RESUMO
Formaldehyde is a key metabolite in natural and synthetic one-carbon metabolism. To facilitate the engineering of formaldehyde-producing enzymes, the development of sensitive, user-friendly, and cost-effective detection methods is required. In this study, we engineered Escherichia coli to serve as a cellular biosensor capable of detecting a broad range of formaldehyde concentrations. Using both natural and promiscuous formaldehyde assimilation enzymes, we designed three distinct E. coli growth biosensor strains that depend on formaldehyde for cell growth. These strains were engineered to be auxotrophic for one or several essential metabolites that could be produced through formaldehyde assimilation. The respective assimilating enzyme was expressed from the genome to compensate the auxotrophy in the presence of formaldehyde. We first predicted the formaldehyde dependency of the biosensors by flux balance analysis and then analysed it experimentally. Subsequent to strain engineering, we enhanced the formaldehyde sensitivity of two biosensors either through adaptive laboratory evolution or modifications at metabolic branch points. The final set of biosensors demonstrated the ability to detect formaldehyde concentrations ranging approximately from 30 µM to 13 mM. We demonstrated the application of the biosensors by assaying the in vivo activity of different methanol dehydrogenases in the most sensitive strain. The fully genomic nature of the biosensors allows them to be deployed as "plug-and-play" devices for high-throughput screenings of extensive enzyme libraries. The formaldehyde growth biosensors developed in this study hold significant promise for advancing the field of enzyme engineering, thereby supporting the establishment of a sustainable one-carbon bioeconomy.
Assuntos
Técnicas Biossensoriais , Escherichia coli , Formaldeído , Engenharia Metabólica , Formaldeído/metabolismo , Técnicas Biossensoriais/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/crescimento & desenvolvimento , Engenharia Metabólica/métodos , Oxirredutases do Álcool/metabolismo , Oxirredutases do Álcool/genética , Biotecnologia/métodosRESUMO
[Fe]-hydrogenase harbors the iron-guanylylpyridinol (FeGP) cofactor, in which the Fe(II) complex contains acyl-carbon, pyridinol-nitrogen, cysteine-thiolate and two CO as ligands. Irradiation with UV-A/blue light decomposes the FeGP cofactor to a 6-carboxymethyl-4-guanylyl-2-pyridone (GP) and other components. Previous in vitro biosynthesis experiments indicated that the acyl- and CO-ligands in the FeGP cofactor can scramble, but whether scrambling occurred during biosynthesis or photolysis was unclear. Here, we demonstrate that the [18 O1 -carboxy]-group of GP is incorporated into the FeGP cofactor by in vitro biosynthesis. MS/MS analysis of the 18 O-labeled FeGP cofactor revealed that the produced [18 O1 ]-acyl group is not exchanged with a CO ligand of the cofactor, indicating that the acyl and CO ligands are scrambled during photolysis rather than biosynthesis, which ruled out any biosynthesis mechanisms allowing acyl/CO ligands scrambling. Time-resolved infrared spectroscopy indicated that an acyl-Fe(CO)3 intermediate is formed during photolysis, in which scrambling of the CO and acyl ligands can occur. This finding also suggests that the light-excited FeGP cofactor has a higher affinity for external CO. These results contribute to our understanding of the biosynthesis and photosensitive properties of this unique H2 -activating natural complex.
Assuntos
Hidrogenase , Proteínas Ferro-Enxofre , Hidrogenase/metabolismo , Ligantes , Espectrometria de Massas em Tandem , Fotólise , Carbono , Proteínas Ferro-Enxofre/químicaRESUMO
2-Hydroxyglutarate (2-HG) is an oncometabolite that accumulates in certain cancers. Gain-of-function mutations in isocitrate dehydrogenase lead to 2-HG accumulation at the expense of alpha-ketoglutarate. Elevated 2-HG levels inhibit histone and DNA demethylases, causing chromatin structure and gene regulation changes with tumorigenic consequences. We investigated the effects of elevated 2-HG levels in Saccharomyces cerevisiae, a yeast devoid of DNA methylation and heterochromatin-associated histone methylation. Our results demonstrate genetic background-dependent gene expression changes and altered H3K4 and H3K36 methylation at specific loci. Analysis of histone demethylase deletion strains indicated that 2-HG inhibits Rph1 sufficiently to induce extensive gene expression changes. Rph1 is the yeast homolog of human KDM4 demethylases and, among the yeast histone demethylases, was the most sensitive to the inhibitory effect of 2-HG in vitro. Interestingly, Rph1 deficiency favors gene repression and leads to further down-regulation of already silenced genes marked by low H3K4 and H3K36 trimethylation, but abundant in H3K36 dimethylation. Our results provide novel insights into the genome-wide effects of 2-HG and highlight Rph1 as its preferential demethylase target.
Assuntos
Histona Desmetilases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Expressão Gênica , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Histonas/metabolismo , Metilação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
NAD is a coenzyme central to metabolism that also serves as a 5'-terminal cap for bacterial and eukaryotic transcripts. Thermal degradation of NAD can generate nicotinamide and ADP-ribose (ADPR). Here, we use LC-MS/MS and NAD captureSeq to detect and identify NAD-RNAs in the thermophilic model archaeon Sulfolobus acidocaldarius and in the halophilic mesophile Haloferax volcanii. None of the four Nudix proteins of S. acidocaldarius catalyze NAD-RNA decapping in vitro, but one of the proteins (Saci_NudT5) promotes ADPR-RNA decapping. NAD-RNAs are converted into ADPR-RNAs, which we detect in S. acidocaldarius total RNA. Deletion of the gene encoding the 5'-3' exonuclease Saci-aCPSF2 leads to a 4.5-fold increase in NAD-RNA levels. We propose that the incorporation of NAD into RNA acts as a degradation marker for Saci-aCPSF2. In contrast, ADPR-RNA is processed by Saci_NudT5 into 5'-p-RNAs, providing another layer of regulation for RNA turnover in archaeal cells.
