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Carbon fluxes rewiring in engineered E. coli via reverse tricarboxylic acid cycle pathway under chemolithotrophic condition

Journal of Biological Engineering volume 19, Article number: 20 (2025) Cite this article

Abstract

Background

A transgenic strain of Escherichia coli has been engineered to directly assimilate gaseous CO2 into its biomass through hydrogen-powered anaerobic respiration. This was achieved by expressing key components of the reverse tricarboxylic acid (rTCA) cycle, including genes encoding α-ketoglutarate: ferredoxin oxidoreductase (KOR) and ATP-dependent citrate lyase (ACL) from Chlorobium tepidum. These enzymes were selected for their essential roles in enabling CO2 fixation and integration into central metabolism.

Results

This study found that KOR alone can support cellular maintenance under chemolithotrophic conditions, while additional expression of ACL enhances CO2 assimilation. Using isotopic 13CO2 tracing, it was demonstrated that KOR alone facilitates CO2 assimilation into TCA metabolites. However, co-expression of ACL with KOR redirected carbon fluxes from TCA cycle toward essential metabolic pathways, particularly those involved in protein and nucleotide biosynthesis. Compared to KOR alone, ACL co-expression significantly increased isotopic enrichments in amino acids (e.g., methionine, threonine, glycine) and nucleotides (e.g., deoxythymidine, deoxycytidine). These results suggest that ACL supports the synthesis of nitrogen-containing metabolites when inorganic nitrogen is sufficient, while KOR alone sustains core metabolic functions under chemolithotrophic conditions.

Conclusions

This study demonstrates a novel strategy to engineer E. coli for CO2 fixation using only one or two heterologous enzymes under chemolithotrophic conditions. These findings reveal the minimal genetic and nutritional requirements for CO2 assimilation and provide insights into metabolic flux partitioning in engineered strains. This research paves the way for sustainable applications in carbon fixation and biotechnological innovation.

Background

The commitment to achieving net zero carbon emissions by 2050 and implementing carbon border adjustment mechanisms have elevated CO2 fixation to a prominent topic, particularly within the field of life sciences [1]. The development of genetic pathways for CO2 fixation and the reconfiguration of carbon fluxes have become a critical issue. Modern synthetic biotechnology and metabolomics advancements have facilitated more effective strategies to redesign and trace the CO2 fluxes into specific target compounds.

Autotrophic bacteria play a vital role in ecosystems by continuously supplying organic carbon to heterotrophs through CO₂ fixation [2]. However, their limited carbon fixation efficiency, slow growth rates, and dependence on extreme environmental conditions restrict their adaptability as cell factories [3]. Chemolithotrophs are a diverse group of microbes capable of deriving all the energy needed for growth by oxidizing inorganic compounds such as hydrogen (H2), hydrogen sulfide (H2S), and reduced metals [4,5,6,7]. By contrast, Escherichia coli presents several advantages for metabolic engineering applications, including rapid growth, substrate versatility, and ease of genetic manipulation [8]. The fixation of CO2 into living matter supports all life on earth. In early earth, the last universal common ancestor (LUCA) and autotrophic microbes, including archaea, could fix CO2 under extreme conditions with minimal inorganic nutrients [9]. At least six types of autotrophic carbon-fixation pathways have been identified across the tree of life, including (1) reductive acetyl-CoA pathway (Wood–Ljungdahl pathway) in some strict anaerobic Proteobacteria, Planctomycetes, spirochaetes and Euryarchaeota [10,11,12,13,14]; (2) 3-hydroxypropionate bicycle in some green non-sulphur bacteria of the family Chloroflexaceae [15,16,17,18]; (3) 3-hydroxypropionic acid/4-hydroxybutyrate cycle in aerobic Crenarchaeota [19]; (4) dicarboxylate-4-hydroxybutyrate cycle in anaerobic Thermoproteales and Desulfurococcales [20,21,22], (5) reductive pentose phosphate cycle (Calvin–Benson–Bassham (CBB) cycle) in plants, algae, cyanobacteria, some aerobic or facultative anaerobic Proteobacteria, Sulfobacillus and Oscillochloris [23,24,25,26]; and (6) reverse tricarboxylic acid (rTCA) cycle (Arnon–Buchanan cycle) in green sulfur bacterium [20, 27,28,29,30].

Among these, the CBB cycle is the most significant autotrophic carbon fixation pathway in plants and algae [2, 31,32,33]. Compared to the CBB cycle, rTCA cycle appears more efficient for CO2 fixation in microorganisms [27, 30, 34], and the rTCA cycle is considered a plausible candidate for the earliest autotrophic metabolism at the origin of life [35]. Its origins are proposed to predate the advent of enzymes, ribonucleic acid (RNA), or cells, with its imprint remaining inherently embedded in the core metabolic structure [36]. Modern theories suggest that the origin of life involved a dynamic process through the intermediacy of five essential universal metabolites—acetate, pyruvate, oxaloacetate, succinate, and α-ketoglutarate—comprising carbon, hydrogen, and oxygen [37, 38]. Muchowska et al. described a purely chemical reaction network promoted by ferrous iron involving aqueous pyruvate and glyoxylate, two products of abiotic CO2 reduction. This network could synthesize nine of the eleven biological tricarboxylic acid (TCA) cycle intermediates, including all five universal metabolic precursors [38]. The observed network overlaps substantially with the TCA and glyoxylate cycles [38] and may represent a prebiotic precursor to these core metabolic pathways. Among these universal pathways, we proposed that the carboxylation of succinyl-CoA to α-ketoglutarate, catalyzed by α-ketoglutarate: ferredoxin oxidoreductase (KOR, encoded by genes of two sub-units: korA and korB), is the most relevant to early autotrophic life for the following reasons: (a) it operates in phylogenetically diverse, extreme-environmental autotrophic bacteria [2]; (b) it is a crucial step in carboxylation within the rTCA cycle, necessary for driving this cycle; and (c) α-ketoglutarate plays a central role in this metabolic network [39]. KOR was selected as the primary entry for carbon incorporation due to its position at the intersection of anabolic and catabolic pathways. Additionally, ATP-dependent citrate lyase (ACL, encoded by genes of two sub-units: aclB and aclA) was included in our carbon-fixing model to catalyze the cleavage of citrate into acetyl-CoA and oxaloacetate, potentially providing key carbon sources for downstream metabolites.

