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A novel method for the quantification of the sulfur-containing metabolites of formaldehyde (thiazolidine carboxylic acid (TCA) and thiazolidine carbonyl glycine (TCG)) and acetaldehyde (methyl thiazolidine carboxylic acid (MTCA) and methyl thiazolidine carbonyl glycine (MTCG)) was developed and validated for human urine and plasma samples. Targeting the sulfur-containing metabolites of formaldehyde and acetaldehyde in contrast to the commonly used biomarkers formate and acetate overcomes the high intra- and inter-individual variance. Due to their involvement in various endogenous processes, formate and acetate lack the required specificity for assessing the exposure to formaldehyde and acetaldehyde, respectively. Validation was successfully performed according to FDA’s Guideline for Bioanalytical Method Validation (2018), showing excellent performance with regard to accuracy, precision, and limits of quantification (LLOQ). TCA, TCG, and MTCG proved to be stable under all investigated conditions, whereas MTCA showed a depletion after 21 months. The method was applied to a set of pilot samples derived from smokers who consumed unfiltered cigarettes spiked with 13C-labeled propylene glycol and 13C-labeled glycerol. These compounds were used as potential precursors for the formation of 13C-formaldehyde and 13C-acetaldehyde during combustion. Plasma concentrations were significantly lower as compared to urine, suggesting urine as suitable matrix for a biomonitoring. A smoking-related increase of unlabeled biomarker concentrations could not be shown due to the ubiquitous distribution in the environment. While the metabolites of 13C-acetaldehyde were not detected, the described method allowed for the quantification of 13C-formaldehyde uptake from cigarette smoking by targeting the biomarkers 13C-TCA and 13C-TCG in urine.Graphical abstract

The folate-driven one-carbon (1C) cycle is a fundamental metabolic hub in cells that enables the synthesis of nucleotides and amino acids and epigenetic modifications. This cycle might also release formaldehyde, a potent protein and DNA crosslinking agent that organisms produce in substantial quantities. Here we show that supplementation with tetrahydrofolate, the essential cofactor of this cycle, and other oxidation-prone folate derivatives kills human, mouse and chicken cells that cannot detoxify formaldehyde or that lack DNA crosslink repair. Notably, formaldehyde is generated from oxidative decomposition of the folate backbone. Furthermore, we find that formaldehyde detoxification in human cells generates formate, and thereby promotes nucleotide synthesis. This supply of 1C units is sufficient to sustain the growth of cells that are unable to use serine, which is the predominant source of 1C units. These findings identify an unexpected source of formaldehyde and, more generally, indicate that the detoxification of this ubiquitous endogenous genotoxin creates a benign 1C unit that can sustain essential metabolism. The mechanism by which formaldehyde, a potent DNA and protein crosslinking agent, is generated from folate is described, with implications for the treatment of certain cancers. Formaldehyde detoxification fuels one-carbon metabolism Folate is an essential cofactor in one-carbon (1C) metabolism, a group of biochemical cycles involved in nucleotide and amino acid synthesis. Ketan Patel and co-workers elucidate the route by which folate derivatives produce intracellular formaldehyde in vivo . Formaldehyde causes DNA and protein crosslinking and is toxic to cells lacking protection against this compound. Endogenous formaldehyde detoxification by the alcohol dehydrogenase ADH5 generates formate, a benign 1C unit, driving nucleotide synthesis. By this route, formaldehyde provides a source of 1C units that can sustain essential metabolism and cell growth in the absence of the amino acid serine. This work shows how formaldehyde fits into key metabolic pathways and provides clues as to how some features of these cycles could be used to target certain cancer cells.

Acetyl-CoA is a fundamental metabolite for all life on Earth, and is also a key starting point for the biosynthesis of a variety of industrial chemicals and natural products. Here we design and construct a Synthetic Acetyl-CoA (SACA) pathway by repurposing glycolaldehyde synthase and acetyl-phosphate synthase. First, we design and engineer glycolaldehyde synthase to improve catalytic activity more than 70-fold, to condense two molecules of formaldehyde into one glycolaldehyde. Second, we repurpose a phosphoketolase to convert glycolaldehyde into acetyl-phosphate. We demonstrated the feasibility of the SACA pathway in vitro, achieving a carbon yield ~50%, and confirmed the SACA pathway by 13 C-labeled metabolites. Finally, the SACA pathway was verified by cell growth using glycolaldehyde, formaldehyde and methanol as supplemental carbon source. The SACA pathway is proved to be the shortest, ATP-independent, carbon-conserving and oxygen-insensitive pathway for acetyl-CoA biosynthesis, opening possibilities for producing acetyl-CoA-derived chemicals from one-carbon resources in the future. The microbial synthesis of carbon-containing compounds from single carbon precursors is desirable, yet designed pathways to achieve this goal overlap with host metabolism. Here the authors design a de novo metabolic pathway to assimilate formaldehyde into acetyl-CoA that does not overlap with known metabolic networks.

