Isotope Labeling Research Articles

Incorporation of the 15N-labeled simulated arthropod rain in the soil food web.

Direct trophic links between aboveground and belowground animal communities are rarely considered in food web models. Most invertebrate animals inhabiting aboveground space eventually become prey of soil predators and scavengers forming a gravity-driven spatial subsidy to detrital food webs, but its importance remains unquantified. We used laboratory-grown 15N-labeled Collembola to trace the incorporation of arthropod rain into soil food webs. Live or euthanized Collembola were supplemented once to field mesocosms in the amount equivalent to the mean daily input of the arthropod rain (19mg d.w. m-2). After the addition of live Collembola, the isotopic label was found most often in predatory Trombidiformes (83% of samples) and Mesostigmata mites (85%), followed by Araneae (58%), Chilopoda (45%), and Coleoptera (29%). Among non-predatory groups, the isotopic label was recorded in Thysanoptera (27%), Collembola (24%), and Oribatida (18%). The 15N-label was also detected in Symphyla, Formicidae, Diplura, Diplopoda, Opiliones, Diptera, Hemiptera, Oligochaeta, and Nematoda. There was a positive correlation between natural 15N abundance and the frequency of the isotopic label among predators, but not among decomposers. In the non-replicated treatment, in which dead collembolans were added, the label was found in predators and decomposers in approximately equal proportions (21-25%). Unlike other forms of the aboveground subsidy (such as leaflitter, frass, or honeydew) that are primarily processed by microorganisms, arthropod rain is assimilated directly by the animals. The high frequency of consumption of the aboveground subsidy suggests that it plays a significant role in maintaining the abundance of soil predators.

Biosynthesis of the Non-canonical C17 Sesquiterpenoids Chlororaphen A and B from Pseudomonas chlororaphis.

Chlororaphens A and B are structurally unique non-canonical C17 sesquiterpenoids from Pseudomonas chlororaphis that are made by two SAM-dependent methyltransferases and a type I terpene synthase. This study addresses the mechanism of their formation in isotopic labelling experiments and DFT calculations. The results demonstrate an astonishing complexity with distribution of labellings within a cyclopentane core that is reversely connected to two acyclic fragments in chlororaphen A and B. In addition, the uptake of up to 14 deuterium atoms from D2O was observed. These findings are explainable by a repeated late stage multistep rearrangement sequence. The absolute configurations of the chlororaphens and their biosynthetic intermediates were elucidated in stereoselective labelling experiments.

Coupling photocatalytic CO2 reduction and CH3OH oxidation for selective dimethoxymethane production

Currently, conventional dimethoxymethane synthesis methods are environmentally unfriendly. Here, we report a photo-redox catalysis system to generate dimethoxymethane using a silver and tungsten co-modified blue titanium dioxide catalyst (Ag.W-BTO) by coupling CO2 reduction and CH3OH oxidation under mild conditions. The Ag.W-BTO structure and its electron and hole transfer are comprehensively investigated by combining advanced characterizations and theoretical studies. Strikingly, Ag.W-BTO achieve a record photocatalytic activity of 5702.49 µmol g−1 with 92.08% dimethoxymethane selectivity in 9 h of ultraviolet-visible irradiation without sacrificial agents. Systematic isotope labeling experiments, in-situ diffuse reflectance infrared Fourier-transform analysis, and theoretical calculations reveal that the Ag and W species respectively catalyze CO2 conversion to *CH2O and CH3OH oxidation to *CH3O. Subsequently, an asymmetric carbon-oxygen coupling process between these two crucial intermediates produces dimethoxymethane. This work presents a CO2 photocatalytic reduction system for multi-carbon production to meet the objectives of sustainable economic development and carbon neutrality.

Regioselective hydroamination of unactivated olefins with diazirines as a diversifiable nitrogen source

Nitrogen-containing compounds, such as amines, hydrazines, and heterocycles, play an indispensable role in medicine, agriculture, and materials. Alkylated derivatives of these compounds, especially in sterically congested environments, remain a challenge to prepare. Here we report a versatile method for the regioselective hydroamination of readily available unactivated olefins with diazirines. Over fifty examples are reported, including the protecting group-free amination of fourteen different natural products. A broad functional group tolerance includes alcohols, ketones, aldehydes, and epoxides. The proximate products of these reactions are diaziridines, which, under mild conditions, are converted to primary amines, hydrazines, and heterocycles. Five target- and diversity-oriented syntheses of pharmaceutical compounds are shown, along with the preparation of a bis-15N diazirine validated in the late-stage isotopic labeling of an RNA splicing modulator candidate. In this work, we report using diazirine (1) as an electrophilic nitrogen source in a regioselective hydroamination reaction, and the diversification of the resulting diaziridines.