The rTCA machinery has been successfully constructed in E. coli through the expression of ten genes encoding four irreversible enzymes of the TCA cycle in wildtype E. coli JM109 (Fig. 1a), derived from the phototrophic green sulfur bacterium Chlorobium tepidum [40]. The recombinant E. coli harboring these ten genes demonstrated CO2 assimilation capabilities, evidenced by a reduction in CO2 release. However, the specific roles of KOR and ACL in redirecting carbon fluxes have yet to be fully elucidated.

Fig. 1
figure 1

a The pathways of central carbon metabolism, TCA cycle, fatty acid synthesis, and genetic engineering strategy. b The growth curves of transgenic E. coli K12 with different gene sets expression under organic-free medium supplied with 0.5% DMSO, 0.00005% thiamine, H2 gas, without any amino acid in the presence or absence of CO2. c Enlarged view of growth curves under organic-free medium in the absence of CO2. The DNA cassettes used to construct expression plasmids pGETS-K and pGETS-KA which encode genes korA and korB, aclB and aclA form C. tepidum. Data are expressed as means standard ± deviation, n = 3 independent biological samples

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This study aimed to identify the minimal gene set and nutritional conditions required for CO2 assimilation in heterotrophic E. coli using the rTCA machinery. The carbon flow and the partitioning among critical metabolic fluxes under such limited conditions was also investigated, providing insights into the essential carboxylation pathways in a heterotrophic host. The study also presents a novel strategy for engineering E. coli to not only assimilates external gaseous CO2 but also to redirects and concentrates carbon flow towards the production of valuable target metabolites.

Materials and methods

Chemicals and materials

Luria-Bertani broth was obtained from Cyrusbioscience, Inc. (New Taipei City, Taiwan). M9 minimal medium was sourced from Becton, Dickinson and Company (East Rutherford, USA). Casein hydrolysate, FeCl2, aspartic acid, sodium nitrate, 13C-CO2, chloroform, methoxymation hydrochloride, N-tert-Butyldimethylsily-N-methyltrifluoroacetamide (MTBSTFA), pentafluorobenzyl bromide (PFBBr), and methanolic sodium hydroxide were procured from Sigma-Aldrich (Merck Group, St. Louis, USA). Dimethyl sulfoxide (DMSO), thiamine and NiCl2 were procured from Acros Organics (Thermo Fisher Scientific Inc., Waltham, USA). MgSO4 was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). CaCl2 and methanol were purchased from Aencore Chem. Pty. Ltd. (City of Whitehorse, Australia). Chloramphenicol was acquired from Bio Basic Inc. (Markham, Canada). Glucose was purchased from Showa Chemical Industry Co., Ltd. (Tokyo, Japan). Tris buffer was obtained from MDBio, Inc. (New Taipei City, Taiwan). [13C3]-pyruvate, [13C3]-lactate, [13C4]-succinate, [13C4]-fumarate, [13C4]-malate, [1,5,6-carboxyl-13C3]-citrate and 13C-formate were sourced from Cambridge Isotope Laboratories, Inc. (Tewksbury, USA). BCl3-methanol solution was purchased from Supelco (Merck Group, Bellefonte, USA). Sodium chloride was obtained from Panreac Química SLU (Barcelona, Spain). HCl was purchased from Biosynth Ltd. (Staad, Switzerland). Formic acid was acquired from J. T. Baker (Mallinckrodt Baker, Inc., Phillipsburg, USA). N, O-bis-[trimethylsilyl] trifluoroacetamide 1% trimethylchlorosilane was sourced from Pierce Biotech (Thermo Fisher Scientific Inc., Waltham, USA). Acetonitrile, n-hexane and acetone were purchased from ECHO Chemicals Co. Ltd. (Miaoli County, Taiwan). DB-225MS, DB-225 J&W GC, CP-Sil and HP-5MS columns were obtained from Agilent Technologies Ltd. (Santa Clara, USA).

Construction of the transgenic strains

The construction of the transgenic strains used in this study followed the methodology described in a previous work employing the Ordered Gene Assembly in the Bacillus subtilis (OGAB) method [41, 42] with modifications detailed in [40]. The designed 3’-tail deoxyribonucleic acid (DNA) cassettes, which contain three complementary nucleotides for the OGAB method, were amplified using the primer pairs KOR-DraIII-f/KOR-PflMI-tail-r and ACL-PfMI-f/ACL-PflMI-tail-r. These primers encode α-ketoglutarate: ferredoxin oxidoreductase and ATP-dependent citrate lyase from plasmid pGETS-KA. Plasmid pGETS-K was constructed with the DNA cassette amplified using the KOR-DraIII-f/KOR-PflMI-tail-r primer pair. Plasmid pGETS-KA was constructed with DNA cassettes amplified using the KOR-DraIII-f/KOR-DraIII-r and ACL-PfMI-f/ACL-PflMI-tail-r primer pairs. The DNA cassettes of the constructed plasmids were verified by sequencing (Genomics, New Taipei City, Taiwan). The constructed strains and primers are listed in Table 1a.

Table 1 (a) The primers employed for constructing the pGETS118-reverse tricarboxylic acid plasmid. (b) Differential gene expression in the constructed E. coli strains K or KA/VC under incubation with CO2 as the sole carbon source
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Microbial incubation and counting

Microbial samples were incubated and collected according to the procedure described previously [40]. The E. coli strains were cultured at 37 °C in Luria-Bertani broth for 12 h, washed with M9 minimal medium and collected. The bacteria were concentrated to an optical density (O.D.) of 5 at 600 nm and inoculated (1%, v/v) into 150 mL of organic-free medium (M9 minimal medium with 0.01 M NaNO3, 2 mM MgSO4, 0.1 mM CaCl2, 0.01 mM NiCl2, 0.01 mM FeCl2 and 20 µg/mL chloramphenicol) in a sealable glass bottle. The medium was prepared in an anaerobic chamber (Coy Laboratory Products, Inc., USA) and sealed with robber and aluminum caps (Shang-Jhan Class Instrument Co., Hsinchu City, Taiwan) for anaerobic experiments.

