBc), the best‐known CO2 fixation pathway, is widely distributed in Nature. While extensively studied, microbial engineering programmes based on the CBBc remains relatively underexplored. In this review, we discuss avenues towards biotechnological exploitation of the CBBc to engineer CO2‐utilizing microbial cell factories, with a focus on chemically‐derived electron donors. We also highlight the advantages and challenges of implementing the CBBc in heterotrophic microbial hosts and its potential to advance a true circular carbon bioeconomy. Moreover, based on the pathway's architecture, we argue about the ideal value‐added products to generate from this metabolic route. Altogether, studying and engineering the CBBc in both natural‐ and synthetic‐autotrophs will enhance our understanding on this CO2 fixation pathway, enabling further exploration of biomanufacturing avenues with CO2 as feedstock.

Recent Advancements and Strategies of Improving CO2 Utilization Efficiency in Bio-Succinic Acid Production

The production of bio-based succinic acid through microbial CO2 fixation and conversion has gained significant attention as a promising approach to mitigate greenhouse gas emissions. However, the low CO2 utilization efficiency limits the efficient biosynthesis of succinic acid. Therefore, it is crucial from environmental and economic perspectives to enhance the efficiency of CO2 utilization in bio-succinic acid production. This review comprehensively covers the introduction of biosynthetic pathways for microbial CO2 fixation and the conversion of CO2 to succinic acid, as well as the challenges associated with CO2 supply and utilization effectiveness. Moreover, strategies including genetic and metabolic engineering for CO2 fixation, extracellular supply methods of CO2 and some potential technical approaches for CO2 capture (such as micro-nano bubbles, CO2 adsorption material and biofilm) are summarized and presented.

Interplay between autotrophic and heterotrophic prokaryotic metabolism in the bathypelagic realm revealed by metatranscriptomic analyses

BackgroundHeterotrophic microbes inhabiting the dark ocean largely depend on the settling of organic matter from the sunlit ocean. However, this sinking of organic materials is insufficient to cover their demand for energy and alternative sources such as chemoautotrophy have been proposed. Reduced sulfur compounds, such as thiosulfate, are a potential energy source for both auto- and heterotrophic marine prokaryotes.MethodsSeawater samples were collected from Labrador Sea Water (LSW, ~ 2000 m depth) in the North Atlantic and incubated in the dark at in situ temperature unamended, amended with 1 µM thiosulfate, or with 1 µM thiosulfate plus 10 µM glucose and 10 µM acetate (thiosulfate plus dissolved organic matter, DOM). Inorganic carbon fixation was measured in the different treatments and samples for metatranscriptomic analyses were collected after 1 h and 72 h of incubation.ResultsAmendment of LSW with thiosulfate and thiosulfate plus DOM enhanced prokaryotic inorganic carbon fixation. The energy generated via chemoautotrophy and heterotrophy in the amended prokaryotic communities was used for the biosynthesis of glycogen and phospholipids as storage molecules. The addition of thiosulfate stimulated unclassified bacteria, sulfur-oxidizing Deltaproteobacteria (SAR324 cluster bacteria), Epsilonproteobacteria (Sulfurimonas sp.), and Gammaproteobacteria (SUP05 cluster bacteria), whereas, the amendment with thiosulfate plus DOM stimulated typically copiotrophic Gammaproteobacteria (closely related to Vibrio sp. and Pseudoalteromonas sp.).ConclusionsThe gene expression pattern of thiosulfate utilizing microbes specifically of genes involved in energy production via sulfur oxidation and coupled to CO2 fixation pathways coincided with the change in the transcriptional profile of the heterotrophic prokaryotic community (genes involved in promoting energy storage), suggesting a fine-tuned metabolic interplay between chemoautotrophic and heterotrophic microbes in the dark ocean.13R5usaxM1iwstSWbHDM6jVideo

Functional expression of RuBisCO reduces CO2 emission during fermentation by engineered Saccharomyces cerevisiae

