Folate-mediated one-carbon (1C) metabolism is a major target of many therapies in human diseases. Studies have focused on the metabolism of serine 3-carbon as it serves as a major source for 1C units. The serine 3-carbon enters the mitochondria transferred by folate cofactors and eventually converted to formate and serves as a major building block for cytosolic 1C metabolism. Abnormal glycine metabolism has been reported in many human pathological conditions. The mitochondrial glycine cleavage system (GCS) catalyzes glycine degradation to CO₂ and ammonium, while tetrahydrofolate (THF) is converted into 5,10-methylene-THF. GCS accounts for a substantial proportion of whole-body glycine flux in humans, yet the particular metabolic route of glycine 2-carbon recycled from GCS during mitochondria glycine decarboxylation in hepatic or bone marrow 1C metabolism is not fully investigated, due to the limited accessibility of human tissues. Labeled glycine at 2-carbon was given to humans and primary cells in previous studies for investigating its incorporations into purines, its interconversion with serine, or the CO₂ production in the mitochondria. Less is known on the metabolic fate of the glycine 2-carbon recycled from the GCS; hence, a model system tracing its metabolic fate would help in this regard. We took the direct approach of isotopic labeling to further explore the in vitro and in vivo metabolic fate of the 2-carbon from [2-¹³C]glycine and [2-¹³C]serine. As the 2-carbon of glycine and serine is decarboxylated and catabolized via the GCS, the original 13C-labeled 2-carbon is transferred to THF and yield methyleneTHF in the mitochondria. In human hepatoma cell-lines, 2-carbon from glycine was found to be incorporated into deoxythymidine (dTMP, dT + 1), M + 3 species of purines (deoxyadenine, dA and deoxyguanine, dG), and methionine (Met + 1). In healthy mice, incorporation of GCS-derived formate from glycine 2-carbon was found in serine (Ser + 2 via cytosolic serine hydroxy methyl transferase), methionine, dTMP, and methylcytosine (mC + 1) in bone marrow DNA. In these experiments, labeled glycine 2-carbon directly incorporates into Ser + 1, A + 2, and G + 2 (at C2 and C8 of purine) in the cytosol. It is noteworthy that since the serine 3-carbon is unlabeled in these experiments, the isotopic enrichments in dT + 1, Ser + 2, dA + 3, dG + 3, and Met + 1 solely come from the 2-carbon of glycine/serine recycled from GCS, re-enters the cytosolic 1C metabolism as formate, and then being used for cytosolic syntheses of serine, dTMP, purine (M + 3) and methionine. Taken together, we established model systems and successfully traced the metabolic fate of mitochondrial GCS-derived formate from glycine 2-carbon in vitro and in vivo. Nutritional supply significantly alters formate generation from GCS. More GCS-derived formate was used in hepatic serine and methionine syntheses, whereas more GCS-derived formate was used in dTMP synthesis in the bone marrow, indicating that the utilization and partitioning of GCS-derived 1C unit are tissue-specific. These approaches enable better understanding concerning the utilization of 1C moiety generated from mitochondrial GCS that can help to further elucidate the role of GCS in human disease development and progression in future applications. More studies on GCS using these approaches are underway.

The enzymatic mechanisms of carbon fixation by autotrophs, such as the reductive tricarboxylic acid cycle (rTCA), have inspired biotechnological approaches to producing bio-based chemicals directly through CO₂. To explore the possibility of constructing an rTCA cycle in Escherichia coli and to investigate their potential for CO₂ assimilation, a total of ten genes encoding the key rTCA cycle enzymes, including α-ketoglutarate:ferredoxin oxidoreductase, ATP-dependent citrate lyase, and fumarate reductase/succinate dehydrogenase, were cloned into E. coli. The transgenic E. coli strain exhibited enhanced growth and the ability to assimilate external inorganic carbon with a gaseous CO₂ supply. Further experiments conducted in sugar-free medium containing hydrogen as the electron donor and dimethyl sulfoxide (DMSO) as the electron acceptor proved that the strain is able to undergo anaerobic respiration, using CO₂ as the major carbon source. The transgenic stain demonstrated CO₂-enhanced growth, whereas the genes involved in chemotaxis, flagellar assembly, and acid-resistance were upregulated under the anaerobic respiration. Furthermore, metabolomic analysis demonstrated that the total concentrations of ATP, ADP, and AMP in the transgenic strain were higher than those in the vector control strain and these results coincided with the enhanced growth. Our approach offers a novel strategy to engineer E. coli for assimilating external gaseous CO₂.

Given the popularity of ketogenic diets, their potential long-term consequences deserve to be more carefully monitored. Mitochondrially derived formate has a critical role in mammalian one-carbon (1C) metabolism and development. The glycine cleavage system (GCS) accounts for another substantial source for mitochondrially derived 1C units. Objective: We investigated how the ketogenic state modulates mitochondrial formate generation and partitioning of 1C metabolic fluxes. Design: HepG2 cells treated with physiological doses (1 mM and 10 mM) of β-hydroxybutyrate (βHB) were utilized as the in vitro ketogenic model. Eight-week male C57BL/6JNarl mice received either a medium-chain fatty-acid-enriched ketogenic diet (MCT-KD) or a control diet AIN 93M for 8 weeks. Stable isotopic labeling experiments were conducted. Results and Conclusions: MCT-KD is effective in weight and fat loss. Deoxythymidine (dTMP) synthesis from the mitochondrial GCS-derived formate was significantly suppressed by βHB and consumption of MCT-KD. Consistently, plasma formate concentrations, as well as the metabolic fluxes from serine and glycine, were suppressed by MCT-KD. MCT-KD also decreased the fractional contribution of mitochondrially derived formate in methionine synthesis from serine. With the worldwide application, people and medical professionals should be more aware of the potential metabolic perturbations when practicing a long-term ketogenic diet.

