Structural and biochemical characterization of the M405S variant of Desulfovibrio vulgaris formate dehydrogenase.

Molybdenum- or tungsten-dependent formate dehydrogenases have emerged as significant catalysts for the chemical reduction of CO2 to formate, with biotechnological applications envisaged in climate-change mitigation. The role of Met405 in the active site of Desulfovibrio vulgaris formate dehydrogenase AB (DvFdhAB) has remained elusive. However, its proximity to the metal site and the conformational change that it undergoes between the resting and active forms suggests a functional role. In this work, the M405S variant was engineered, which allowed the active-site geometry in the absence of methionine Sδ interactions with the metal site to be revealed and the role of Met405 in catalysis to be probed. This variant displayed reduced activity in both formate oxidation and CO2 reduction, together with an increased sensitivity to oxygen inactivation.

The formate-hydrogen axis and its impact on the physiology of enterobacterial fermentation.

Formic acid (HCOOH) and dihydrogen (H2) are characteristic products of enterobacterial mixed-acid fermentation, with H2 generation increasing in conjunction with a decrease in extracellular pH. Formate and acetyl-CoA are generated by radical-based and coenzyme A-dependent cleavage of pyruvate catalysed by pyruvate formate-lyase (PflB). Formate is also the source of H2, which is generated along with carbon dioxide through the action of the membrane-associated, cytoplasmically-oriented formate hydrogenlyase (FHL-1) complex. Synthesis of the FHL-1 complex is completely dependent on the cytoplasmic accumulation of formate. Consequently, formate determines its own disproportionation into H2 and CO2 by the FHL-1 complex. Cytoplasmic formate levels are controlled by FocA, a pentameric channel that translocates formic acid/formate bidirectionally between the cytoplasm and periplasm. Each protomer of FocA has a narrow hydrophobic pore through which neutral formic acid can pass. Two conserved amino acid residues, a histidine and a threonine, at the center of the pore control directionality of translocation. The histidine residue is essential for pH-dependent influx of formic acid. Studies with the formate analogue hypophosphite and amino acid variants of FocA suggest that the mechanisms of formic acid efflux and influx differ. Indeed, current data suggest, depending on extracellular formate levels, two separate uptake mechanisms exist, both likely contributing to maintain pH homeostasis. Bidirectional formate/formic acid translocation is dependent on PflB and influx requires an active FHL-1 complex. This review describes the coupling of formate and H2 production in enterobacteria.

One-carbon unit supplementation fuels purine synthesis in tumor-infiltrating T cells and augments checkpoint blockade.

Nucleotides perform important metabolic functions, carrying energy and feeding nucleic acid synthesis. Here, we use isotope tracing-mass spectrometry to quantitate contributions to purine nucleotides from salvage versus de novo synthesis. We further explore the impact of augmenting a key precursor for purine synthesis, one-carbon (1C) units. We show that tumors and tumor-infiltrating T cells (relative to splenic or lymph node T cells) synthesize purines de novo. Shortage of 1C units for T cell purine synthesis is accordingly a potential bottleneck for anti-tumor immunity. Supplementing 1C units by infusing formate drives formate assimilation into purines in tumor-infiltrating T cells. Orally administered methanol functions as a formate pro-drug, with deuteration enabling kinetic control of formate production. Safe doses of methanol raise formate levels and augment anti-PD-1 checkpoint blockade in MC38 tumors, tripling durable regressions. Thus, 1C deficiency can gate antitumor immunity and this metabolic checkpoint can be overcome with pharmacological 1C supplementation.

Cycling back to folate metabolism in cancer.

Metabolic changes contribute to cancer initiation and progression through effects on cancer cells, the tumor microenvironment and whole-body metabolism. Alterations in serine metabolism and the control of one-carbon cycles have emerged as critical for the development of many tumor types. In this Review, we focus on the mitochondrial folate cycle. We discuss recent evidence that, in addition to supporting nucleotide synthesis, mitochondrial folate metabolism also contributes to metastasis through support of antioxidant defense, mitochondrial protein synthesis and the overflow of excess formate. These observations offer potential therapeutic opportunities, including the modulation of formate metabolism through dietary interventions and the use of circulating folate cycle metabolites as biomarkers for cancer detection.

Systems metabolic engineering of Corynebacterium glutamicum to assimilate formic acid for biomass accumulation and succinic acid production.