Industrial biotechnology based on yeast fermentation is a promising strategy that can alleviate global warming and climate change. However, Saccharomyces cerevisiae, widely used in bioprocesses, releases a large amount of carbon dioxide (CO2) during fermentation. This study developed a mixotrophic CO2-fixing S. cerevisiae to achieve carbon neutrality and sustainability in bioprocess. A CO2-fixation pathway was constructed in a xylose-utilizing S. cerevisiae by heterologous expression of ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) and phosphoribulokinase (PRK). Furthermore, a delta-integration strategy was utilized, and the RuBisCO gene copy number was increased to 10 copies to improve the efficiency of CO2-fixation. An additional Cas9-based genome editing was performed to overexpress other CO2-fixation related genes. The resulting CO2-fixing yeast, SJ03, exhibited the highest RuBisCO activity. During anaerobic xylose fermentation, ethanol concentration was increased by 17% and ethanol yield was increased by 16% compared to the control strain. In addition, CO2 emissions decreased by 7%. These results suggest that overexpression of the CO2-fixation pathway coupled with xylose utilization in S. cerevisiae might reduce CO2 emission in bioprocesses.

Effect of electron donors on CO2 fixation from a model cement industry flue gas by non-photosynthetic microbial communities in batch and continuous reactors.

The aim of this work was to evaluate the effect of different inorganic compounds as electron donors for the capture of CO2 from a model cement flue gas CO2 /O2 /N2 (4.2:13.5:82.3% v/v) using a non-photosynthetic microbial community. The inoculum obtained from a H2 -producing reactor was acclimated to CO2 consumption achieving 100% of CO2 removal after 45 days. Na2 S, MnCl2 , NaNO2 , NH4 Cl, Na2 S2 O3 , and FeCl2 were used as energy source for CO2 fixation by the acclimated microbial community showing different efficiencies, being Na2 S the best electron donor evaluated (100% of CO2 consumption) and FeCl2 the less effective (28% of CO2 consumption). In all treatments, acetate and propionate were the main endpoint metabolites. Moreover, scaling the process to a continuous laboratory biotrickling filter using Na2 S as energy source showed a CO2 consumption of up to 77%. Analysis of the microbial community showed that Na2 S and FeCl2 exerted a strong selection on the microbial members in the community showing significant differences (PERMANOVA, p = 0.0001) compared to the control and the other treatments. Results suggest that the CO2 fixing pathways used by the microbial community in all treatments were the 3-hydroxypropionate-4-hydroxybutyrate cycle and the Wood-Ljungdahl pathway.

Engineering Enzymes for Environmental Sustainability.

The development and implementation of sustainable catalytic technologies is key to delivering our net-zero targets. Here we review how engineered enzymes, with a focus on those developed using directed evolution, can be deployed to improve the sustainability of numerous processes and help to conserve our environment. Efficient and robust biocatalysts have been engineered to capture carbon dioxide (CO2 ) and have been embedded into new efficient metabolic CO2 fixation pathways. Enzymes have been refined for bioremediation, enhancing their ability to degrade toxic and harmful pollutants. Biocatalytic recycling is gaining momentum, with engineered cutinases and PETases developed for the depolymerization of the abundant plastic, polyethylene terephthalate (PET). Finally, biocatalytic approaches for accessing petroleum-based feedstocks and chemicals are expanding, using optimized enzymes to convert plant biomass into biofuels or other high value products. Through these examples, we hope to illustrate how enzyme engineering and biocatalysis can contribute to the development of cleaner and more efficient chemical industry.

Engineering Enzymes for Environmental Sustainability.

The development and implementation of sustainable catalytic technologies is key to delivering our net-zero targets. Here we review how engineered enzymes, with a focus on those developed using directed evolution, can be deployed to improve the sustainability of numerous processes and help to conserve our environment. Efficient and robust biocatalysts have been engineered to capture carbon dioxide (CO2) and have been embedded into new efficient metabolic CO2 fixation pathways. Enzymes have been refined for bioremediation, enhancing their ability to degrade toxic and harmful pollutants. Biocatalytic recycling is gaining momentum, with engineered cutinases and PETases developed for the depolymerization of the abundant plastic, polyethylene terephthalate (PET). Finally, biocatalytic approaches for accessing petroleum-based feedstocks and chemicals are expanding, using optimized enzymes to convert plant biomass into biofuels or other high value products. Through these examples, we hope to illustrate how enzyme engineering and biocatalysis can contribute to the development of cleaner and more efficient chemical industry.

