C1 Assimilation Research Articles

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.

The Role of Serine-Glyoxylate Aminotransferase and Malyl-CoA Lyase in the Metabolism of Methylococcus capsulatus Bath.

The genome of aerobic methanotroph Methylococcus capsulatus Bath possesses genes of three biochemical pathways of C1-carbon assimilation: the ribulose monophosphate cycle, the Calvin-Benson-Bassham cycle, and the partial serine cycle. Numerous studies have demonstrated that during methanotrophic growth cells of Methylococcus capsulatus Bath express key enzymes of these routes. In this study, the role of the serine cycle key enzymes, serine-glyoxylate aminotransferase (Sga) and malyl-CoA lyase (Mcl) in metabolism of Methylococcus capsulatus Bath was investigated by gene inactivation. The Δsga mutant obtained by double homologous recombination showed a prolonged lag phase, and after the lag period, the growth rate became similar to that of the wild type strain. The elevated intracellular levels of glutamate, serine, glycine, alanine, methionine, leucine, and succinate suggested significant metabolic changes in the mutant cells. Deletion of the mcl gene resulted in very poor growth and glycine only partially improved growth of the mutant strain. Cells of Δmcl mutant possess lower content of histidine, but enhanced level of alanine, leucine, and lysine than those of the wild type strain. Our data imply the importance of the serine cycle enzymes in metabolism of the methanotroph as well as relationships of the three C1 assimilation pathways in the gammaproteobacterial methanotrophs.

Integrated rational and evolutionary engineering of genome-reduced Pseudomonas putida strains promotes synthetic formate assimilation

Formate is a promising, water-soluble C1 feedstock for biotechnology that can be efficiently produced from CO2—but formatotrophy has been engineered in only a few industrially-relevant microbial hosts. We addressed the challenge of expanding the feedstock range of bacterial hosts by adopting Pseudomonas putida as a robust platform for synthetic formate assimilation. Here, the metabolism of a genome-reduced variant of P. putida was radically rewired to establish synthetic auxotrophies that could be functionally complemented by expressing components of the reductive glycine (rGly) pathway. We adopted a modular engineering approach, dividing C1 assimilation in segments composed of both heterologous activities (sourced from Methylobacterium extorquens) and native biochemical reactions. Modular expression of rGly pathway elements enabled growth on formate as carbon source and acetate (predominantly for energy supply), and adaptive laboratory evolution of two lineages of engineered P. putida formatotrophs lead to doubling times of ca. 15 h. We likewise identified emergent metabolic features for assimilation of C1 units in these evolved P. putida populations. Taken together, our results consolidate the landscape of useful microbial platforms that can be implemented for C1-based biotechnological production towards a formate bioeconomy.

Insights into C1 and C3 assimilation pathways in type I methanotrophic bacterium from co-production of 1,2-propanediol and lactate

Methanotrophic bacteria are attractive hosts for mining metabolic pathways of C1 assimilation to produce value-added products. Herein, the type I methanotroph Methylotuvimicrobium alcaliphilum 20Z was employed to explore the carbon flux from methane and methanol via the EMP pathway to produce 1,2-propanediol (1,2-PDO). The production of 1,2-PDO on methane was found to be mainly restricted by the lower carbon flux toward the EMP pathway. The co-utilization of C1 substrates and glycerol (C3) could contribute to enhance 1,2-PDO. Lactate was co-produced in much higher amounts than 1,2-PDO. This unexpected product was probably derived from lactaldehyde by inherent aldehyde dehydrogenases. The 1,2-PDO production without increased accumulation of lactate was observed via establishing the acetol–based pathway by propane utilization with the overexpression of pmoD. This is the first study to provide experimental insights into the operation of metabolic routes for 1,2-PDO and lactate co-production from C1 and C3 compounds in methanotrophs.

Chem-bio interface design for rapid conversion of CO2 to bioplastics in an integrated system

Chem-bio interface design for rapid conversion of CO2 to bioplastics in an integrated system

Metabolic engineering strategies for microbial utilization of C1 feedstocks

The use of abundant and cheap one carbon (C1) feedstocks to produce value-added chemicals is an important approach for achieving carbon neutrality and tackling environmental problems. The conversion of C1 feedstocks to high-value chemicals is dependent on efficient C1 assimilation pathways and microbial chassis adapted for efficient incorporation. Here, we opted to summarize the natural and synthetic C1 assimilation pathways and their key factors for metabolizing C1 feedstock. Accordingly, we discussed the metabolic engineering strategies for enabling the microbial utilization of C1 feedstocks for the bioproduction of value-added chemicals. In addition, we highlighted future perspectives of C1-based biomanufacturing for achieving a low-carbon footprint for the biosynthesis of chemicals.