Formate as an ideal mediator between the physicochemical and biological realms can be obtained from electrochemical reduction of CO2 and used to produce bio-chemicals. Yet, limitations arise when employing natural formate-utilizing microorganisms due to restricted product range and low biomass yield. This study presents a breakthrough: engineered Corynebacterium glutamicum strains (L2-L4) through modular engineering. L2 incorporates the formate-tetrahydrofolate cycle and reverse glycine cleavage pathway, L3 enhances NAD(P)H regeneration, and L4 reinforces metabolic flux. Metabolic modeling elucidates C1 assimilation, guiding strain optimization for co-fermentation of formate and glucose. Strain L4 achieves an OD600 of 0.5 and produces 0.6 g/L succinic acid. 13C-labeled formate confirms C1 assimilation, and further laboratory evolution yields 1.3 g/L succinic acid. This study showcases a successful model for biologically assimilating formate in C. glutamicum that could be applied in C1-based biotechnological production, ultimately forming a formate-based bioeconomy.

A remarkable peroxidase-like behavior of the catalase KatA from the pathogenic bacteria Helicobacter pylori: The oxidation reaction with formate as substrate and the stabilization of an [Fe(IV) = O Trp•] intermediate assessed by multifrequency EPR spectroscopy.

We have characterized the catalytic cycle of the Helicobacter pylori KatA catalase (HPC). H. pylori is a human and animal pathogen responsible for gastrointestinal infections. Multifrequency (9-285 GHz) EPR spectroscopy was applied to identify the high-valent intermediates (5 ≤ pH ≤ 8.5). The broad (2000 G) 9-GHz EPR spectrum consistent with the [Fe(IV) = O Por•+] intermediate was detected, and showed a clear pH dependence on the exchange-coupling of the radical (delocalized over the porphyrin moiety) due to the magnetic interaction with the ferryl iron. In addition, Trp• (for pH ≤ 6) and Tyr• (for 5 ≤ pH ≤ 8.5) species were distinguished by the advantageous resolution of their g-values in the 285-GHz EPR spectrum. The unequivocal identification of the high-valent intermediates in HPC by their distinct EPR spectra allowed us to address their reactivity towards substrates. The stabilization of an [Fe(IV) = O Trp•] species in HPC, unprecedented in monofunctional catalases and possibly involved in the oxidation of formate to the formyloxyl radical at pH ≤ 6, is reminiscent of intermediates previously identified in the catalytic cycle of bifunctional catalase-peroxidases. The 2e- oxidation of formate by the [Fe(IV) = O Por•+] species, both at basic and acidic pH conditions, involving a 1H+/2e- oxidation in a cytochrome P450 peroxygenase-like reaction is proposed. Our findings demonstrate that moonlighting by the H. pylori catalase includes formate oxidation, an enzymatic reaction possibly related to the unique strategy of the neutrophile bacterium for gastric colonization, that is the release of CO2 to regulate the pH in the acidic environment.

Optical coherence tomography-guided Brillouin microscopy highlights regional tissue stiffness differences during anterior neural tube closure in the Mthfd1l murine mutant.

Neurulation is a highly synchronized biomechanical process leading to the formation of the brain and spinal cord, and its failure leads to neural tube defects (NTDs). Although we are rapidly learning the genetic mechanisms underlying NTDs, the biomechanical aspects are largely unknown. To understand the correlation between NTDs and tissue stiffness during neural tube closure (NTC), we imaged an NTD murine model using optical coherence tomography (OCT), Brillouin microscopy and confocal fluorescence microscopy. Here, we associate structural information from OCT with local stiffness from the Brillouin signal of embryos undergoing neurulation. The stiffness of neuroepithelial tissues in Mthfd1l null embryos was significantly lower than that of wild-type embryos. Additionally, exogenous formate supplementation improved tissue stiffness and gross embryonic morphology in nullizygous and heterozygous embryos. Our results demonstrate the significance of proper tissue stiffness in normal NTC and pave the way for future studies on the mechanobiology of normal and abnormal embryonic development.

Chlamydomonas reinhardtii chloroplast factory construction for formate bioconversion.