Serpentinization as the source of energy, electrons, organics, catalysts, nutrients and pH gradients for the origin of LUCA and life.

Serpentinization in hydrothermal vents is central to some autotrophic theories for the origin of life because it generates compartments, reductants, catalysts and gradients. During the process of serpentinization, water circulates through hydrothermal systems in the crust where it oxidizes Fe (II) in ultramafic minerals to generate Fe (III) minerals and H2. Molecular hydrogen can, in turn, serve as a freely diffusible source of electrons for the reduction of CO2 to organic compounds, provided that suitable catalysts are present. Using catalysts that are naturally synthesized in hydrothermal vents during serpentinization H2 reduces CO2 to formate, acetate, pyruvate, and methane. These compounds represent the backbone of microbial carbon and energy metabolism in acetogens and methanogens, strictly anaerobic chemolithoautotrophs that use the acetyl-CoA pathway of CO2 fixation and that inhabit serpentinizing environments today. Serpentinization generates reduced carbon, nitrogen and - as newer findings suggest - reduced phosphorous compounds that were likely conducive to the origins process. In addition, it gives rise to inorganic microcompartments and proton gradients of the right polarity and of sufficient magnitude to support chemiosmotic ATP synthesis by the rotor-stator ATP synthase. This would help to explain why the principle of chemiosmotic energy harnessing is more conserved (older) than the machinery to generate ion gradients via pumping coupled to exergonic chemical reactions, which in the case of acetogens and methanogens involve H2-dependent CO2 reduction. Serpentinizing systems exist in terrestrial and deep ocean environments. On the early Earth they were probably more abundant than today. There is evidence that serpentinization once occurred on Mars and is likely still occurring on Saturn's icy moon Enceladus, providing a perspective on serpentinization as a source of reductants, catalysts and chemical disequilibrium for life on other worlds.

Insights on the SWEET Gene Role in Soluble Sugar Accumulation via the CO2 Fixation Pathway in Forage Maize Under Salt Stress

AbstractRising soil and water salinity endanger plant growth and crop productivity, putting global food security at risk. As plants are sessile, their adaptation to rapidly changing environments is slow, endangering their survival. As a result, mitigation efforts should shift to developing smart crops capable of withstanding dynamic and heterogeneously distributed salinity. Recent breakthroughs in bioinformatics and high throughput genomics can cost-effectively accelerate the introduction of superior varieties for saline regions. Sugar plays an essential role in biomass accumulation and is thus a viable target for forage crop improvement programs. Sugars Will Eventually be Exported Transporter (SWEET) gene family transcribes for source-sink carbon allocation in the form of sugar in higher plants. However, little is known about SWEET’s role in maize's phenotypes of agronomic interest for forage production. Here, through a genome-wide analysis, we identified and characterized 19 SWEET genes that are expressed across various shoot phenotypes. Eleven of the genes are salt-responsive, and ZmSWEET7 is most abundant in high-sugar-yielding varieties compared to low-sugar varieties. Homologous overexpression of the ZmSWEET7 increases the maximum quantum yield of photosystem II photochemistry (FV/FM), CO2 assimilation rate (A), soluble sugar content, and dry matter, with the quantum yield for CO2 fixation efficiency (phiCO2) showing the most significant increase. There is a strong positive association between phiCO2 and soluble sugar content, dry matter, and FV/FM in ZmSWEET7 overexpressing mutants compared to the wild. These findings indicate that ZmSWEET7-mediated CO2 fixation efficiency rather than assimilation rate plays a positive pleiotropic role in C accumulation in the form of sugar or dry matter via increased FV/FM. This work lays a strong foundation for salt-tolerant forage maize genetic improvement.

Unfolding the secrets of microbiome (Symbiodiniaceae and bacteria) in cold-water coral.