The headspace for each bottle (100 mL) was filled with hydrogen and CO2 (90% H2, 10% CO2). To elucidate the role of korAB and aclBA genes, two transgenic strains, namely strain KA (korAB + aclBA) and strain K (korAB), were cultured in the organic-free medium. The CO2 gas was refilled every 12 h during incubation.

In the isotopic labeling experiment, gaseous CO2 was replaced with 13CO2. Bacterial growth was assessed by measuring optical density (GeneQuant 1300, GE Healthcare, Little Chalfont, Buckinghamshire, UK) or counting colony-forming units (cfu).

Enzyme assays

The E. coli strains were incubated in M9 medium supplied with 2 g/L glucose, 2 mM MgSO4, 0.1 mM CaCl2, 50 µg/mL thiamine, 20 µg/mL chloramphenicol, and 1 g/L casein hydrolysate under anaerobic conditions. The headspace of the bottle was purged with H2 and CO2 and sealed with rubber to maintain the anaerobic conditions. For anaerobic respiration conditions, glucose and casein hydrolysate were replaced with 10 mM sodium nitrate, 0.01 mM NiCl2, and 0.01 mM FeCl2. Approximately 4 × 109 bacteria cells were collected from 150 mL of glucose medium or 750 mL anaerobic respiration medium and sonicated in Tris buffer (100 mM Tris-HCl at pH 8.4, 3 mM dithioerythritol). The crude proteins were collected from the supernatant by centrifugation at 10,000 ×g. Methyl viologen assays were performed in an anaerobic chamber filled with 9% H2, 15% CO2, and 76% N2 gas at 35 °C, and optical densities were measured. Enzyme activities were assessed using a Jasco V-630 UV-Vis spectrophotometer (JASCO, Tokyo, Japan) equipped with a temperature controller (Water Bath D-606, Deng Yng, New Taipei City, Taiwan).

α-Ketoglutarate oxidoreductase activity assays

The activity of α-ketoglutarate oxidoreductase was assessed using a modified method described previously [43]. The enzyme activity was determined by monitoring succinyl-CoA reduction (reduced methyl viologen: succinyl-CoA oxidoreductase). The assay was conducted in a 1 mL reaction mixture containing 100 mM Tris-HCl at pH 8.4, 2 mM MgCl2, 4 mM methyl viologen, and 1 mM succinyl-CoA. Dithionite was added from a 1 M stock solution until the methyl viologen-containing assay solutions exhibited a faint blue color. Optical density changes were measured at 578 nm (methyl viologen, ε578 = 9.8 × 103 M−1 cm−1) after the addition of the succinyl-CoA solutions.

Isocitrate dehydrogenase activity assay

Isocitrate dehydrogenase activity was measured by the reductive carboxylation of α-ketoglutarate [44]. The assay was performed in a reaction mixture containing 100 mM potassium phosphate buffer (pH 6.5), 0.6 mM α-ketoglutarate, 0.5 mM Nicotinamide adenine dinucleotide phosphate (NADPH), 1 mM MnCl2, and 20 mM NaHCO3. Optical density changes were measured at 340 nm (NADPH, ε340 = 6.22 × 103 M−1 cm−1) following the addition of NADPH.

Isocitrate lyase activity assay

Isocitrate lyase activity was determined by the formation of glyoxylate-phenylhydrazone (ε324 = 16.8 × 103 M−1 cm−1) [45], coupled with intrinsic aconitase. The assay was conducted in a reaction mixture containing 100 mM Tris-HCl (pH 7.1), 7.5 mM MgCl2, 5 mM phenylhydrazine-HCl, 3 mM cysteine-HCl, and 2 mM sodium citrate. Optical density changes were measured at 324 nm following the addition of sodium citrate.

Succinyl-CoA synthetase activity assay

Succinyl-CoA synthetase activity was measured by monitoring the formation of thioester bonds of succinyl-CoA (ε235 = 4 × 103 M−1 cm−1) [46]. The assay was conducted in a reaction mixture containing 100 mM Tris-HCl (pH 7.1), 2 mM 2-mercaptoethanol, 10 mM MgCl2, 0.375 mM adenosine triphosphate (ATP), 0.1 mM coenzyme A, and 50 mM sodium succinate dibasic. Optical density changes were measured at 235 nm following the addition of sodium succinate dibasic.

Determination of TCA cycle metabolite enrichments and concentration

The TCA cycle metabolites from microbial samples were extracted using methanol and chloroform with an internal standard mixture ([13C3]-pyruvate, [13C3]-lactate, [13C4]-succinate, [13C4]-fumarate, [13C4]-malate and [1,5,6-carboxyl-13C3]-citrate). The polar metabolites (upper phase) were freeze-dried and derivatized using methoxymation hydrochloride and MTBSTFA. The metabolite derivatives were separated on a DB-225MS column (30 m × 0.25 mm) and analyzed in electron ionization mode by gas chromatography-mass spectrometry (GC-MS) using a model 6890 gas chromatograph and model 5973 mass spectrometer (Agilent Technologies Ltd., Santa Clara, USA). The results were quantitated by referencing the standard curve for each TCA cycle metabolite. The coefficient of determination (r2) for the standard curve was consistently greater than 0.99 [47, 48].

Extraction and quantitation of short-chain fatty acids in microbes

The short-chain fatty acids (SCFAs) from microbial samples were extracted and derivatized using 100 mM PFBBr in acetone in the presence of a working buffer (0.5 M phosphate buffer (pH 8.0) and a standard solution of sodium 13C-formate (1 mM sodium 13C-formate) in HPLC-grade water. n-hexane was used to separate polar and nonpolar metabolites from the extracted samples. The SCFAs in the organic phase were converted to alkylated derivatives and separated using a DB-225 J&W GC column (30 m × 0.25 mm). Mass spectrometry (MS) was performed in electron impact ionization mode, monitoring m/z at 226, 227, 240, 254, and 268 for formate, internal standard, acetate, propionate, and butyrate, respectively. The SCFAs were quantified by GC-MS using electron ionization mode. The results were quantitated by referencing a standard curve with known concentrations of SCFA standards plotted against the 12C/13C-formate ratio on the y-axis. The coefficient of determination for the standard curve was consistently greater than 0.99 [49].