Synthesis of isotope‐substituted conjugated ladder polymers

AbstractConjugated ladder polymers (cLPs) represent an intriguing class of macromolecules, characterized by their multi‐stranded structure, with continuous fused π‐conjugated rings forming the backbone. Isotope substitution, such as deuteration and carbon‐13 labeling, offers unique approaches to address the significant challenges associated with elucidating the structure and solution phase dynamics of these polymers. For instance, selective deuteration can highlight parts of the polymer by controlling the scattering length density of specific molecular sections, thereby enhancing the contrast for neutron scattering experiments. In this context, deuteration of side‐chains in cLPs represents a promising approach to uncover the elusive polymer physics properties of their backbone. The synthesis of two distinct types of cLPs with perdeuterated side‐chains are reported here. During the synthesis, 13C isotope labeling was also employed to verify the low levels of defects in the synthesized polymers. Demonstrating these synthetic successes lays the foundation for rigorous characterization of the defects, conformation, and dynamics of cLPs.

Isotopomer labeling and oxygen dependence of hybrid nitrous oxide production

Abstract. Nitrous oxide (N2O) is a potent greenhouse gas and ozone depletion agent, with a significant natural source from marine oxygen-deficient zones (ODZs). Open questions remain, however, about the microbial processes responsible for this N2O production, especially hybrid N2O production when ammonia-oxidizing archaea are present. Using 15N-labeled tracer incubations, we measured the rates of N2O production from ammonium (NH4+), nitrite (NO2-), and nitrate (NO3-) in the eastern tropical North Pacific ODZ and the isotopic labeling of the central (α) and terminal (β) nitrogen (N) atoms of the N2O molecule. We observed production of both doubly and singly labeled N2O from each tracer, with the highest rates of labeled N2O production at the same depths as the near-surface N2O concentration maximum. At most stations and depths, the production of 45N2Oα and 45N2Oβ were statistically indistinguishable, but at a few depths there were significant differences in the labeling of the two nitrogen atoms in the N2O molecule. Implementing the rates of labeled N2O production in a time-dependent numerical model, we found that N2O production from NO3- dominated at most stations and depths, with rates as high as 1600 ± 200 pM N2O d−1. Hybrid N2O production, one of the mechanisms by which ammonia-oxidizing archaea produce N2O, had rates as high as 230 ± 80 pM N2O d−1 that peaked in both the near-surface and deep N2O concentration maxima. Based on the equal production of 45N2Oα and 45N2Oβ in the majority of our experiments, we infer that hybrid N2O production likely has a consistent site preference, despite drawing from two distinct substrate pools. We also found that the rates and yields of hybrid N2O production were enhanced at low dissolved oxygen concentrations ([O2]), with hybrid N2O yields as high as 20 % at depths where [O2] was below detection (880 nM) but nitrification was still active. Finally, we identified a few incubations with [O2] up to 20 µM where N2O production from NO3- was still active. A relatively high O2 tolerance for N2O production via denitrification has implications for the feedbacks between marine deoxygenation and greenhouse gas cycling.

Unveiling the In Situ Evolution of Li2O-Rich Solid Electrolyte Interface on CoOx Embedded Carbon Fibers as Li Anode Host.

Modification of three-dimensional (3D) carbon hosts with metal oxides has been considered as advantageous for the formation of Li2O-rich solid electrolyte interface (SEI), which can show fast Li+ diffusion, and meanwhile alleviate dendrite problems caused by fragility and nonuniformity of native SEIs. However, the lack of convincing experimental evidence has made it difficult to unveil the true origin of oxygen in Li2O-rich SEIs until now. Herein, CoOx embedded carbon nanofibers (CNF-CoOx) are successfully prepared as high-performance Li anode hosts. By employing 18O isotope labeling, the role of CoOx during SEI evolution is elucidated, revealing that CoOx contributes significantly to Li2O formation by delivering oxygen. Benefiting from the rich Li2O content, the as-formed SEIs greatly improve the Li+ migration kinetics, and therefore, the CNF-CoOx@Li anode can exhibit excellent cycling stability in half, symmetrical, and full cells.