Recent deep-ocean exploration has uncovered a variety of cold-water coral (CWC) ecosystems around the world ocean, but it remains unclear how microbiome is associated with these corals at a molecular levels. This study utilized metabarcoding, tissue section observation, and metatranscriptomes to investigate the microbiome (Symbiodiniaceae and bacteria) of CWC species (Narella versluysi, Heterogorgia uatumani, and Muriceides sp.) from depths ranging from 260 m to 370 m. Warm-water coral (WWC) species (Acropora pruinosa, Pocillopora damicornis, and Galaxea fascicularis) were used as control groups. Results revealed that CWC host diverse bacteria and Symbiodiniaceae cells were observed in endoderm of CWC tissues. Several new candidate bacterial phyla were found in both CWC and WWC, including Coralsanbacteria, Coralqiangbacteria, Coralgsqaceae, Coralgongineae, etc. Both the 16S rRNA gene sequencing and metatranscriptomes revealed that Actinobacteria and Proteobacteria were abundant bacterial phyla in CWC. At the gene transcription level, the CWC-associated Symbiodiniaceae community showed a low-level transcription of genes involved in photosynthesis, CO2 fixation, glycolysis, citric acid cycle, while bacteria associated with CWC exhibited a high-level transcription of genes for carbon fixation via the Wood-Lijungdahl pathway, short chain fatty acids production, nitrogen, and sulfur cycles. IMPORTANCE This study shed new light on the functions of both Symbiodiniaceae and bacteria in cold-water coral (CWC). The results demonstrated that Symbiodiniaceae can survive and actively transcribe genes in CWC, suggesting a possible symbiotic or parasitic relationship with the host. This study also revealed complete non-photosynthetic CO2 fixation pathway of bacteria in CWC, as well as their roles in short chain fatty acids production and assimilation of host-derived organic nitrogen and sulfur. These findings highlight the important role of bacteria in the carbon, nitrogen sulfur cycles in CWC, which were possibly crucial for CWC survival in in deep-water environments.

When green carbon plants meet synthetic biology

AbstractRecycling 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.

De novo artificial synthesis of hexoses from carbon dioxide

Developing artificial “CO2-sugar” platforms is meaningful for addressing challenges posed by land scarcity and climate change to the supply of dietary sugar. However, upcycling CO2 into complex polyoxygenated carbohydrates involves several major challenges, including achieving enantioselective and thermodynamically driven transformation and expanding product repertoires while reducing energy consumption. We present a versatile chemoenzymatic roadmap based on aldol condensation, iso/epimerization, and dephosphorylation reactions for asymmetric CO2 and H2 assembly into sugars with perfect stereocontrol. In particular, we developed a minimum ATP consumption and the shortest pathway for bottom-up biosynthesis of the fundamental precursor, fructose-6-phosphate, which is valuable for synthesizing structure-diverse sugars and derivatives. Engineering bottleneck–associated enzyme catalysts aided in the thermodynamically driven synthesis of several energy-dense and functional hexoses, such as glucose and D-allulose, featuring higher titer (63 mmol L−1) and CO2-product conversion rates (25 mmol C L−1 h−1) compared to established in vitro CO2-fixing pathways. This chemical–biological platform demonstrated greater carbon conversion yield than the conventional “CO2-bioresource-sugar” process and could be easily extended to precisely synthesize other high-order sugars from CO2.

Functional and biotechnological potential of microbiome associated with soils colonised by cyanobacteria in drylands