Endophytic bacteria are a group of microorganisms that can intercellularly colonize plant hosts without causing apparent damage or disease. Our previous works found that a pyrroloquinoline quinone (PQQ)-producing endophyte could promote plant growth and systemic tolerance. To demonstrate this PQQ-producing endophyte’s beneficial role in plants, a set of five PQQ synthesis genes from Gluconobacter oxydans was introduced into both Escherichia coli JM109 and Bacillus subtilis RM125, a BsuM-deficient mutant of laboratory strain B. subtilis 168. Interestingly, both strains harboring the PQQ synthesis genes exhibited significantly higher optimal optical density than control strains. In a carbon flux analysis, both strains showed a noticeable increase in their citric acid, alpha-ketoglutaric acid, and succinic acid levels. Conversely, in E. coli, pyruvic acid, malic acid, and fumaric acid levels decreased. These results suggest that PQQ impacts various host species differently. Furthermore, the presence of PQQ in fermentation broth was also confirmed in the RM125 PQQ synthesis recombinant strain. Subsequent experiments by inoculating those Bacillus strains revealed that the laboratory host strain could function as an endophyte, and the PQQ transgenic strain could further promote the growth of Arabidopsis thaliana and increase the number of siliques. These findings confirm PQQ’s vital role in endophyte-mediated plant growth promotion and also suggest the potential of B. subtilis transformed with PQQ genes as an engineered endophyte for studying PQQ’s biological functions in plants. This research is a step forward in understanding how specific substances can beneficially influence plant growth and systemic tolerance through endophytic mechanisms.

Fusarium wilt has a substantial impact on global banana production, posing a threat to food security worldwide. However, breeding new Fusarium-resistant cultivars is difficult and time-consuming. Alternatively, endophytic biostimulants that could combat such pervasive plant diseases provide possible novel solutions. Our prior research demonstrated that a pyrroloquinoline quinone (PQQ)–producing endophytic bacterium, Burkholderia seminalis 869T2, can enhance the growth of various plant species and protect bananas from Fusarium wilt in the field. PQQ is a peptide-derived redox cofactor known to stimulate mitochondrial biogenesis and metabolism in animals, but its molecular roles, especially in plants, remain to be elucidated. In this study, multi-omics approaches were employed to explore the potential mechanisms through which PQQ influences banana plants. The result of in situ imaging mass spectrometry revealed that the endophytic metabolite PQQ does not function through direct antagonism against Fusarium. The follow-up transcriptomic profiling shows it could regulate plant respiration, TCA cycle, oxidative phosphorylation, NAD/NADP–dependent dehydrogenases, MAPK signalling, and various phytohormone signalling pathways. Furthermore, PQQ appeared to trigger plant systemic immunity, thereby enhancing plant health and resistance to biotic stress. Beyond that, the complete genome of 869T2 was determined for follow-up comparative genomics analyses, revealing its genetic contexts, potential evolutionary events of PQQ operons among the Burkholderia species, and the absence of human virulence-facilitating genes within those PQQ-producing agricultural isolates. In summary, this study facilitates our understanding of PQQ in plant-microbe mutualisms and provides scientific evidence for its future application in agriculture. Significance Statement Fusarium wilt is caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), a notorious soil-borne pathogen that attacks bananas’ vascular system, which critically threatens global banana production and food security. A potential PQQ-producing endophytic strain has been confirmed to protect bananas through in planta biocontrol, reducing the morbidity of Banana Fusarium Wilt (BFW) disease in the field and promoting the growth of banana plants simultaneously. Our results revealed that the endophytic metabolite PQQ does not function through direct antagonism but triggers plant systemic immunity and coordinates energetic metabolisms, thereby improving the overall health of host plants and enhancing their resistance against Fusarium wilt.

Pyrroloquinoline quinone (PQQ) is a redox cofactor derived from prokaryotes that participates in various biological processes involving dehydrogenase enzymes. Previous field trials identified a PQQ‐producing endophyte, Burkholderia seminalis 869T2, which enhances banana growth and reduces Fusarium wilt incidence from 24.5% to 3.4%. While more recent studies have confirmed its agricultural benefits across multiple plant species, the underlying molecular mechanisms remain unclear. Here, integrated omics and imaging mass spectrometry were employed to investigate the role of PQQ in planta . Our results indicate that PQQ achieves these outcomes by modulating key aspects of plant energy metabolism, including the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and NAD/NADP pathways. In addition, PQQ appears to influence phytohormone signalling pathways and trigger systemic plant resistance. Consistent with these molecular responses, exogenous PQQ enhanced root and shoot development and improved resistance to Fusarium infection. Collectively, these findings indicate that the endophyte functions as a biostimulant through PQQ production, coordinating plant metabolism and defence to counter pathogen invasion. This study provides mechanistic insight into plant–endophyte mutualism and highlights the potential of both PQQ and PQQ‐producing endophytes as biostimulants for sustainable agricultural applications.