Deuteration of Arenes via Pd-Catalyzed C-H Activation: A Lesson in Nondirected C-H Activation, Isotopic Labeling, and NMR Characterization.

Isotopic labeling is an important tool in medicinal research, metabolomics, and for understanding reaction mechanisms. In this context, transition metal-catalyzed C-H activation has emerged as a key technology for deuterium incorporation via hydrogen isotope exchange. A detailed and easy-to-implement experimental procedure for a nondirected arene deuteration has been developed that exclusively uses commercial equipment and chemicals. The protocol is ideally suited for students and other prospective applicants who are not experts in catalysis. The degree of deuterium incorporation was analyzed via different means like mass spectrometry and 1H and 2H nuclear magnetic resonance (NMR). A hands-on understanding of quantitative NMR, as well as the influence of H/D exchange on experimental spectra, was conveyed by comparative NMR spin simulations. Students were measurably familiarized with the concepts of C-H activation, isotope effects, and basics in experimental catalysis.

Exploring formation of turanose in honey via stable isotope labelling and high-resolution mass spectrometry analysis

Turanose, an isomer of sucrose, naturally exists in honey. Previous study indicated that turanose content increased gradually in acacia honey as honeybees brewed honey in the hive. However, it is unclear how turanose is generated in honey. We hypothesised that turanose was produced by enzymes from honeybees and performed a series of simulation experiments to prove this hypothesis. We found turanose in honey was produced by honeybees processing sucrose. Furthermore, we determined that sugar composition of simulated nectar influenced the turanose concentration in honey: when sucrose concentration was below 5%, turanose was difficult to form, whereas high concentration of fructose and limited glucose were beneficial in producing turanose. Using 13C-labelled sucrose tests combined with proteomics analysis, we identified that α-glucosidase converted sucrose to turanose through an intermolecular isomerisation process. This study reveals the formation mechanism of turanose in honey and assists in the scientific control and improvement of honey quality.

Global protein turnover quantification in Escherichia coli reveals cytoplasmic recycling under nitrogen limitation

Protein turnover is critical for proteostasis, but turnover quantification is challenging, and even in well-studied E. coli, proteome-wide measurements remain scarce. Here, we quantify the turnover rates of ~3200 E. coli proteins under 13 conditions by combining heavy isotope labeling with complement reporter ion quantification and find that cytoplasmic proteins are recycled when nitrogen is limited. We use knockout experiments to assign substrates to the known cytoplasmic ATP-dependent proteases. Surprisingly, none of these proteases are responsible for the observed cytoplasmic protein degradation in nitrogen limitation, suggesting that a major proteolysis pathway in E. coli remains to be discovered. Lastly, we show that protein degradation rates are generally independent of cell division rates. Thus, we present broadly applicable technology for protein turnover measurements and provide a rich resource for protein half-lives and protease substrates in E. coli, complementary to genomics data, that will allow researchers to study the control of proteostasis.

Selective Increase in CO2 Electroreduction to Ethanol Activity at Nanograin-Boundary-Rich Mixed Cu(I)/Cu(0) Sites via Enriching Co-Adsorbed CO and Hydroxyl Species.

Selective producing ethanol from CO2 electroreduction is highly demanded, yet the competing ethylene generation route is commonly more thermodynamically preferred. Herein, we reported an efficient CO2-to-ethanol conversion (53.5 % faradaic efficiency at -0.75 V versus reversible hydrogen electrode (vs. RHE)) over an oxide-derived nanocubic catalyst featured with abundant "embossment-like" structured grain-boundaries. The catalyst also attains a 23.2 % energy efficiency to ethanol within a flow cell reactor. In situ spectroscopy and electrochemical analysis identified that these dualphase Cu(I) and Cu(0) sites stabilized by grain-boundaries are very robust over the operating potential window, which maintains a high concentration of co-adsorbed *CO and hydroxyl (*OH) species. Theoretical calculations revealed that the presence of *OHad not only promote the easier dimerization of *CO to form *OCCO (ΔG~0.20 eV) at low overpotentials but also preferentially favor the key *CHCOH intermediate hydrogenation to *CHCHOH (ethanol pathway) while suppressing its dehydration to *CCH (ethylene pathway), which is believed to determine the remarkable ethanol selectivity. Such imperative intermediates associated with the bifurcation pathway were directly distinguished by isotope labelling in situ infrared spectroscopy. Our work promotes the understanding of bifurcating mechanism of CO2ER-to-hydrocarbons more deeply, providing a feasible strategy for the design of efficient ethanol-targeted catalysts.