The photosynthetic autotrophic production of microalgae is limited by the effective supply of carbon and light energy, and the production efficiency is lower than the theoretical value. Represented by methanol, C1 compounds have been industrially produced by artificial photosynthesis with a solar energy efficiency over 10%, but the complexity of artificial products is weak. Here, based on a construction of chloroplast factory, green microalgae Chlamydomonas reinhardtii CC137c was modified for the bioconversion of formate for biomass production. By screening the optimal combination of chloroplast transport peptides, the cabII-1 cTP1 fusion formate dehydrogenase showed significant enhancement on the conversion of formate with a better performance in the maintenance of light reaction activity. This work provided a new way to obtain bioproducts from solar energy and CO2 with potentially higher-than-nature efficiency by the artificial-natural hybrid photosynthesis.

Using RNA-Stable isotope probing to investigate methane oxidation metabolites and active microbial communities in methane oxidation coupled to denitrification.

The active denitrifying communities performing methane oxidation coupled to denitrification (MOD) were investigated using samples from an aerobic reactor (∼20% O2 and 2% CH4) and a microaerobic reactor (2% O2, 2% CH4) undertaking denitrification. The methane oxidation metabolites excreted in the reactors were acetate, methanol, formate and acetaldehyde. Using anaerobic batch experiments supplemented with exogenously supplied 13C-labelled metabolites, the active denitrifying bacteria were identified using 16S rRNA amplicon sequencing and RNA-stable isotope probing (RNA-SIP). With the aerobic reactor (AR) samples, the maximum NO3- removal rates were 0.43 mmol g-1 d-1, 0.40 mmol g-1 d-1, 0.33 mmol g-1 d-1 and 0.10 mmol g-1 d-1 for exogenously supplied acetate, formate, acetaldehyde and methanol batch treatments respectively, while with the microaerobic reactor (MR) samples, the maximum NO3- removal rates were 0.41 mmol g-1 d-1, 0.33 mmol g-1 d-1, 0.38 mmol g-1 d-1 and 0.14 mmol g-1 d-1 for exogenously supplied acetate, formate, acetaldehyde and methanol batch treatments respectively. The RNA-SIP experiments with 13C-labelled acetate, formate, and methanol identified Methyloversatilis, and Hyphomicrobium as the active methane-driven denitrifying bacteria in the AR samples, while Pseudoxanthomonas, Hydrogenophaga and Hyphomicrobium were the active MOD bacteria in the MR samples. Collectively, all the data indicate that formate is a key cross-feeding metabolite excreted by methanotrophs and consumed by denitrifiers performing MOD.

Engineering a solar formic acid/pentose (SFAP) pathway in Escherichia coli for lactic acid production.

Microbial CO2 fixation into lactic acid (LA) is an important approach for low-carbon biomanufacturing. Engineering microbes to utilize CO2 and sugar as co-substrates can create efficient pathways through input of moderate reducing power to drive CO2 fixation into product. However, to achieve complete conservation of organic carbon, how to engineer the CO2-fixing modules compatible with native central metabolism and merge the processes for improving bioproduction of LA is a big challenge. In this study, we designed and constructed a solar formic acid/pentose (SFAP) pathway in Escherichia coli, which enabled CO2 fixation merging into sugar catabolism to produce LA. In the SFAP pathway, adequate reducing equivalents from formate oxidation drive glucose metabolism shifting from glycolysis to the pentose phosphate pathway. The Rubisco-based CO2 fixation and sequential reduction of C3 intermediates are conducted to produce LA stoichiometrically. CO2 fixation theoretically can bring a 20% increase of LA production compared with sole glucose feedstock. This SFAP pathway in the integration of photoelectrochemical cell and an engineered Escherichia coli opens an efficient way for fixing CO2 into value-added bioproducts.

Inhibition of iron oxidation in Acidithiobacillus ferrooxidans by low-molecular-weight organic acids: Evaluation of performance and elucidation of mechanisms.

The catalytic role of Acidithiobacillus ferrooxidans (A. ferrooxidans) in iron biooxidation is pivotal in the formation of Acid Mine Drainage (AMD), which poses a significant threat to the environment. To control AMD generation, treatments with low-molecular-weight organic acids are being studied, yet their exact mechanisms are unclear. In this study, AMD materials, organic acids, and molecular methods were employed to gain a deeper understanding of the inhibitory effects of low-molecular-weight organic acids on the biooxidation of iron by A. ferrooxidans. The inhibition experiments of A. ferrooxidans on the oxidation of Fe2+ showed that to attain a 90 % inhibition efficacy within 72 h, the minimum concentrations required for formic acid, acetic acid, propionic acid, and lactic acid are 0.5, 6, 4, and 10 mmol/L, respectively. Bacterial imaging illustrated the detrimental effects of these organic acids on the cell envelope structure. This includes severe damage to the outer membrane, particularly from formic and acetic acids, which also caused cell wall damage. Coupled with alterations in the types and quantities of protein, carbohydrate, and nucleic acid content in extracellular polymeric substances (EPS), indicate the mechanisms underlying these inhibitory treatments. Transcriptomic analysis revealed interference of these organic acids with crucial metabolic pathways, particularly those related to energy metabolism. These findings establish a comprehensive theoretical basis for understanding the inhibition of A. ferrooxidans' biooxidation by low-molecular-weight organic acids, offering a novel opportunity to effectively mitigate the generation of AMD at its source.