Maternal Administration Of Probiotic Lactobacilli Since Pre-Conception Programs Offspring Gut Microbiota Functions

ObjectivesProbiotics intake may be considered beneficial by prospective and pregnant mothers but their effects on offspring development are underexplored. The maternal gut microbiota is a determinant of early offspring gut microbial colonization patterns and pre-conception may be a critical period. Our aim was to assess the programming effects of a probiotic Lacticaseibacillus rhamnosus R0011 and Lactobacillus helveticus R0052 mixture, which is known to support intestinal homeostasis in infancy, on the temporal dynamics of offspring gut microbiota functions.MethodsFemale C57BL/6 specific pathogen free mice received sterile or probiotic-supplemented water starting one week before mating and until weaning (postnatal day, PND, 21). Offspring remained on their respective mother's treatment from PND 21 to 36. Offspring cecal microbiota was profiled at PND 14, 21, and 36 by 16S rRNA (V3-V4) gene amplicon sequencing and metabolic pathways were inferred with PICRUSt2 and the MetaCyc database. Data analysis and clustering were conducted with DESeq2-Wald test and hclust in R.Results355 MetaCyc pathways were predicted and collapsed into 48 functional classes; 10 of these accounted for more than 80% of the total pathway relative abundance, including various biosynthetic as well as carbohydrate and protein degradation pathways. With time, 113 and 55 pathways were altered (q-values < 0.01) in the control and probiotic groups, respectively, but only control samples clustered by age. At PND 14 and 21,195 and 39 pathways were differentially represented in the probiotic group vs. controls, but none at PND 36. Age-dependent pathways altered by probiotic supplementation belonged to biosynthesis (Amino Acid, Nucleoside and Nucleotide, Cofactor, Electron Carrier, and Vitamin) and degradation (Carbohydrate, Nucleoside and Nucleotide, Secondary Metabolite Degradation, C1 Compound Utilization and Assimilation) functional classes.ConclusionsMaternal probiotic supplementation accelerates the establishment of gut microbiota metabolic functions necessary to process host diet as well as for the synthesis of metabolites relevant for host health in the juvenile offspring.Funding SourcesNSERC; Lallemand Health Solutions; Lawson Family Chair in Microbiome Research.

Biosynthesis of Polyhydroxyalkanoate Terpolymer from Methanol via the Reverse β-Oxidation Pathway in the Presence of Lanthanide.

Methylorubrum extorquens AM1 is the attractive platform for the production of value-added products from methanol. We previously demonstrated that M. extorquens equipped with PHA synthase with broad substrate specificity synthesized polyhydroxyalkanoates (PHAs) composed of (R)-3-hydroxybutyrate and small fraction of (R)-3-hydroxyvalerate (3HV) and (R)-3-hydroxyhexanoate (3HHx) units on methanol. This study further engineered M. extorquens for biosynthesis of PHAs with higher 3HV and 3HHx composition focusing on the EMC pathway involved in C1 assimilation. The introduction of ethylmalonyl-CoA decarboxylase, catalyzing a backward reaction in the EMC pathway, aiming to increase intracellular propionyl/butyryl-CoA precursors did not affect PHA composition. Reverse β-oxidation pathway and subsequent (R)-specific hydration of 2-enoyl-CoA were then enhanced by heterologous expression of four genes derived from Ralstonia eutropha for the conversion of propionyl/butyryl-CoAs to the corresponding (R)-3-hydroxyacyl-CoA monomers. The resulting strains produced PHAs with higher 3HV and 3HHx compositions, while the methylotrophic growth was severely impaired. This growth impairment was interestingly restored by the addition of La3+ without a negative impact on PHA biosynthesis, suggesting the activation of the EMC pathway by La3+. The engineered M. extorquens synthesized PHA terpolymer composed of 5.4 mol% 3HV and 0.9% of 3HHx with 41% content from methanol as a sole carbon source in the presence of La3+.