Determination of fatty acid profile in microbes

Fatty acids from the microbial samples were extracted using a chloroform/methanol mixture (1:2) on ice, along with an internal standard (C19:0 in ethanol) [50]. The nonpolar metabolites were freeze-dried and esterified using methanolic sodium hydroxide at 100 °C. They were then extracted using BCl3-methanol solution (12%), saturated aqueous sodium chloride, and n-hexane [51, 52]. The fatty acids were converted to methyl ester derivatives and separated on a CP-Sil column (50 m × 0.25 mm). Their composition was determined by GC-MS in electron ionization mode [52,53,54,55].

Determination of amino acid profile and enrichments

Microbial pellets were collected from the broth by centrifugation (10,000 ×g) at 4 °C after incubation. The protein pellets were hydrolyzed using 6 N HCl under vacuum. Amino acids were then converted to heptafluorobutyryl n-propyl ester derivatives and separated on an HP-5MS column (30 m × 0.25 mm). Isotopic enrichment of amino acids was determined using GC-MS in electron capture negative ionization mode, as described previously [56, 57].

Determination of deoxyribonucleic acid metabolites enrichments

The microbial pellets were collected from the broth by centrifugation (10,000 ×g) at 4 °C after incubation. DNA samples were dried and hydrolyzed in formic acid under vacuum, then derivatized by N, O-bis-[trimethylsilyl] trifluoroacetamide 1% trimethylchlorosilane and acetonitrile. The resulting trimethylsilane-base derivatives were separated on an HP-5MS column. Isotopic enrichments in deoxyadenylate (dAMP, dA), deoxyguanylate (dGMP, dG), deoxythymidine (dTMP, dT), and deoxycytidine (dCMP, dC) were determined in positive ionization mode by GC-MS, as described previously [56, 58].

Statistical analysis

Comparisons of the means between the control and the experimental strains were conducted using the Student’s t-test and Mann-Whitney U test. To examine statistical difference among the transgenic strains, a one-way ANOVA model followed by Tukey’s post hoc test was employed. All statistical analyses were performed using SYSTAT 11.0 for Windows™ (Systat software Inc., Richmond, CA, USA). The results were deemed statistically significant if p values were < 0.05, and borderline significance was considered for p values < 0.1.

Results and discussion

The present study provides new insights into the role of KOR in supporting transgenic E. coli under chemolithotrophic mode through hydrogen-powered anaerobic respiration. KOR expression facilitated CO₂ assimilation and helped retain carbon fluxes within the TCA cycle. Furthermore, the additional expression of ACL preferentially redirected carbon fluxes from the TCA cycle and further channeled them into essential metabolic pathways, including those for protein and nucleotide biosynthesis. These pathways likely play critical roles in sustaining essential reactions under nutrient-limited conditions. The implications of these findings are discussed below.

α-ketoglutarate: ferredoxin oxidoreductase (KOR) supported cellular maintenance of Escherichia coli under chemolithotrophic mode

Transcriptome analysis under chemolithotrophic mode was conducted using CO2 as the sole carbon source after two transgenic E. coli strains were constructed. The heterologous genes were well-expressed in two transgenic strains K (korAB genes) and KA (korAB and aclBA genes) (Table 1b). In enzymatic activity assays, α-ketoglutarate oxidoreductase activity encoded by KOR was significantly higher in the K (49.6 ± 12.2 nmol/min/mg protein, p = 0.002) and KA (47.0 ± 16.6 nmol/min/mg protein, p = 0.008) strains compared to that of the vector control strain (strain VC, < 0.01), confirming the functionality of the heterologous enzymes in these transgenic strains (Additional file 1: Table S1).

The activities of isocitrate dehydrogenase and isocitrate lyase showed no significant difference between strains VC and K due to their natural presence in E. coli [45, 59]. However, four enzymes including α-Ketoglutarate oxidoreductase, isocitrate dehydrogenase, isocitrate lyase, and succinyl-CoA synthetase exhibited higher activity in strain KA compared to strain VC. Notably, succinyl-CoA synthetase activity was significantly higher in the strain KA (9072 ± 522) compared to strain K (5457 ± 674, p = 0.008). This increase in activity may be attributed to enhanced acetyl-CoA production driven by ACL expression. Acetyl-CoA, a substrate of malate synthase A (encoded by the aceB gene), promotes glyoxylate consumption, which is also a product of isocitrate lyase. As a result, isocitrate lyase activity was marginally higher in strain KA (17.3 ± 3.5, p = 0.073) than in strain VC, though not significantly different in strain K (23.2 ± 12.6, p = 0.280). The promotion of isocitrate lyase likely increased succinate levels, which serve as the substrate for succinyl-CoA synthetase. This cascade of metabolic changes may explain the significantly enhanced succinyl-CoA synthetase activity observed in strain KA.

To evaluate the role of rTCA machinery under chemolithotrophic conditions, two transgenic E. coli strains (K and KA) and a strain with an empty vector (VC) were cultured in an organic-free medium (devoid of organic compounds) under conditions with and without CO2 supply. The medium was supplemented with hydrogen as the electron donor and nitrate as the electron acceptor for anaerobic respiration. None of the transgenic strains could survive without CO2 (Fig. 1c, hollow symbols). However, KOR-expressing strains (K and KA) showed an increase in microbial counts under chemolithotrophic conditions with CO2 supply, in contrast to the VC strain (Fig. 1b, solid symbols). These findings indicate that KOR expression can support cellular maintenance under chemolithotrophic conditions through hydrogen-powered anaerobic respiration.

In previous studies, we introduced four rTCA enzymes into E. coli JM109, which successfully assimilated CO2 with glucose supplementation [40, 60]. Yu et al. introduced five heterologous enzymes (fructose-1,6-bisphosphatase, phosphoribulokinase, fructose-bisphosphate aldolase, ribulose bisphosphate carboxylase, transketolase, and glyceraldehyde-3-phosphate dehydrogenase) from the CBB cycle that enabled CO2 fixation in the presence of a glucose supply [61]. Gleizer et al. engineered an evolved E. coli strain with three additional enzymes including: ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), phosphoribulokinase through the RuBisCO system, and formate dehydrogenase, enabling autotrophic growth using formate as an energy source [62]. These approaches demonstrated that engineered carbon-fixing E. coli strains require multiple heterologous enzymes and supplementation with glucose or formate to accomplish the carbon fixation recycling system and maintain cell viability.