Drylands are areas under continuous degradation and desertification frequently covered by cyanobacterial biocrusts. Several studies have indicated that soil microorganisms contribute to nutrient cycling. Nevertheless, little is still known about how those could potentially be participating according to their taxonomic groups. In addition, biocrust-associated communities still remain largely unexplored at taxonomic and functional levels and, therefore, biotechnologically underexploited in comparison with other terrestrial ecosystems. This work aimed at revealing the ecological and biotechnological potential of soil communities using a shotgun sequencing approach in cyanobacterial-colonised biocrusts in the Tabernas Desert regarding the cycles of carbon, nitrogen, phosphorus, iron, sulfur, and potassium. The abundance and successful adaptation of major groups Rubrobacter and Conexibacter was explained by the presence of metabolic diversity regarding glycolytic and CO2 fixation pathways but also by exhibiting a rich pool of hydrolytic enzymes. Besides, Rubrobacter representatives might potentially be driving sulfate mineralisation. Others, such as Caulobacter representatives, were also suggested to have a potential key role in K+ uptake. Furthermore, iron uptake was found to potentially be mediated via synthesis and release of siderophores, mostly derived from pseudomonads. Importantly, this work revealed a meaningful biotechnological niche in which the recovery of novel isolates is a very desirable step forward for the industry and a step forward in unwrapping the potential role of those microorganisms in soil restoration as well as in the functioning of arid soils as CO2 sinks.

Insights into Enzyme Reactions with Redox Cofactors in Biological Conversion of CO2.

Carbon dioxide (CO2) is the most abundant component of greenhouse gases (GHGs) and directly creates environmental issues such as global warming and climate change. Carbon capture and storage have been proposed mainly to solve the problem of increasing CO2 concentration in the atmosphere; however, more emphasis has recently been placed on its use. Among the many methods of using CO2, one of the key environmentally friendly technologies involves biologically converting CO2 into other organic substances such as biofuels, chemicals, and biomass via various metabolic pathways. Although an efficient biocatalyst for industrial applications has not yet been developed, biological CO2 conversion is the needed direction. To this end, this review briefly summarizes seven known natural CO2 fixation pathways according to carbon number and describes recent studies in which natural CO2 assimilation systems have been applied to heterogeneous in vivo and in vitro systems. In addition, studies on the production of methanol through the reduction of CO2 are introduced. The importance of redox cofactors, which are often overlooked in the CO2 assimilation reaction by enzymes, is presented; methods for their recycling are proposed. Although more research is needed, biological CO2 conversion will play an important role in reducing GHG emissions and producing useful substances in terms of resource cycling.

Recent Advances in Engineering Heterotrophic Microorganisms for Reinforcing CO2 Fixation Based on Calvin–Benson–Bassham Cycle

Global climate change caused by greenhouse gas emission is the fundamental challenge facing mankind. Biological carbon dioxide fixation has recently attracted much attention. The Calvin–Benson–Bassham (CBB) cycle is a major carbon fixation pathway in the biosphere, and it is the most intensively studied because of its prominence and key role in nature as the primary pathway for net CO2 fixation. It was recently shown that expressing the enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphoribulokinase (PRK) in Escherichia coli, Saccharomyces cerevisiae, Methylobacterium extorquens, Kluyveromyces marxianus, and Pichia pastoris could achieve CO2 fixation for the production of value-added chemicals. In addition, expressing the synthetic CBB cycle allowed strains to grow autotrophically on CO2. Here, we review the current understanding of the CBB cycle for CO2 fixation, summarize research progress in the assembly of the CBB cycle in heterotrophic microorganisms or developing synthetic autotrophs, and present the strategies to enhance carbon fixation efficiency of the CBB cycle in heterotrophic microorganisms. Recent progress in such applications in reinforcing CO2 fixation for the improved production of various value-added chemicals is also summarized. The challenges encountered in this process and future prospects are further discussed.

Exploring alternative pathways for the in vitro establishment of the HOPAC cycle for synthetic CO2 fixation.

Nature has evolved eight different pathways for the capture and conversion of CO2, including the Calvin-Benson-Bassham cycle of photosynthesis. Yet, these pathways underlie constrains and only represent a fraction of the thousands of theoretically possible solutions. To overcome the limitations of natural evolution, we introduce the HydrOxyPropionyl-CoA/Acrylyl-CoA (HOPAC) cycle, a new-to-nature CO2-fixation pathway that was designed through metabolic retrosynthesis around the reductive carboxylation of acrylyl-CoA, a highly efficient principle of CO2 fixation. We realized the HOPAC cycle in a step-wise fashion and used rational engineering approaches and machine learning-guided workflows to further optimize its output by more than one order of magnitude. Version 4.0 of the HOPAC cycle encompasses 11 enzymes from six different organisms, converting ~3.0 mM CO2 into glycolate within 2 hours. Our work moves the hypothetical HOPAC cycle from a theoretical design into an established in vitro system that forms the basis for different potential applications.