Outcome of Posterior Fossa Decompression and Duroplasty in Adult Chiari Malformation

OBJECTIVE: Evaluate of Chiari malformations outcome by the method of decompression with duraplasty. METHOD: This was a prospective descriptive study, duration: 03-03- 2013 to 3-2019 conducted at neuro spinal &amp; cancer care institute, Karachi, the study included 91 patients of CM I, including criteria after diagnosis with the help of MRI cervical spine, a patient having a headache, neck pain, limbs numbness and syringomyelia. Patients excluded from the study are those previously operated previously, history of cervical trauma, operated for a brain tumour. RESULTS:&nbsp; we had 91 CM patients, 58 (63.7%) were male, and 33 (36.26%) were female patients and the means age for this surgical procedure was 32 years old. Moreover, all of the patients were gone through the Posterior Fossa Decompression, C1 Laminectomy and Duraplasty. For duraplasty, we took autologous cervical fascia, 1patient had C1 assimilation. 12 (13.1%) patients had hydrocephalus; Ventriculoperitoneal (VP) shunt was placed. Headache was in 91 patients with duration resolved in 87 (95.6%) patients, neck pain was in 82 (90.1%) patient resolved in 74 (90.2%) cases, syrinx was in 36 (39.5%) cases improved in 28 (77.8%) cases, cerebellar sign improved in 40 (83.3%) out of 48 (52.7%) patient, limb numbness resolved in 56 (71.7%) out of 78 (85.7%) cases swallowing difficulty was 31( 34.0%)cases and improved in 24 (77.4%) cases and weakness in upper limbs was relieved in 31 (34%) patients. &nbsp;

Engineering a Highly Efficient Carboligase for Synthetic One-Carbon Metabolism.

One of the biggest challenges to realize a circular carbon economy is the synthesis of complex carbon compounds from one-carbon (C1) building blocks. Since the natural solution space of C1–C1 condensations is limited to highly complex enzymes, the development of more simple and robust biocatalysts may facilitate the engineering of C1 assimilation routes. Thiamine diphosphate-dependent enzymes harbor great potential for this task, due to their ability to create C–C bonds. Here, we employed structure-guided iterative saturation mutagenesis to convert oxalyl-CoA decarboxylase (OXC) from Methylobacterium extorquens into a glycolyl-CoA synthase (GCS) that allows for the direct condensation of the two C1 units formyl-CoA and formaldehyde. A quadruple variant MeOXC4 showed a 100 000-fold switch between OXC and GCS activities, a 200-fold increase in the GCS activity compared to the wild type, and formaldehyde affinity that is comparable to natural formaldehyde-converting enzymes. Notably, MeOCX4 outcompetes all other natural and engineered enzymes for C1–C1 condensations by more than 40-fold in catalytic efficiency and is highly soluble in Escherichia coli. In addition to the increased GCS activity, MeOXC4 showed up to 300-fold higher activity than the wild type toward a broad range of carbonyl acceptor substrates. When applied in vivo, MeOXC4 enables the production of glycolate from formaldehyde, overcoming the current bottleneck of C1–C1 condensation in whole-cell bioconversions and paving the way toward synthetic C1 assimilation routes in vivo.

Biocatalytic C-C Bond Formation for One Carbon Resource Utilization.

The carbon-carbon bond formation has always been one of the most important reactions in C1 resource utilization. Compared to traditional organic synthesis methods, biocatalytic C-C bond formation offers a green and potent alternative for C1 transformation. In recent years, with the development of synthetic biology, more and more carboxylases and C-C ligases have been mined and designed for the C1 transformation in vitro and C1 assimilation in vivo. This article presents an overview of C-C bond formation in biocatalytic C1 resource utilization is first provided. Sets of newly mined and designed carboxylases and ligases capable of catalyzing C-C bond formation for the transformation of CO2, formaldehyde, CO, and formate are then reviewed, and their catalytic mechanisms are discussed. Finally, the current advances and the future perspectives for the development of catalysts for C1 resource utilization are provided.