Formaldehyde (FA) is a ubiquitous endogenous and environmental metabolite that is thought to exert cytotoxicity through DNA and DNA-protein crosslinking, likely contributing to the onset of the human DNA repair condition Fanconi Anaemia. Mutations in the genes coding for FA detoxifying enzymes underlie a human inherited bone marrow failure syndrome (IBMFS), even in the presence of functional DNA repair, raising the question of whether FA causes relevant cellular damage beyond genotoxicity. Here, we report that FA triggers cellular redox imbalance in human cells and in Caenorhabditis elegans . Mechanistically, FA reacts with the redox-active thiol group of glutathione (GSH), altering the GSH:GSSG ratio and causing oxidative stress. FA cytotoxicity is prevented by the enzyme alcohol dehydrogenase 5 (ADH5/GSNOR), which metabolizes FA-GSH products, lastly yielding reduced GSH. Furthermore, we show that GSH synthesis protects human cells from FA, indicating an active role of GSH in preventing FA toxicity. These findings might be relevant for patients carrying mutations in FA-detoxification systems and could suggest therapeutic benefits from thiol-rich antioxidants like N-acetyl-L-cysteine. Formaldehyde (FA) is known to exert cytotoxicity through DNA damage. Here, the authors show that FA also triggers cellular redox imbalance by reacting with glutathione (GSH), and that FA cytotoxicity is prevented by GSH synthesis and by ADH5, an enzyme that metabolizes FA-GSH products.

Endogenous DNA damage can perturb transcription, triggering a multifaceted cellular response that repairs the damage, degrades RNA polymerase II and shuts down global transcription 1 – 4 . This response is absent in the human disease Cockayne syndrome, which is caused by loss of the Cockayne syndrome A (CSA) or CSB proteins 5 – 7 . However, the source of endogenous DNA damage and how this leads to the prominent degenerative features of this disease remain unknown. Here we find that endogenous formaldehyde impedes transcription, with marked physiological consequences. Mice deficient in formaldehyde clearance ( Adh5 −/− ) and CSB ( Csb m/m ; Csb is also known as Ercc6 ) develop cachexia and neurodegeneration, and succumb to kidney failure, features that resemble human Cockayne syndrome. Using single-cell RNA sequencing, we find that formaldehyde-driven transcriptional stress stimulates the expression of the anorexiogenic peptide GDF15 by a subset of kidney proximal tubule cells. Blocking this response with an anti-GDF15 antibody alleviates cachexia in Adh5 −/− Csb m/m mice. Therefore, CSB provides protection to the kidney and brain against DNA damage caused by endogenous formaldehyde, while also suppressing an anorexic endocrine signal. The activation of this signal might contribute to the cachexia observed in Cockayne syndrome as well as chemotherapy-induced anorectic weight loss. A plausible evolutionary purpose for such a response is to ensure aversion to genotoxins in food. Endogenous formaldehyde accumulation reveals Cockayne syndrome in mice and stimulates production of the anorexiogenic peptide GDF15 in proximal tubule cells.

Formaldehyde (FA) has long been considered as a toxin and carcinogen due to its damaging effects to biological macromolecules, but its beneficial roles have been increasingly appreciated lately. Real-time monitoring of this reactive molecule in living systems is highly desired in order to decipher its physiological and/or pathological functions, but a genetically encoded FA sensor is currently lacking. We herein adopt a structure-based study of the underlying mechanism of the FA-responsive transcription factor HxlR from Bacillus subtilis , which shows that HxlR recognizes FA through an intra-helical cysteine-lysine crosslinking reaction at its N-terminal helix α1, leading to conformational change and transcriptional activation. By leveraging this FA-induced intra-helical crosslinking and gain-of-function reorganization, we develop the genetically encoded, reaction-based FA sensor—FAsor, allowing spatial-temporal visualization of FA in mammalian cells and mouse brain tissues. In order to understand the role of formaldehyde in living systems, real-time monitoring is required. Here the authors report a genetically encoded, reaction-based formaldehyde sensor (FAsor) that enables visualisation of formaldehyde in mammalian cells and tissues.

Background Pichia pastoris ( Komagataella phaffii ) is a model organism widely used for the recombinant expression of eukaryotic proteins, and it can metabolize methanol as its sole carbon and energy source. Methanol is oxidized to formaldehyde by alcohol oxidase (AOX). In the dissimilation pathway, formaldehyde is oxidized to CO 2 by formaldehyde dehydrogenase (FLD), S-hydroxymethyl glutathione hydrolase (FGH) and formate dehydrogenase (FDH). Results The transcriptome and metabolome of P. pastoris were determined under methanol cultivation when its dissimilation pathway cut off. Firstly, Δfld and Δfgh were significantly different compared to the wild type (GS115), with a 60.98% and 23.66% reduction in biomass, respectively. The differential metabolites between GS115 and Δfld were mainly enriched in ABC transporters, amino acid biosynthesis, and protein digestion and absorption. Secondly, comparative transcriptome between knockout and wild type strains showed that oxidative phosphorylation, glycolysis and the TCA cycle were downregulated, while alcohol metabolism, proteasomes, autophagy and peroxisomes were upregulated. Interestingly, the down-regulation of the oxidative phosphorylation pathway was positively correlated with the gene order of dissimilation pathway knockdown. In addition, there were significant differences in amino acid metabolism and glutathione redox cycling that raised our concerns about formaldehyde sorption in cells. Conclusions This is the first time that integrity of dissimilation pathway analysis based on transcriptomics and metabolomics was carried out in Pichia pastoris . The blockage of dissimilation pathway significantly down-regulates the level of oxidative phosphorylation and weakens the methanol assimilation pathway to the point where deficiencies in energy supply and carbon fixation result in inefficient biomass accumulation and genetic replication. In addition, transcriptional upregulation of the proteasome and autophagy may be a stress response to resolve formaldehyde-induced DNA–protein crosslinking.