Exploiting a heterologous construction of the 3-hydroxypropionic acid carbon fixation pathway with mesaconate as an indicator in Saccharomyces cerevisiae

The 3-Hydroxypropionic acid (3-HP) pathway is one of the six known natural carbon fixation pathways, in which the carbon species used is bicarbonate. It has been considered to be the most suitable pathway for aerobic CO2 fixation among the six natural carbon fixation pathways. Mesaconate is a high value-added derivative in the 3-HP pathway and can be used as a co-monomer to produce fire-retardant materials and hydrogels. In this study, we use mesaconate as a reporting compound to evaluate the construction and optimization of the sub-part of the 3-HP pathway in Saccharomyces cerevisiae. Combined with fine-tuning of the malonyl-CoA reductase (MCR-C and MCR-N) expression level and optimization of 3-Hydroxypropionyl-CoA synthase, the 3-HP sub-pathway was optimized using glucose or ethanol as the substrate, with the productions of mesaconate reaching 90.78 and 61.2 mg/L, respectively.Graphical

Enhancing the Substrate Specificity of Clostridium Succinyl-CoA Reductase for Synthetic Biology and Biocatalysis.

Succinyl-CoA reductase (SucD) is an acylating aldehyde reductase that catalyzes the NADPH-dependent reduction of succinyl-CoA to succinic semialdehyde. The reaction sequence from succinate to crotonyl-CoA is of particular interest for several new-to-nature CO2-fixation pathways, such as the crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle, in which SucD plays a key role. However, pathways like the CETCH cycle feature several CoA-ester intermediates, which could be potentially side substrates for this enzyme. Here, we show that the side reaction for most CETCH cycle metabolites is relatively small (<2%) with the exception of mesaconyl-C1-CoA (16%), which represents a competing substrate in this pathway. We addressed this promiscuity by solving the crystal structure of a SucD of Clostridium kluyveri in complex with NADP+ and mesaconyl-C1-CoA. We further identified two residues (Lys70 and Ser243) that coordinate mesaconyl-C1-CoA at the active site. We targeted those residues with site-directed mutagenesis to improve succinyl-CoA over mesaconyl-C1-CoA reduction. The best resulting SucD variant, K70R, showed a strongly reduced side activity for mesaconyl-C1-CoA, but the substitution also reduced the specific activity for succinyl-CoA by a factor of 10. Transferring the same mutations into a SucD homologue from Clostridium difficile similarly decreases the side reaction of this enzyme for mesaconyl-C1-CoA from 12 to 2%, notably without changing the catalytic efficiency for succinyl-CoA. Overall, our structure-based engineering efforts provided a highly specific enzyme of interest for several applications in biocatalysis and synthetic biology.

Metabolic homeostasis and growth in abiotic cells

Metabolism constitutes the core chemistry of life. How it began on the early Earth and whether it had a cellular origin are still uncertain. A leading hypothesis for life's origins postulates that metabolism arose from geochemical CO2-fixing pathways, driven by inorganic catalysts and energy sources, long before enzymes or genes existed. The acetyl-CoA pathway and the reductive tricarboxylic acid cycle are considered ancient reaction networks that hold relics of early carbon-fixing pathways. Although transition metals can promote many steps of these pathways, whether they form a functional metabolic network in abiotic cells has not been demonstrated. Here, we formulate a nonenzymatic carbon-fixing network from these pathways and determine its functional feasibility in abiotic cells by imposing fundamental physicochemical constraints. Using first principles, we show that abiotic cells can sustain a steady carbon-fixing cycle that performs a systemic function over a relatively narrow range of conditions. Furthermore, we find that in all feasible steady states, the operation of the cycle elevates the osmotic pressure, leading to volume expansion. These results suggest that achieving homeostatic metabolic states under prebiotic conditions was possible, but challenging, and volume growth was a fundamental property of early metabolism.