Metabolic diversification of anaerobic methanotrophic archaea in a deep-sea cold seep

Anaerobic methanotrophic archaea (ANME) can assimilate methane and govern the greenhouse effect of deep-sea cold seeps. In this study, a total of 13 ANME draft genomes representing five ANME types (ANME-1a, ANME-1b, ANME-2a, ANME-2b and ANME-2c), in size between 0.8 and 1.8 Mbp, were obtained from the Jiaolong cold seep in the South China Sea. The small metagenome-assembled genomes (MAGs) contained all the essential pathways for methane oxidization and carbon dioxide fixation. All genes related to nitrate and sulfate reduction were absent from the MAGs, indicating their syntrophic dependence on partner organisms. Aside from acetate secretion and sugar storage, propanoate synthesis pathway, as an alternative novel carbon flow, was identified in all the MAGs and transcriptionally active. Regarding type-specific features of the MAGs, the genes encoding archaellum and bacteria-derived chemotaxis were specific to ANME-2, perhaps for fitness under fluctuation of methane and sulfate concentration flux. Our genomic and transcriptomic results strongly suggested that ANME could carry out simple carbon metabolism from C1 assimilation to C3 biosynthesis in the SCS cold seep, which casts light on a novel approach for synthetic biology.

Short Segment Rigid Fixation for a Patient of Basilar Invagination and Assimilated C1 With Quadriparesis

Background &amp; Importance: Atlas assimilation is the congenital Atlas fusion with the base of the occipital bone. Case Presentation: A patient was presented with neck pain, dysaesthesia and tingling of hands and feet and difficulty in walking for 4 years. On examinations, the patient had a short neck with rigidity in neck muscles and there were exaggerated jerks with bilateral Babinski sign. Cervical spinal x-ray indicated basilar invagination with the absence of a C1 arch and CT scan of the cervical spine showed assimilation of C1 with basilar invagination. Occipitocervical stabilization has been commonly proposed for the operational fusion. The iliac crest interposing bone graft was performed for fusion and stabilization of the occipitocervical region. The patient was treated surgically and soon after operation became painless and was able to move easily. Conclusion: In C1 assimilation with basilar invagination, bicortical occipital screws and C2 pedicle screws with interposition bone graft can be the way to optimize the shortest segmental stabilization and fusion procedure for occipitocervical fusion.

Metabolic role of pyrophosphate-linked phosphofructokinase pfk for C1 assimilation in Methylotuvimicrobium alcaliphilum 20Z

BackgroundMethanotrophs is a promising biocatalyst in biotechnological applications with their ability to utilize single carbon (C1) feedstock to produce high-value compounds. Understanding the behavior of biological networks of methanotrophic bacteria in different parameters is vital to systems biology and metabolic engineering. Interestingly, methanotrophic bacteria possess the pyrophosphate-dependent 6-phosphofructokinase (PPi-PFK) instead of the ATP-dependent 6-phosphofructokinase, indicating their potentials to serve as promising model for investigation the role of inorganic pyrophosphate (PPi) and PPi-dependent glycolysis in bacteria. Gene knockout experiments along with global-omics approaches can be used for studying gene functions as well as unraveling regulatory networks that rely on the gene product.ResultsIn this study, we performed gene knockout and RNA-seq experiments in Methylotuvimicrobium alcaliphilum 20Z to investigate the functional roles of PPi-PFK in C1 metabolism when cells were grown on methane and methanol, highlighting its metabolic importance in C1 assimilation in M. alcaliphilum 20Z. We further conducted adaptive laboratory evolution (ALE) to investigate regulatory architecture in pfk knockout strain. Whole-genome resequencing and RNA-seq approaches were performed to characterize the genetic and metabolic responses of adaptation to pfk knockout. A number of mutations, as well as gene expression profiles, were identified in pfk ALE strain to overcome insufficient C1 assimilation pathway which limits the growth in the unevolved strain.ConclusionsThis study first revealed the regulatory roles of PPi-PFK on C1 metabolism and then provided novel insights into mechanism of adaptation to the loss of this major metabolic enzyme as well as an improved basis for future strain design in type I methanotrophs.

Ancylobacter lacus sp. nov. and Ancylobacter plantiphilus sp. nov., Novel Aerobic Facultative Methylotrophic Bacteria