Selective Increase in CO2 Electroreduction to Ethanol Activity at Nanograin‐Boundary‐Rich Mixed Cu(I)/Cu(0) Sites via Enriching Co‐Adsorbed CO and Hydroxyl Species

AbstractSelective producing ethanol from CO2 electroreduction is highly demanded, yet the competing ethylene generation route is commonly more thermodynamically preferred. Herein, we reported an efficient CO2‐to‐ethanol conversion (53.5 % faradaic efficiency at −0.75 V versus reversible hydrogen electrode (vs. RHE)) over an oxide‐derived nanocubic catalyst featured with abundant “embossment‐like” structured grain‐boundaries. The catalyst also attains a 23.2 % energy efficiency to ethanol within a flow cell reactor. In situ spectroscopy and electrochemical analysis identified that these dualphase Cu(I) and Cu(0) sites stabilized by grain‐boundaries are very robust over the operating potential window, which maintains a high concentration of co‐adsorbed *CO and hydroxyl (*OH) species. Theoretical calculations revealed that the presence of *OHad not only promote the easier dimerization of *CO to form *OCCO (ΔG~0.20 eV) at low overpotentials but also preferentially favor the key *CHCOH intermediate hydrogenation to *CHCHOH (ethanol pathway) while suppressing its dehydration to *CCH (ethylene pathway), which is believed to determine the remarkable ethanol selectivity. Such imperative intermediates associated with the bifurcation pathway were directly distinguished by isotope labelling in situ infrared spectroscopy. Our work promotes the understanding of bifurcating mechanism of CO2ER‐to‐hydrocarbons more deeply, providing a feasible strategy for the design of efficient ethanol‐targeted catalysts.

Atomic-Level Asymmetric Tuning of the Co1-N3P1 Catalyst for Highly Efficient N-Alkylation of Amines with Alcohols.

Despite the extensive development of non-noble metals for the N-alkylation of amines with alcohols, the exploitation of catalysts with high selectivity, activity, and stability still faces challenges. The controllable modification of single-atom sites through asymmetric coordination with a second heteroatom offers new opportunities for enhancing the intrinsic activity of transition metal single-atom catalysts. Here, we prepared the asymmetric N/P hybrid coordination of single-atom Co1-N3P1 by absorbing the Co-P complex on ZIF-8 using a concise impregnation-pyrolysis process. The catalyst exhibits ultrahigh activity and selectivity in the N-alkylation of aniline and benzyl alcohol, achieving a turnover number (TON) value of 3480 and a turnover frequency (TOF) value of 174-h. The TON value is 1 order of magnitude higher than the reported catalysts and even 37-fold higher than that of the homogeneous catalyst CoCl2(PPh3)2. Furthermore, the catalyst maintains its high activity and selectivity even after 6 cycles of usage. Controlling experiments and isotope labeling experiments confirm that in the asymmetric Co1-N3P1 system, the N-alkylation of aniline with benzyl alcohol proceeds via a transfer hydrogenation mechanism involving the monohydride route. Theoretical calculations prove that the superior activity of asymmetric Co1-N3P1 is attributed to the higher d-band energy level of Co sites, which leads to a more stable four-membered ring transition state and a lower reaction energy barrier compared to symmetrical Co1-N4.

Water residence time controls seasonal nitrous oxide budget in a semi-enclosed bay: Insights from an improvement estimation method

This study developed an estimation method for the N2O budget using 15N stable isotope labeling techniques, a dual-layer model and a box model, which was used to elucidate the underlying dynamics of N2O accumulation in Zhanjiang Bay. The results showed that although the net input of N2O during the rainy season was 2.36 times higher than that during the dry season, the overall N2O concentration was only 66.6 % of that during the dry season due to the extended water residence time in the dry season. Our findings highlighted that water residence time was the key factor for the N2O emission, and a longer water residence time was unfavorable for the efflux of N2O through hydrodynamic processes and was more conducive to the production and accumulation of N2O within the bay. This research enhanced our comprehension of N2O dynamics and provided crucial insights for refining nitrogen management strategies and mitigation efforts.