One-carbon (C1) substrates, such as methanol or formate, are attractive feedstocks for circular bioeconomy. These substrates are typically converted into formaldehyde, serving as the entry point into metabolism. Here, we design an erythrulose monophosphate (EuMP) cycle for formaldehyde assimilation, leveraging a promiscuous dihydroxyacetone phosphate dependent aldolase as key enzyme. In silico modeling reveals that the cycle is highly energy-efficient, holding the potential for high bioproduct yields. Dissecting the EuMP into four modules, we use a stepwise strategy to demonstrate in vivo feasibility of the modules in E. coli sensor strains with sarcosine as formaldehyde source. From adaptive laboratory evolution for module integration, we identify key mutations enabling the accommodation of the EuMP reactions with endogenous metabolism. Overall, our study demonstrates the proof-of-concept for a highly efficient, new-to-nature formaldehyde assimilation pathway, opening a way for the development of a methylotrophic platform for a C1-fueled bioeconomy in the future. One-carbon substrates are attractive feedstocks for circular bioeconomy. Here, the authors design an erythrulose monophosphate (EuMP) cycle for formaldehyde assimilation, demonstrate the activity of the core reactions in E. coli , and show its integration with pathway reactions existed in pentose phosphate pathway and glycolysis.

Formalin-fixed paraffin embedding (FFPE) of tissues has been assumed to alter the metabolite content or chemical state, hampering metabolomics studies. Here, Ly et al . describe reproducible metabolomic analysis of FFPE samples by mass spectrometry imaging. Formalin-fixed and paraffin-embedded (FFPE) tissue specimens are the gold standard for histological examination, and they provide valuable molecular information in tissue-based research. Metabolite assessment from archived tissue samples has not been extensively conducted because of a lack of appropriate protocols and concerns about changes in metabolite content or chemical state due to tissue processing. We present a protocol for the in situ analysis of metabolite content from FFPE samples using a high-mass-resolution matrix-assisted laser desorption/ionization fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FT-ICR-MSI) platform. The method involves FFPE tissue sections that undergo deparaffinization and matrix coating by 9-aminoacridine before MALDI-MSI. Using this platform, we previously detected ∼1,500 m / z species in the mass range m / z 50–1,000 in FFPE samples; the overlap compared with fresh frozen samples is 72% of m/z species, indicating that metabolites are largely conserved in FFPE tissue samples. This protocol can be reproducibly performed on FFPE tissues, including small samples such as tissue microarrays and biopsies. The procedure can be completed in a day, depending on the size of the sample measured and raster size used. Advantages of this approach include easy sample handling, reproducibility, high throughput and the ability to demonstrate molecular spatial distributions in situ . The data acquired with this protocol can be used in research and clinical practice.

Synthetic methylotrophy offers opportunities for sustainable chemical and biofuel production. While recently established methylotrophic E. coli can grow on methanol, undesirable formate accumulation occurs during growth and bioproduction. Here, we show that NAD-dependent methanol dehydrogenase Mdh2 from Cupriavidus necator inherently overoxidizes methanol to formate, a trait we find to be widespread among NAD-dependent Mdh enzymes. In contrast, Mdh/Mdh1 enzymes from Bacillus methanolicus exclusively oxidize methanol to formaldehyde without overoxidation, as we validate in vitro for Mdh Bm MGA3 with and without activator protein Act. Since only formaldehyde is assimilated via the ribulose monophosphate pathway, this explains the physiological role of Mdh/Mdh1 paralogs in natural methylotrophs and highlights the importance of selecting appropriate Mdh variants for synthetic methylotrophy. We demonstrate methanol-dependent growth using non-overoxidizing Mdh Bm MGA3, strongly reducing formate accumulation and carbon loss. Our findings reveal a characteristic of NAD-dependent Mdh enzymes and provide insights for engineering synthetic methylotrophs. Formate produced by synthetic methylotrophic E. coli can lead to carbon loss and negatively impact bioproduction efficiency. Here, the authors report the production of formate as a widespread property of NAD-dependent methanol dehydrogenases and identify Mdhs without this overoxidation activity.