Role of the Escherichia coli FocA and FocB formate channels in controlling proton/potassium fluxes and hydrogen production during osmotic stress in energy-limited, stationary phase fermenting cells.

Escherichia coli FocA and FocB formate channels export formate or import it for further disproportionation by the formate hydrogenlyase (FHL) complex to H2 and CO2. Here, we show that under pH and osmotic stress FocA and FocB play important roles in regulating proton and potassium fluxes and couple this with H2 production in stationary-phase cells. Using whole-cell assays with glucose as electron donor, a focB mutant showed a 50 % decrease in VH2, while N'N'-dicyclohexylcarbodiimide (DCCD) treatment of osmotically stressed cells underlined the role of FOF1 ATPase in H2 production. At pH 7.5 and under osmotic stress FocB contributed to the proton flux but not to the potassium flux. At pH 5.5 both formate channels contributed to the proton and potassium fluxes. Particulalry, a focA mutant had 40 % lower potassium flux whereas the proton flux increased approximately two-fold. Moreover, at pH 5.5H2 production was totally inhibited by DCCD in the focA mutant. Taken together, our results suggest that depending on external pH, the formate channels play an important role in osmoregulation by helping to balance proton/potassium fluxes and H2 production, and thus assist the proton FOF1-ATPase in maintenance of ion gradients in fermenting stationary-phase cells.

Deciphering the molecular mechanism and regulation of formaldehyde detoxification in Mycobacterium smegmatis.

The build-up of formaldehyde, a highly reactive molecule is cytotoxic and must be eliminated for the organism's survival. Formaldehyde detoxification system is found in nearly all organisms including both pathogenic and non-pathogenic mycobacteria. MscR, a formaldehyde dehydrogenase from Mycobacterium smegmatis (Msm), is an indispensable part of this system and forms a bicistronic operon with its downstream uncharacterized gene, fmh. We here show that Fmh, a putative metallo-beta-lactamase, is essential in tolerating higher amounts of formaldehyde when co-overexpressed with mscR in vivo. Our NMR studies indicate that MscR, along with Fmh, enhances formate production through a mycothiol (MSH)-dependent pathway, emphasizing the importance of Fmh in detoxifying formaldehyde. Although another aldehyde dehydrogenase, MSMEG_1543, induces upon formaldehyde addition, it is not involved in its detoxification. We also show that the expression of the mscR operon is constitutive and remains unchanged upon formaldehyde addition, as displayed by the promoter activity of PmscR and by the transcript and protein levels of MscR. Furthermore, we establish the role of a thiol-responsive sigma factor SigH in formaldehyde detoxification. We show that SigH, and not SigE, is crucial for formaldehyde detoxification, even though it does not directly regulate mscR operon expression. In addition, sensitivity to formaldehyde in sigH-knockout could be alleviated by overexpression of mscR. Taken together, our data demonstrate the importance of MSH-dependent pathways in detoxifying formaldehyde in a mycobacterial system. An absence of such MSH-dependent proteins in eukaryotes and its complete conservation in M. tuberculosis, the causative agent of tuberculosis, further unravel new drug targets for this pathogen.IMPORTANCEExtensive research has been done on formaldehyde detoxification in different bacteria. However, our current understanding of the mechanisms underlying this process in mycobacteria remains exceedingly little. We previously showed that MscR, a formaldehyde dehydrogenase from Mycobacterium smegmatis, plays a pivotal role in this detoxification pathway. Here, we present a potential S-formyl-mycothiol hydrolase named Fmh, thought to be a metallo-beta-lactamase, which functions along with mycothiol (MSH) and MscR to enhance formate production within this detoxification pathway. Co-expression of Fmh with MscR significantly enhances the efficiency of formaldehyde detoxification in M. smegmatis. Our experiments establish that Fmh catalyzes the final step of this detoxification pathway. Although an alternative sigma factor SigH was found to be involved in formaldehyde detoxification, it did not directly regulate the expression of mscR. Since formaldehyde detoxification is essential for bacterial survival, we envisage this process to be a potential drug target for M. tuberculosis eradication.