Isotopologues of potassium 2,2,2-trifluoroethoxide for applications in positron emission tomography and beyond

The 2,2,2-trifluoroethoxy group increasingly features in drugs and potential tracers for biomedical imaging with positron emission tomography (PET). Herein, we describe a rapid and transition metal-free conversion of fluoroform with paraformaldehyde into highly reactive potassium 2,2,2-trifluoroethoxide (CF3CH2OK) and demonstrate robust applications of this synthon in one-pot, two-stage 2,2,2-trifluoroethoxylations of both aromatic and aliphatic precursors. Moreover, we show that these transformations translate easily to fluoroform that has been labeled with either carbon-11 (t1/2 = 20.4 min) or fluorine-18 (t1/2 = 109.8 min), so allowing the appendage of complex molecules with a no-carrier-added 11C- or 18F- 2,2,2-trifluoroethoxy group. This provides scope to create candidate PET tracers with radioactive and metabolically stable 2,2,2-trifluoroethoxy moieties. We also exemplify syntheses of isotopologues of potassium 2,2,2-trifluoroethoxide and show their utility for stable isotopic labeling which can be of further benefit for drug discovery and development.

Spectroscopic Characterization and Photochemistry of the Atmospherically Relevant Methanesulfenic Acid.

Methanesulfenic acid, CH3SOH, is a fleeting intermediate in the ·OH-initiated oxidation reactions of dimethyl sulfide (DMS) and dimethyl disulfide (DMDS) in the atmosphere. Herein, we report the characterization and photochemistry of CH3SOH in Ar- and N2-matrices at 10 K. The characterization of CH3SOH with matrix-isolation IR and UV-vis spectroscopy is supported by D and 13C isotope labeling experiments and quantum chemical calculations. In line with the observed absorption at 260 nm for CH3SOH, its photolysis at 254 nm leads to dissociation by yielding the novel water complex H2CS···H2O, which exhibits a five-membered ring structure with intermolecular S···HO and CH···O hydrogen bonding interactions. Upon further irradiation at 193 nm, the H2CS···H2O complex undergoes dehydrogenation to form CS···H2O, which can further convert to HC(O)SH under irradiation at 254 nm. When the photolysis of CH3SOH was performed in an O2-doped Ar-matrix, methanesulfonic acid (MSA, CH3SO3H) was obtained as the oxidation product.

Metal-Metal Redox Exchange to Produce Heterometallic Manganese-Cobalt Oxo Cubanes via a "Dangler" Intermediate.

Pendent metals bound to heterocubanes are components of well-known active sites in enzymes that mediate difficult chemical transformations. Investigations into the specific role of these metal ions, sometimes referred to as "danglers", have been hindered by a paucity of rational synthetic routes to appropriate model structures. To generate pendent metal ions bonded to an oxo cubane through a carboxylate bridge, the cubane Co4(μ3-O)4(OAc)4(t-Bupy)4 (OAc = acetate, t-Bupy = 4-tert-butylpyridine) was exposed to various metal acetate complexes. Reaction with Cu(OAc)2 gave the structurally characterized (by X-ray diffraction) dicopper dangler Cu2Co4(μ4-O)2(μ3-O)2(OAc)6(Cl)2(t-Bupy)4. In contrast, the analogous reaction with Mn(OAc)2 produced the MnIV-containing cubane cation [MnCo3(μ3-O)4(OAc)4(t-Bupy)4]+ by way of a metal-metal exchange that gives Co(OAc)2 and [CoIII(μ-OH)(OAc)]n oligomers as byproducts. Additionally, reaction of the formally CoIV cubane complex [Co4(μ3-O)4(OAc)4(t-Bupy)4][PF6] with Mn(OAc)2 gave the corresponding Mn-containing cubane in 80% yield. A mechanistic examination of the related metal-metal exchange reaction between Co4(μ3-O)4(OBz)4(py)4 (OBz = benzoate) and [Mn(acac)2(py)2][PF6] by ultraviolet-visible (UV-vis) spectroscopy provided support for a process involving rate-determining association of the reactants and electron transfer through a μ-oxo bridge in the adduct intermediate. The rates of exchange correlate with the donor strength of the cubane pyridine and benzoate ligand substituents; more electron-donating pyridine ligands accelerate metal-metal exchange, while both electron-donating and -withdrawing benzoate ligands can accelerate exchange. These experiments suggest that the basicity of the cubane oxo ligands promotes metal-metal exchange reactivity. The redox potentials of the Mn and cubane starting materials and isotopic labeling studies suggest an inner-sphere electron-transfer mechanism in a dangler intermediate.