This study is the first to successfully engineer a carbon-fixing E. coli capable of sustaining cellular maintenance under chemolithotrophic conditions by introducing a single enzyme (KOR) from rTCA cycle, with CO₂ as the sole carbon source and hydrogen as the energy source. By limiting the heterologous enzyme expression to KOR only, this work achieved carbon fixation, cellular maintenance, and the production of essential metabolites under chemolithotrophic conditions.

Constraint-based flux balance analysis of the reverse tricarboxylic acid cycle machinery in Escherichia coli

In-silico modeling was employed to explore potential pathways for CO2 assimilation in E. coli via the rTCA machinery. The predicted pathways for CO2 assimilation in the E. coli strains are illustrated in Additional file 1: Fig. S1. Additional file 1: Tables S2-S4 present the stoichiometric matrix results used in the flux balance analysis aimed at maximizing the biomass reaction rate. The model predicted that CO2 assimilated by KOR predominantly follows the pathway from glyoxylate to tartronate semialdehyde to glycerate pathway, regulated by glyoxylate carboligase (encoded by the gcl gene). To validate the importance of this pathway for transgenic cell survival, this study assessed the cell growth of the gcl gene knock-out strains under organic-free conditions in the presence of CO2. None of the gcl gene knock-out strains could survive under these conditions (Additional file 1: Fig. S1b), highlighting the essential role of this pathway. However, further isotopic labeling experiments are needed to quantify the actual metabolic fluxes in transgenic strains.

Expression of α-ketoglutarate: ferredoxin oxidoreductase (KOR) significantly enhanced the incorporation of CO2 in tricarboxylic acid cycle metabolites

Strains K and KA were cultured under organic-free conditions with 13CO2 supplementation. The potential and actual incorporations of 13CO2 into TCA cycle metabolites are depicted in Fig. 2. Compared to strain KA, strain K exhibited significantly higher enrichments of 13CO2 in citrate (p = 0.004), succinate (p = 0.008), pyruvate (p = 0.016), α-ketoglutarate (p = 0.005), and malate (p = 0.007). Increased TCA metabolite concentrations were also observed compared to strain VC, especially in fumarate (p = 0.089), malate (p = 0.013), and citrate (p = 0.025).

These results indicate that KOR expression alone effectively enhances the retention of carbon fluxes within the TCA cycle, whereas the addition of ACL might divert the carbon flow away from the TCA (Fig. 2; Table 2). Thus, KOR alone and not KOR + ACL is more effective in maintaining CO2 fluxes within the TCA cycle compared to KOR + ACL. Further investigation was conducted to explore the role of ACL.

Fig. 2
figure 2

An overview of the potential metabolic fate of carbon during CO2 carboxylation in TCA cycle. Isotopic labeling experiments from 13CO2 demonstrate the preferential TCA cycle metabolites synthesis and metabolites concentration in transgenic E. coli K12. Data are expressed as means standard ± deviation, n = 3-4/group. A two-tailed t-test was used for statistical analysis, **** indicates p < 0.005 (highly statistically significantly difference); *** indicates p < 0.01; ** indicates p < 0.05 (statistically significantly difference); * indicates p < 0.1 (borderline statistically significantly difference). Strains were supplemented with 10% 13CO2 and 90% H2, re-filled 10% of 13CO2 per 12 hr for 48 hr incubation. Isotopic enrichments derived from 13CO2 are detected in the M + 1 specie of various organic acid. The labeled 13CO2 are marked in red asterisk * marks. Hetero-expressed enzymes: KOR & ACL are marked in purple and yellow, respectively

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Table 2 Isotopic labeling experiments from 13CO2 demonstrate the preferential TCA cycle metabolites synthesis and metabolites concentration in transgenic E. Coli K12
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The TCA cycle is a crucial metabolic pathway that integrates carbohydrate, fat, and protein metabolism [63]. Compared to strain VC, the transgenic strain K expressing KOR demonstrated the supporting cellular maintenance under chemolithotrophic conditions. This finding underscores the importance of KOR within the TCA cycle for CO2 assimilation. The fact that a single enzyme (encoded by two genes) can drive the core reactions in the TCA cycle is noteworthy. α-ketoglutarate, the product of the KOR reaction, is one of the five universal metabolic precursors, as shown through chemical reactions [38], suggesting that the reactions encoded by the korAB genes closely resemble metabolic processes. The in-silico modeling of strain K proposed an alternative metabolic pathway where glyoxylate is converted to glycerate 2-phosphate. The glyoxylate shunt is an anabolic pathway within the TCA cycle in E. coli, which synthesizes components of proteins and the pyrimidines under limited carbon source incubation [64, 65]. This pathway may support cell survival under nutrient-scarce conditions. Metabolic studies further confirmed that KOR expression enhanced the metabolic flux toward glyoxylate, accumulating key metabolites such as citrate, malate, and fumarate. With only one exogenous enzyme for CO2 fixation, E. coli can activate the glyoxylate shunt and replenish critical TCA metabolites. The activation of the glyoxylate shunt by KOR expression may mimic the metabolic pathways of ancient microbes [38].

Additional ATP-dependent citrate lyase (ACL) expression rewired carbon flux of CO2

Although the in-silico model predicted that CO2 flux rates in the TCA cycle would be higher in the strain expressing both KOR and ACL compared to the strain expressing KOR alone (Additional file 1: Table S4), the experimental results were different. In the experiments using a stable isotope tracer, strain K retained a significantly higher amount of labeled CO2 in α-ketoglutarate + 1, succinate + 1, malate + 1, and citrate + 1, resulting in increased succinate production compared to strain KA (Fig. 2; Table 2). This discrepancy could be due to differences between the modeling and actual situations. The flux balance analysis and models used [66] did not account for cofactors (nicotinamide adenine dinucleotide (NAD+), NADP+, ATP, and others), water, or H+, assuming these were sufficiently supplied and did not limit biomass synthesis. Additionally, the anaplerotic reactions from the TCA cycle were not included in the current model, and the actual experiments provided complementary insights to the in-silico model.