Accelerated Molecular Dynamics and AlphaFold Uncover a Missing Conformational State of Transporter Protein OxlT.

Transporter proteins change their conformations to carry their substrate across the cell membrane. The conformational dynamics is vital to understanding the transport function. We have studied the oxalate transporter (OxlT), an oxalate:formate antiporter from Oxalobacter formigenes, significant in avoiding kidney stone formation. The atomic structure of OxlT has been recently solved in the outward-open and occluded states. However, the inward-open conformation is still missing, hindering a complete understanding of the transporter. Here, we performed a Gaussian accelerated molecular dynamics simulation to sample the extensive conformational space of OxlT and successfully predicted the inward-open conformation where cytoplasmic substrate formate binding was preferred over oxalate binding. We also identified critical interactions for the inward-open conformation. The results were complemented by an AlphaFold2 structure prediction. Although AlphaFold2 solely predicted OxlT in the outward-open conformation, mutation of the identified critical residues made it partly predict the inward-open conformation, identifying possible state-shifting mutations.

Theoretical hypothesis in a direct electron transfer between non-interacting Fe-S proteins within an artificial fusion.

Reduction of CO2 to formate utilizing formate dehydrogenases (FDHs) has been attempted biologically and electrochemically. However, the conversion efficiency is very low due to the low energy potential of electron donors and/or electron competition with other electron acceptors. To overcome such a low conversion efficiency, I focused on a direct electron transfer between two unrelated redox enzymes for the efficient reduction of CO2 and utilized the quantum mechanical magnetic properties of the [Fe-S] ([iron-sulfur]) cluster to develop a novel electron path. Using this electron path, we connected non-interacting carbon monoxide dehydrogenase and FDH, constructing a synthetic carbon monoxide:formate oxidoreductase as a single functional enzyme complex in the previous study. Here, a theoretical hypothesis that can explain the direct electron transfer phenomenon based on the magnetic properties of the [Fe-S] cluster is proposed.

Genome-scale metabolic model of the versatile bacterium Paracoccus denitrificans Pd1222.

A genome scale metabolic model of the bacterium Paracoccus denitrificans has been constructed. The model containing 972 metabolic genes, 1,371 reactions, and 1,388 unique metabolites has been reconstructed. The model was used to carry out quantitative predictions of biomass yields on 10 different carbon sources under aerobic conditions. Yields on C1 compounds suggest that formate is oxidized by a formate dehydrogenase O, which uses ubiquinone as redox co-factor. The model also predicted the threshold methanol/mannitol uptake ratio, above which ribulose biphosphate carboxylase has to be expressed in order to optimize biomass yields. Biomass yields on acetate, formate, and succinate, when NO3- is used as electron acceptor, were also predicted correctly. The model reconstruction revealed the capability of P. denitrificans to grow on several non-conventional substrates such as adipic acid, 1,4-butanediol, 1,3-butanediol, and ethylene glycol. The capacity to grow on these substrates was tested experimentally, and the experimental biomass yields on these substrates were accurately predicted by the model.IMPORTANCEParacoccus denitrificans has been broadly used as a model denitrifying organism. It grows on a large portfolio of carbon sources, under aerobic and anoxic conditions. These characteristics, together with its amenability to genetic manipulations, make P. denitrificans a promising cell factory for industrial biotechnology. This paper presents and validates the first functional genome-scale metabolic model for P. denitrificans, which is a key tool to enable P. denitrificans as a platform for metabolic engineering and industrial biotechnology. Optimization of the biomass yield led to accurate predictions in a broad scope of substrates.

Functionally redundant formate dehydrogenases enable formate-dependent growth in Methanococcus maripaludis.