Metal-ligand dual-site single-atom nanozyme mimicking urate oxidase with high substrates specificity

In nature, coenzyme-independent oxidases have evolved in selective catalysis using isolated substrate-binding pockets. Single-atom nanozymes (SAzymes), an emerging type of non-protein artificial enzymes, are promising to simulate enzyme active centers, but owing to the lack of recognition sites, realizing substrate specificity is a formidable task. Here we report a metal-ligand dual-site SAzyme (Ni-DAB) that exhibited selectivity in uric acid (UA) oxidation. Ni-DAB mimics the dual-site catalytic mechanism of urate oxidase, in which the Ni metal center and the C atom in the ligand serve as the specific UA and O2 binding sites, respectively, characterized by synchrotron soft X-ray absorption spectroscopy, in situ near ambient pressure X-ray photoelectron spectroscopy, and isotope labeling. The theoretical calculations reveal the high catalytic specificity is derived from not only the delicate interaction between UA and the Ni center but also the complementary oxygen reduction at the beta C site in the ligand. As a potential application, a Ni-DAB-based biofuel cell using human urine is constructed. This work unlocks an approach of enzyme-like isolated dual sites in boosting the selectivity of non-protein artificial enzymes.

Enhanced dehydroaromatization of methane with methanol on Mo-ZSM-5/ZSM-11 intergrown zeolite materials

Methane dehydroaromatization (MDA) represents an appealing route for the direct utilization of abundant methane resources. Currently, the major challenge of MDA reaction lies in the rapid catalyst deactivation via coke formation, hindering large-scale industrialization of the process. Herein we report an enhanced conversion route of MDA in the presence of methanol on Mo-based zeolite materials. Compared to regular ZSM-5, ZSM-11, and their mechanically mixed counterparts, Mo embedded in ZSM-5/ZSM-11 intergrown matrix exhibits higher methane conversion (16.0%) and aromatics selectivity (62.5%) while limiting coke production to 12.1% under the co-feeding of methane and methanol (nCH4/nCH3OH = 30). If no methanol addition, coke selectivity is up to 45.1% in MDA reaction. Additionally, hydrogen enriched syngas (nH2/nCO = 6) was obtained as a co-product of the methane and methanol co-aromatization. NH3-adsorbed 1H MAS NMR, Co2+ titrated UV-vis and Raman spectroscopy characterizations reveal that a proper amount of Brönsted acid sites and the binuclear Mo anchored on the Al pairs at the intersection cavity may account for the superior reaction performance of the intergrown zeolites. A catalytic mechanism was also proposed via 13CH3OH isotopic labeling tracer and in-situ13C solid-state NMR studies. Methanol is first decomposed into CO/H2, while CO is hydrogenated to CH4 and converted into CO2 via the water-gas shift reaction. Alternatively, methanol is converted into CO/CO2 through formate intermediates. Subsequently, CO2 reduces coke deposition via the reverse Boudart reaction, which improves the catalytic performance.

Production of succinate with two CO2 fixation reactions from fatty acids in Cupriavidus necator H16

BackgroundBiotransformation of CO2 into high-value-added carbon-based products is a promising process for reducing greenhouse gas emissions. To realize the green transformation of CO2, we use fatty acids as carbon source to drive CO2 fixation to produce succinate through a portion of the 3-hydroxypropionate (3HP) cycle in Cupriavidus necator H16.ResultsThis work can achieve the production of a single succinate molecule from one acetyl-CoA molecule and two CO2 molecules. It was verified using an isotope labeling experiment utilizing NaH13CO3. This implies that 50% of the carbon atoms present in succinate are derived from CO2, resulting in a twofold increase in efficiency compared to prior methods of succinate biosynthesis that relied on the carboxylation of phosphoenolpyruvate or pyruvate. Meanwhile, using fatty acid as a carbon source has a higher theoretical yield than other feedstocks and also avoids carbon loss during acetyl-CoA and succinate production. To further optimize succinate production, different approaches including the optimization of ATP and NADPH supply, optimization of metabolic burden, and optimization of carbon sources were used. The resulting strain was capable of producing succinate to a level of 3.6 g/L, an increase of 159% from the starting strain.ConclusionsThis investigation established a new method for the production of succinate by the implementation of two CO2 fixation reactions and demonstrated the feasibility of ATP, NADPH, and metabolic burden regulation strategies in biological carbon fixation.