Many biosynthetic reactions utilize the TCA cycle molecules as substrates; however, the model did not incorporate the metabolic pathways for amino acid and protein, fatty acid, or nucleotide biosynthesis. The experiments showed that while the transgenic E. coli expressing ACL accumulated fewer CO2 fluxes within the TCA cycle compared to the KOR-only strain, wild-type E. coli did not express ACL, and this cannot cleave citrate into acetyl-CoA and oxaloacetate. The additional expression of ACL in strain K appeared to redirect CO2 flux toward oxaloacetate and aspartate. Given that aspartate is a proteinogenic amino acid [67] vital for protein synthesis in E. coli [68], this study further examined the incorporation of CO2 into cellular proteins.

“Co-expression of ATP-Dependent Citrate Lyase (ACL) and α-Ketoglutarate: Ferredoxin Oxidoreductase (KOR) exhibited limited effect on fatty acid production

ACL catalyzes the ATP-dependent and coenzyme A (CoA)-dependent conversion of citrate to oxaloacetate and acetyl-CoA, which are key precursors for the biosynthesis [69] of fatty acids, cholesterol, and acetylcholine [70, 71]. The possible role of ACL expression in promoting fatty acid synthesis in the transgenic strains was investigated under anaerobic conditions. However, the experiments showed that the concentrations of fatty acid (Additional file 1: Fig. S2a) and SCFAs (Additional file 1: Fig. S2b) did not differ between strains VC and KA. Additionally, the concentration of butyrate (ng/log cfu) in strain KA (63.37 ± 9.42) was lower than strain VC (79.94 ± 3.02, p = 0.044), suggesting that ACL expression did not promote fatty acid synthesis under such a scarce environment.

As for the short-chain fatty acids, strain K exhibited a trend of lower levels of formate, acetate, and propionate compared to strain VC and KA (p < 0.1), while butyrate levels were significantly lower (p < 0.05). This may indicate that the expression of KOR helps keep CO₂-derived fluxes within the TCA cycle at the expanse of reduced synthesis of SCFAs. A previous study showed that E. coli can resorb acetate for converting it to acetyl-CoA via acetyl-CoA synthetase (Acs) and utilized it to energy and biosynthetic components through the TCA cycle and the glyoxylate shunt [72]. This aligns with the observed TCA cycle metabolite profile in strain K. To sustain KOR activity, strain K appears to use SCFAs as substrates to replenish its carbon source, reflecting a metabolic adaptation to maintain carbon flux under chemolithotrophic conditions.

Co-expression of KOR and ACL restored SCFA levels to values comparable to those of strain VC. Although additional ACL expression in the presence of KOR did modestly enhance SCFA, it did not enhance long chain fatty acid synthesis. Rather, additional ACL expression help re-direct CO2 metabolic fluxes into nucleotide and protein synthesis.

ATP-dependent citrate lyase (ACL) and α-ketoglutarate: ferredoxin oxidoreductase (KOR) expression assisted 13CO2 carbon fixation in protein biosynthesis

ACL cleaves citrate into oxaloacetate and acetyl-CoA, both of which are directly linked to fatty acid turnover in E. coli [73]. The similar contents of fatty acid among strains VC, K, and KA suggested that the CO2 assimilated by KOR was not used for fatty acid with the addition of ACL and that ACL may divert the flow of carbon into other critical metabolic pathways. Consequently, carbon fluxes in cellular protein biosynthesis were experimentally tracked (Fig. 3). As data shown in Fig. 3; Table 3, strain KA exhibited enhanced amino acid enrichments of cellular protein hydrolysates compared to strain K, including threonine + 1, phenylalanine + 1, glycine + 1, isoleucine + 1, methionine + 1, serine + 1, glutamate + 1, cysteine + 1, and alanine + 1. These results suggested that the expression of heterologous KOR with additional ACL, facilitated an increase in CO2 incorporation in protein biosynthesis.

Fig. 3
figure 3

Incorporation of CO2 into cellular proteins. The intermediates of the TCA cycle, oxaloacetate and α-ketoglutarate, are metabolized to produce various amino acids, which are subsequently incorporated into cellular proteins. Isotopic labeling experiments using 13CO2 demonstrate the preferential synthesis of proteinogenic amino acids in transgenic E. coli K12. Data are presented as means ± standard deviation, with n = 3 independent biological samples. A two-tailed t-test was used for statistical analysis, **** indicates p < 0.005 (highly statistically significantly difference); *** indicates p < 0.01; ** indicates p < 0.05 (statistically significantly difference); * indicates p < 0.1 (borderline statistically significantly difference). Strains were supplemented with 10% 13CO2 and 90% H2, with 10% of 13CO2 replenished every 12 h during the 48 h incubation. Isotopic enrichments derived from 13CO2 are detected in the M + 1 specie of various amino acids. The labeled 13CO2 are marked in red asterisk * marks. Hetero-expressed enzymes KOR and ACL are indicated in purple and yellow, respectively

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Table 3 Isotopic labeling experiments from 13CO2 demonstrate the preferential proteinic amino acid synthesis in transgenic E. Coli K12
Full size table

Expressing ACL in strain K may have been expected to increase fatty acid synthesis due to the greater availability of precursors for fatty acid elongation. However, in this study, the addition of ACL in strain K did not enhance fatty acid production (Additional file 1: Fig. S2). This outcome may be influenced by the presence of inorganic nitrogen (NaNO₃) in the culture medium. Nitrogen limitation has been shown to promote fatty acid accumulation in various organisms [74,75,76]. It is plausible that when nitrogen is abundant, carbon fluxes are more likely to support the synthesis of nitrogen-containing compounds rather than lipid accumulation. These results suggest that carbon flow in strain KA may be redirected toward other metabolic pathways, such as the production of oxaloacetate, a precursor of aspartate. Aspartate is a key compound involved in the synthesis of various metabolites, including amino acids and nucleotides [77, 78]. This redirection aligns with the observed role of ACL in promoting protein biosynthesis over fatty acid production under chemolithotrophic conditions in the presence of NaNO₃. Consequently, the effects of KOR and ACL expression on CO₂ incorporation into nucleotides were further investigated.