Novel aerobic facultatively methylotrophic bacteria were isolated from the water of a freshwater lake (strain F30LT), soil sample of rhizosphere of white clover Trifolium repens L. (strain 1TCT), and rhizosphere of carrot Daucus carota L. (strain Dau2). The isolates were gram-negative, non-spore-forming, nonmotile, pleomorphic/rod-shaped cells, propagating by binary fission; they were facultative methylotrophs, using a broad range of polycarbon compounds, apart from methanol. Optimum growth occurred at 25−29 °C and pH 7.0‒7.5, 0.5 vol % СН3OH, and 0.05 wt % NaCl. The strains employ the ribulose bisphosphate pathway for C1 assimilation. The predominant phospholipids were phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. In the whole-cell fatty acid pattern of strains, C18:1 ω7c, C19: 0 ω8c cyclo, C16:0 and С18:0 acids were predominant. The major ubiquinone was Q-10. According to the 16S rRNA gene sequencing, strain F30LT exhibited high similarity to Ancylobacter dichloromethanicus DM16T (97.8%), while strains 1ТСT and Dau2 were closely related to A. oerskovii DSM 18746T (98.6%). The 16S rRNA gene sequence similarity between strains 1TCT and Dau2 was 99.4%. DNA–DNA hybridization of the strain F30LT with A. dichloromethanicus DM16T revealed only 22% homology; homology for strain 1ТСT and A. oerskovii DSM 18746T was 27%, while DNA‒DNA homology between strains 1TCT and Dau2 was 77%. Based on the data obtained, strain F30LT was identified as a new species within the genus Ancylobacter, Ancylobacter lacus sp. nov. (VKM В-3280Т = DSM 106439T). Strains 1ТСТ and Dau2 (=ВКМ В-3227 = CCUG71988) represent a novel species of the same genus, for which the name Ancylobacterplantiphilus sp. nov. is proposed, with the type strain 1ТСТ (=VKM В-3219Т = DSM 106438T).

Efficient C1 Elongation by Reversing α-Oxidation

Single carbon (C1) feedstocks are attractive for bioconversion, but native C1 assimilation pathways are difficult to engineer.Chou etal.developed a novel process for elongating the C1 compound formyl-CoA. Besides demonstrating a new approach for C1 bioconversion, this work paves the way for engineering synthetic methylotrophy into chassis organisms.

Systematic design and in vitro validation of novel one-carbon assimilation pathways

The utilization of one-carbon (C1) assimilation pathways to produce chemicals and fuels from low-cost C1 compounds could greatly reduce the substrate-related production costs, and would also alleviate the pressure of the resource supply for bio-manufacturing. However, the natural C1 assimilation pathways normally involve ATP consumption or the loss of carbon resources as CO2, resulting in low product yields, making the design of novel pathways highly pertinent. Here we present several new ATP-independent and carbon-conserving C1 assimilation cycles with 100% theoretical carbon yield, which were discovered by computational analysis of metabolic reaction set with 6578 natural reactions from MetaCyc database and 73 computationally predicted aldolase reactions from ATLAS database. Then, kinetic evaluation of these cycles was conducted and the cycles without kinetic traps were chosen for further experimental verification. Finally, we used the two engineered enzymes Gals and TalBF178Y for the artificial reactions to construct a novel C1 assimilation pathway in vitro and optimized the pathway to achieve 88% carbon yield. These results demonstrate the usefulness of computational design in finding novel metabolic pathways for the efficient utilization of C1 compounds and shedding light on other promising pathways.

A comparative transcriptome analysis of the novel obligate methanotroph Methylomonas sp. DH-1 reveals key differences in transcriptional responses in C1 and secondary metabolite pathways during growth on methane and methanol

BackgroundMethanotrophs play an important role in biotechnological applications, with their ability to utilize single carbon (C1) feedstock such as methane and methanol to produce a range of high-value compounds. A newly isolated obligate methanotroph strain, Methylomonas sp. DH-1, became a platform strain for biotechnological applications because it has proven capable of producing chemicals, fuels, and secondary metabolites from methane and methanol. In this study, transcriptome analysis with RNA-seq was used to investigate the transcriptional change of Methylomonas sp. DH-1 on methane and methanol. This was done to improve knowledge about C1 assimilation and secondary metabolite pathways in this promising, but under-characterized, methane-bioconversion strain.ResultsWe integrated genomic and transcriptomic analysis of the newly isolated Methylomonas sp. DH-1 grown on methane and methanol. Detailed transcriptomic analysis indicated that (i) Methylomonas sp. DH-1 possesses the ribulose monophosphate (RuMP) cycle and the Embden–Meyerhof–Parnas (EMP) pathway, which can serve as main pathways for C1 assimilation, (ii) the existence and the expression of a complete serine cycle and a complete tricarboxylic acid (TCA) cycle might contribute to methane conversion and energy production, and (iii) the highly active endogenous plasmid pDH1 may code for essential metabolic processes. Comparative transcriptomic analysis on methane and methanol as a sole carbon source revealed different transcriptional responses of Methylomonas sp. DH-1, especially in C1 assimilation, secondary metabolite pathways, and oxidative stress. Especially, these results suggest a shift of central metabolism when substrate changed from methane to methanol in which formaldehyde oxidation pathway and serine cycle carried more flux to produce acetyl-coA and NADH. Meanwhile, downregulation of TCA cycle when grown on methanol may suggest a shift of its main function is to provide de novo biosynthesis, but not produce NADH.ConclusionsThis study provides insights into the transcriptomic profile of Methylomonas sp. DH-1 grown on major carbon sources for C1 assimilation, providing in-depth knowledge on the metabolic pathways of this strain. These observations and analyses can contribute to future metabolic engineering with the newly isolated, yet under-characterized, Methylomonas sp. DH-1 to enhance its biochemical application in relevant industries.