Methanogens are essential for the complete remineralization of organic matter in anoxic environments. Most cultured methanogens are hydrogenotrophic, using H2 as an electron donor to reduce CO2 to CH4, but in the absence of H2 many can also use formate. Formate dehydrogenase (Fdh) is essential for formate oxidation, where it transfers electrons for the reduction of coenzyme F420 or to a flavin-based electron bifurcating reaction catalyzed by heterodisulfide reductase (Hdr), the terminal reaction of methanogenesis. Furthermore, methanogens that use formate encode at least two isoforms of Fdh in their genomes, but how these different isoforms participate in methanogenesis is unknown. Using Methanococcus maripaludis, we undertook a biochemical characterization of both Fdh isoforms involved in methanogenesis. Both Fdh1 and Fdh2 interacted with Hdr to catalyze the flavin-based electron bifurcating reaction, and both reduced F420 at similar rates. F420 reduction preceded flavin-based electron bifurcation activity for both enzymes. In a Δfdh1 mutant background, a suppressor mutation was required for Fdh2 activity. Genome sequencing revealed that this mutation resulted in the loss of a specific molybdopterin transferase (moeA), allowing for Fdh2-dependent growth, and the metal content of the proteins suggested that isoforms are dependent on either molybdenum or tungsten for activity. These data suggest that both isoforms of Fdh are functionally redundant, but their activities in vivo may be limited by gene regulation or metal availability under different growth conditions. Together these results expand our understanding of formate oxidation and the role of Fdh in methanogenesis.

Engineering and evolution of the complete Reductive Glycine Pathway in Saccharomyces cerevisiae for formate and CO2 assimilation.

Using captured CO2 and C1-feedstocks like formate and methanol derived from electrochemical activation of CO2 are key solutions for transforming industrial processes towards a circular carbon economy. Engineering formate and CO2-based growth in the biotechnologically relevant yeast Saccharomyces cerevisiae could boost the emergence of a formate-mediated circular bio-economy. This study adopts a growth-coupled selection scheme for modular implementation of the Reductive Glycine Pathway (RGP) and subsequent Adaptive Laboratory Evolution (ALE) to enable formate and CO2 assimilation for biomass formation in yeast. We first constructed a serine biosensor strain and then implemented the serine synthesis module of the RGP into yeast, establishing glycine and serine synthesis from formate and CO2. ALE improved the RGP-dependent growth by 8-fold. 13C-labeling experiments reveal glycine, serine, and pyruvate synthesis via the RGP, demonstrating the complete pathway activity. Further, we re-established formate and CO2-dependent growth in non-evolved biosensor strains via reverse-engineering a mutation in GDH1 identified from ALE. This mutation led to significantly more 13C-formate assimilation than in WT without any selection or overexpression of the RGP. Overall, we demonstrated the activity of the complete RGP, showing evidence for carbon transfer from formate to pyruvate coupled with CO2 assimilation.

Valorization of single-carbon chemicals by using carboligases as key enzymes.

Single-carbon (C1) biorefinery plays a key role in the consumption of global greenhouse gases and a circular carbon economy. Thereby, we have focused on the valorization of C1 compounds (e.g. methanol, formaldehyde, and formate) into multicarbon products, including bioplastic monomers, glycolate, and ethylene glycol. For instance, methanol, derived from the oxidation of CH4, can be converted into glycolate, ethylene glycol, or erythrulose via formaldehyde and glycolaldehyde, employing C1 and/or C2 carboligases as essential enzymes. Escherichia coli was engineered to convert formate, produced from CO via CO2 or from CO2 directly, into glycolate. Recent progress in the design of biotransformation pathways, enzyme discovery, and engineering, as well as whole-cell biocatalyst engineering for C1 biorefinery, was addressed in this review.

Discovery and remodeling of Vibrio natriegens as a microbial platform for efficient formic acid biorefinery.

Formic acid (FA) has emerged as a promising one-carbon feedstock for biorefinery. However, developing efficient microbial hosts for economically competitive FA utilization remains a grand challenge. Here, we discover that the bacterium Vibrio natriegens has exceptional FA tolerance and metabolic capacity natively. This bacterium is remodeled by rewiring the serine cycle and the TCA cycle, resulting in a non-native closed loop (S-TCA) which as a powerful metabolic sink, in combination with laboratory evolution, enables rapid emergence of synthetic strains with significantly improved FA-utilizing ability. Further introduction of a foreign indigoidine-forming pathway into the synthetic V. natriegens strain leads to the production of 29.0 g · L-1 indigoidine and consumption of 165.3 g · L-1 formate within 72 h, achieving a formate consumption rate of 2.3 g · L-1 · h-1. This work provides an important microbial chassis as well as design rules to develop industrially viable microorganisms for FA biorefinery.