ATP-dependent citrate lyase (ACL) and α-ketoglutarate: ferredoxin oxidoreductase (KOR) expression assisted 13CO2 carbon fixation in nucleotides biosynthesis

Nucleotides, essential for RNA and DNA synthesis and as the primary energy donors for cellular processes [79], were investigated for their biosynthesis in the presence of ACL and KOR. L-aspartate, a substrate of pyrimidine biosynthesis catalyzed by the allosteric enzyme, aspartate transcarbamoylase, is regulated both homotrophically by L-aspartate and heterotrophically by nucleotide effectors such as ATP, CTP (cytidine triphosphate) [80], and UTP (uridine triphosphate) in the presence of CTP [81,82,83]. It was speculated that ACL also promote nucleotides biosynthesis by increasing the supply of aspartate, a substrate for nucleotides biosynthesis. Although E. coli naturally incorporates CO2 slightly in nucleotide biosynthesis (Fig. 4) [78, 84], it cannot use CO2 as the sole carbon source for cell growth. When the selected carbon-fixing genes were introduced to the rTCA cycle, the transgenic E. coli strains could incorporate more CO2 into nucleotide structures under nutrient-scarce environments.

Fig. 4
figure 4

Incorporation of CO2 into nucleotides. Effect of hetero-expressed enzymes promote CO2 incorporation into nucleotides in E. coli K12. Data are expressed as means standard ± deviation, n = 3 independent biological samples. A two-tailed t-test was used for statistical analysis, **** indicates p < 0.005 (highly statistically significantly difference); *** indicates p < 0.01; ** indicates p < 0.05 (statistically significantly difference); * indicates p < 0.1 (borderline statistically significantly difference). Strains were supplemented with 10% 13CO2 and 90% H2, re-filled 10% of 13CO2 per 12 hr for 48 hr incubation. Isotopic enrichments derived from 13CO2 are detected in the M + 1 or M + 2 specie of various nucleotides. The labeled 13CO2 are marked in red asterisk * marks. Hetero-expressed enzymes: KOR & ACL are marked in purple and yellow, respectively. In deoxythymidine and inosine-5'-monophosphate biosynthesis pathway, 13CO2 was not incorporated into nucleotide through reverse TCA cycle which are marked in pink asterisk * marks. (Asp: aspartate; Gly: glycine; Gln: glutamine; For: formate; PPRP: phosphoribosyl pyrophosphate; UTP: Uridine triphosphate; IMP: inosine-5'-monophosphate; dC: deoxycytidine; dT: deoxythymidine; dG: deoxyguanosine; dA: deoxyadenosine)

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A comparison of nucleotide biosynthesis between the two transgenic strains revealed differences. Compared to strains VC and K, the isotopic enrichments in deoxythymidine + 1 (dT + 1) increased by 29% and 48%, respectively, in strain KA. Similarly, the isotopic enrichments in deoxycytidine + 1 (dC + 1) increased by 26% and 38%, respectively, in strain KA compared to strains VC and K. Additionally, in the purine biosynthesis pathway, the isotopic enrichments in deoxyadenosine + 1 (dA + 1) increased by 134% and 29%, respectively, in strain KA compared to strains VC and K, while enrichment in dA + 2 was increased by 401% in strain KA compared to strain VC. The enrichments of deoxyguanosine + 1 (dG + 1) increased by 115% and 26% in strain KA compared to strains VC and K, respectively, with dG + 2 being significantly enriched in strain KA compared to that in strain VC (Fig. 4; Table 4). These results suggested that the co-expressions of KOR, and KOR with ACL promoted the incorporation of CO2 for the biosynthesis of pyrimidines and purine in E. coli, with the highest CO2 incorporation was detected in strain KA.

Table 4 Effect of hetero-expressed enzymes promote CO2 incorporation into nucleotides in E. Coli K12
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In E. coli and other organisms, CO2, formate, and glycine are involved in synthesizing the purine ring [85] (Fig. 4). The model indicated an advantage of CO2 incorporation into purines upon KOR expression. Furthermore, the additional expression of ACL significantly impacted purine synthesis from CO2. These findings align with the increased enrichment of glycine in cellular proteins and cytoplasmic amino acids. Glycine is a crucial source for purine formation [84], suggesting that ACL and KOR facilitate CO2 incorporation into purines by improving glycine availability. For pyrimidines, CO2, aspartate, glutamate, and acetate serve as carbon sources [84] (Fig. 4). Compared to purines, the effects of KOR and ACL on pyrimidines biosynthesis were relatively moderate. These findings are consistent with the similar enrichment of aspartate in cellular protein and cytoplasmic amino acids. Notably, KA exhibited higher enrichments of deoxy-cytosine + 1 and deoxy-thymidine + 1 compared to K and VC, which may partially result from increased CO2 incorporations into glutamate.

In this study, carbon-fixing E. coli transgenic strains capable of assimilating CO₂ via rTCA machinery were successfully constructed. The results demonstrate that expressing either KOR alone or KOR in combination with ACL is sufficient to support cellular maintenance and CO₂ assimilation in E. coli under chemolithotrophic conditions. The engineered metabolic pathways in these transgenic strains represent the minimal and essential requirements for sustaining cellular life, closely reflecting the core metabolic functions necessary for survival.

However, achieving indefinite growth of E. coli with CO₂ as the sole carbon source remains a significant challenge. According to a previous study [2], α-ketoglutarate: ferredoxin oxidoreductase (KOR) catalyzes the carboxylation of succinyl-CoA to α-ketoglutarate and coenzyme A, utilizing CO₂ and two moles of reduced ferredoxin as substrates. We hypothesize that the primary limitation lies in the insufficient availability of reduced ferredoxin to sustain KOR activity under nutrient-scarce conditions.

To address this bottleneck and move closer to fully autotrophic growth, future research should focus on increasing the availability of reduced ferredoxin. Potential strategies include engineering pathways to enhance ferredoxin reduction or exploring methods for external supplementation of reducing power. These efforts could pave the way for transgenic strains capable of achieving complete autotrophic growth in fully organic-free environments.

Conclusion

This study is the first to successfully engineer a carbon-fixing E. coli strain capable of cellular maintenance under chemolithotrophic conditions with hydrogen-powered anaerobic respiration by adding only one enzyme. The expression of KOR facilitates CO2 assimilation and carbon fluxes retention within the TCA cycle (Fig. 5a). Moreover, the additional expression of ACL further directs carbon fluxes into essential cellular processes such as protein and nucleotide biosynthesis (Fig. 5b). The metabolic pathways assembled in the transgenic E. coli strains are deemed essential and represent a minimal configuration for cell living, closely reflecting core metabolism functions.