Comprehensive Drilling of C1-2 Facets and Multiplanar Realignment for Atlanto-Axial Dislocation and Basilar Invagination: 2-Dimensional Operative Video.

The management for congenital atlantoaxial dislocation (AAD) and basilar invagination (BI) has significantly changed in the last 2 decades. Authors operate these cases through a direct posterior approach by opening and manipulating the C1-2 joints (irrespective of C1 assimilation), modifying Goel's technique. The joints in these cases are often oblique in both sagittal and coronal planes giving rise to anteroposterior and vertical slip of C1 on C2. Asymmetry on either side gives rise to coronal/lateral angular tilt along with rotational component. The authors have described comprehensive drilling of the facets (osteotomies) in sagittal and coronal planes to release the joints. Metallic spacers with graft window packed with bone chips are inserted to compensate for the drilled bone. Screws are inserted in C1 lateral mass and C2 pedicle. A rod is fastened between the screw heads and further multiplanar realignment can be achieved by manipulating the rod. The technique obviates the need for transoral decompression and the C1-2 joints are closely fused. The facetal osteotomies coupled with manipulation for realignment in all planes provides a composite solution for even the extremely complex lateral dislocation or complete spondyloptosis with severely deformed C1-2 joints that may be difficult with techniques described earlier. There is no need to include occipital squama and multiple cervical vertebrae in the construct, irrespective of the C1 assimilation. The authors have operated over 200 cases of irreducible CAAD/BI with good outcome and have illustrated their technique in this video. Proper informed consent was obtained from the patient.

Assimilation of formic acid and CO2 by engineered Escherichia coli equipped with reconstructed one-carbon assimilation pathways

Gaseous one-carbon (C1) compounds or formic acid (FA) converted from CO2 can be an attractive raw material for bio-based chemicals. Here, we report the development of Escherichia coli strains assimilating FA and CO2 through the reconstructed tetrahydrofolate (THF) cycle and reverse glycine cleavage (gcv) pathway. The Methylobacterium extorquens formate-THF ligase, methenyl-THF cyclohydrolase, and methylene-THF dehydrogenase genes were expressed to allow FA assimilation. The gcv reaction was reversed by knocking out the repressor gene (gcvR) and overexpressing the gcvTHP genes. This engineered strain synthesized 96% and 86% of proteinogenic glycine and serine, respectively, from FA and CO2 in a glucose-containing medium. Native serine deaminase converted serine to pyruvate, showing 4.5% of pyruvate-forming flux comes from FA and CO2 The pyruvate-forming flux from FA and CO2 could be increased to 14.9% by knocking out gcvR, pflB, and serA, chromosomally expressing gcvTHP under trc, and overexpressing the reconstructed THF cycle, gcvTHP, and lpd genes in one vector. To reduce glucose usage required for energy and redox generation, the Candida boidinii formate dehydrogenase (Fdh) gene was expressed. The resulting strain showed specific glucose, FA, and CO2 consumption rates of 370.2, 145.6, and 14.9 mg⋅g dry cell weight (DCW)-1⋅h-1, respectively. The C1 assimilation pathway consumed 21.3 wt% of FA. Furthermore, cells sustained slight growth using only FA and CO2 after glucose depletion, suggesting that combined use of the C1 assimilation pathway and C. boidinii Fdh will be useful for eventually developing a strain capable of utilizing FA and CO2 without an additional carbon source such as glucose.