Fig. 5
figure 5

An overview of the potential metabolic fate of 13CO2 during the TCA cycle, cellular protein biosynthesis, nucleotide biosynthesis, and the impact on fatty acid production in transgenic E. coli K12 strains expressed (a) KOR or (b) KOR with ACL compared to vector control strain, respectively. A two-tailed t-test was used for statistical analysis, **** indicates p < 0.005 (highly statistically significantly difference); *** indicates p < 0.01; ** indicates p < 0.05 (statistically significantly difference); * indicates p < 0.1 (borderline statistically significantly difference). Two heterologous enzymes, KOR and ACL, play distinct roles in this carbon fixing system. KOR primarily fixes CO2 through the rTCA cycle, maintaining carbon flow within the TCA cycle. ACL further redirects and shifts the carbon flux of CO2 out of TCA cycle towards cellular protein and nucleotide biosynthesis. Metabolites from carbohydrate and TCA cycle are denoted in black, amino acids in blue, fatty acids in orange, and labeled 13CO2 in red. Hetero-expressed enzymes, KOR and ACL, are represented in purple and yellow, respectively. (SAFA: saturated fatty acids; MUFA: mono-unsaturated fatty acids; TFA: total fatty acid; PPRP: phosphoribosyl pyrophosphate; dC: deoxycytidine; dT: deoxythymidine; dG: deoxyguanosine; dA: deoxyadenosine)

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Data availability

No datasets were generated or analysed during the current study.

Abbreviations

rTCA:

reverse tricarboxylic acid

KOR:

α-ketoglutarate: ferredoxin oxidoreductase

ACL:

ATP-dependent citrate lyase

FR:

fumarate reductase

SDH:

succinate dehydrogenase

LUCA:

last universal common ancestor

CBB:

Calvin-Benson-Bassham

RNA:

ribonucleic acid

TCA:

tricarboxylic acid

MTBSTFA:

N-tert-Butyldimethylsily-N-methyltrifluoroacetamide

PFBBr:

pentafluorobenzyl bromide

DMSO:

Dimethyl sulfoxide

OGAB:

Ordered Gene Assembly in the Bacillus subtilis

DNA:

deoxyribonucleic acid

O.D.:

optical density

cfu:

colony-forming units

NADP:

Nicotinamide adenine dinucleotide phosphate

ATP:

adenosine triphosphate

GC-MS:

gas chromatography-mass spectrometry

SCFAs:

short-chain fatty acids

MS:

Mass spectrometry

dA:

deoxyadenylate

dG:

deoxyguanylate

dT:

deoxythymidine

dC:

deoxycytidine

VC:

vector control

CoA:

coenzyme A

CTP:

cytidine triphosphate

UTP:

uridine triphosphate

RuBisCO:

ribulose-1,5-bisphosphate carboxylase/oxygenase

NAD:

nicotinamide adenine dinucleotide

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Funding

This work was supported by National Science and Technology Council (NSTC) 110-2320-B-005-008-MY3 (E.-P.I.C.), 110-2320-B-005-003-MY3 (E.-P.I.C.), 110-2321-B-005-008 (E.-P.I.C., C.-C.H., Y.-T.Y.), 107-2621-M-005-008-MY3 (E.-P.I.C., C.-C.H., Y.-T.Y.), 107-2621-M-005-007-MY3 (C.-C.H.), 107-2321-B-005-009 (E.-P.I.C., C.-C.H.), 107-2320-B-005-003-MY3 (E.-P.I.C., C.-C.H.), 106-2221-E-005-007-MY3 (C.-C.H), 104-2621-M-005-003-MY3 (C.-C.H.). It is also funded in part by the “Innovation and Development Center of Sustainable Agriculture (IDCSA)” and the “Advanced Plant and Food Crop Biotechnology Center (APFCBC) from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) (E.-P.I.C., C.-C.H.) in Taiwan.

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Authors and Affiliations

  1. Doctoral Program in Microbial Genomics, National Chung Hsing University and Academia Sinica, Taichung City and Taipei City, 402 and 115, Taiwan

    Jian-Hau Peng, Chieh-Chen Huang & En-Pei Isabel Chiang

  2. Department of Life Sciences, National Chung Hsing University, Taichung City, 402, Taiwan

    Shou-Chen Lo, Yu-Ning Yu, Yu-Chieh Chen, An-I Tsai, Dong-Yan Wu, Chu-Han Huang, Tien-Tsai Su & Chieh-Chen Huang

  3. Department of Electrical Engineering, National Tsing Hua University, Hsinchu City, 30013, Taiwan

    Ya-Tang Yang

  4. Advanced Plant and Food Crop Biotechnology Center, National Chung Hsing University, Taichung City, 402, Taiwan

    Jian-Hau Peng, Chieh-Chen Huang & En-Pei Isabel Chiang

  5. Innovation and Development Center of Sustainable Agriculture, National Chung Hsing University, Taichung City, 402, Taiwan

    Jian-Hau Peng, Chieh-Chen Huang & En-Pei Isabel Chiang

  6. Department of Food Science and Biotechnology, National Chung Hsing University, Taichung City, 402, Taiwan

    Jian-Hau Peng, Yu-Ning Yu & En-Pei Isabel Chiang

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Contributions

J.H.P. performed the experiments, analyzed the data, and prepared the manuscript. Y.N.Y., S.C.L., and Y.C.C. performed the experiments and analyzed the data. E.P.I.C., and C.C.H. analyzed the data and prepared the manuscript. Y.T.Y., A.I.T., and T.T.S. analyzed data. D.Y.W., C.H.Y., and C.H.H. performed experiments. All authors have read and agreed to the published version of the manuscript.

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Correspondence to Chieh-Chen Huang or En-Pei Isabel Chiang.

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Peng, JH., Lo, SC., Yu, YN. et al. Carbon fluxes rewiring in engineered E. coli via reverse tricarboxylic acid cycle pathway under chemolithotrophic condition. J Biol Eng 19, 20 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13036-025-00489-w

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Journal of Biological Engineering

ISSN: 1754-1611