Assuntos
NAD , RNA , NAD/metabolismo , Adenosina Difosfato Ribose/metabolismo , Archaea/metabolismo , Cromatografia Líquida , Espectrometria de Massas em TandemRESUMO
Lower respiratory tract infections caused by Streptococcus pneumoniae (Spn) are a leading cause of death globally. Here we investigate the bronchial epithelial cellular response to Spn infection on a transcriptomic, proteomic and metabolic level. We found the NAD+ salvage pathway to be dysregulated upon infection in a cell line model, primary human lung tissue and in vivo in rodents, leading to a reduced production of NAD+. Knockdown of NAD+ salvage enzymes (NAMPT, NMNAT1) increased bacterial replication. NAD+ treatment of Spn inhibited its growth while growth of other respiratory pathogens improved. Boosting NAD+ production increased NAD+ levels in immortalized and primary cells and decreased bacterial replication upon infection. NAD+ treatment of Spn dysregulated the bacterial metabolism and reduced intrabacterial ATP. Enhancing the bacterial ATP metabolism abolished the antibacterial effect of NAD+. Thus, we identified the NAD+ salvage pathway as an antibacterial pathway in Spn infections, predicting an antibacterial mechanism of NAD+.
Assuntos
Infecções Bacterianas , Nicotinamida-Nucleotídeo Adenililtransferase , Infecções Respiratórias , Humanos , NAD/metabolismo , Proteômica , Citocinas/metabolismo , Linhagem Celular , Trifosfato de Adenosina , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismoRESUMO
Nature has evolved eight different pathways for the capture and conversion of CO2, including the Calvin-Benson-Bassham cycle of photosynthesis. Yet, these pathways underlie constrains and only represent a fraction of the thousands of theoretically possible solutions. To overcome the limitations of natural evolution, we introduce the HydrOxyPropionyl-CoA/Acrylyl-CoA (HOPAC) cycle, a new-to-nature CO2-fixation pathway that was designed through metabolic retrosynthesis around the reductive carboxylation of acrylyl-CoA, a highly efficient principle of CO2 fixation. We realized the HOPAC cycle in a step-wise fashion and used rational engineering approaches and machine learning-guided workflows to further optimize its output by more than one order of magnitude. Version 4.0 of the HOPAC cycle encompasses 11 enzymes from six different organisms, converting ~3.0 mM CO2 into glycolate within 2 hours. Our work moves the hypothetical HOPAC cycle from a theoretical design into an established in vitro system that forms the basis for different potential applications.
Assuntos
Dióxido de Carbono , Procedimentos de Cirurgia Plástica , Fotossíntese , Engenharia , Aprendizado de MáquinaRESUMO
Formate can be envisioned at the core of a carbon-neutral bioeconomy, where it is produced from CO2 by (electro-)chemical means and converted into value-added products by enzymatic cascades or engineered microbes. A key step in expanding synthetic formate assimilation is its thermodynamically challenging reduction to formaldehyde. Here, we develop a two-enzyme route in which formate is activated to formyl phosphate and subsequently reduced to formaldehyde. Exploiting the promiscuity of acetate kinase and N-acetyl-γ-glutamyl phosphate reductase, we demonstrate this phosphate (Pi)-based route in vitro and in vivo. We further engineer a formyl phosphate reductase variant with improved formyl phosphate conversion in vivo by suppressing cross-talk with native metabolism and interface the Pi route with a recently developed formaldehyde assimilation pathway to enable C2 compound formation from formate as the sole carbon source in Escherichia coli. The Pi route therefore offers a potent tool in expanding the landscape of synthetic formate assimilation.
Assuntos
Formiatos , Fosfatos , Carbono , Escherichia coli/genética , FormaldeídoRESUMO
Ethylene glycol (EG) is a promising next generation feedstock for bioprocesses. It is a key component of the ubiquitous plastic polyethylene terephthalate (PET) and other polyester fibers and plastics, used in antifreeze formulations, and can also be generated by electrochemical conversion of syngas, which makes EG a key compound in a circular bioeconomy. The majority of biotechnologically relevant bacteria assimilate EG via the glycerate pathway, a wasteful metabolic route that releases CO2 and requires reducing equivalents as well as ATP. In contrast, the recently characterized ß-hydroxyaspartate cycle (BHAC) provides a more efficient, carbon-conserving route for C2 assimilation. Here we aimed at overcoming the natural limitations of EG metabolism in the industrially relevant strain Pseudomonas putida KT2440 by replacing the native glycerate pathway with the BHAC. We first prototyped the core reaction sequence of the BHAC in Escherichia coli before establishing the complete four-enzyme BHAC in Pseudomonas putida. Directed evolution on EG resulted in an improved strain that exhibits 35% faster growth and 20% increased biomass yield compared to a recently reported P. putida strain that was evolved to grow on EG via the glycerate pathway. Genome sequencing and proteomics highlight plastic adaptations of the genetic and metabolic networks in response to the introduction of the BHAC into P. putida and identify key mutations for its further integration during evolution. Taken together, our study shows that the BHAC can be utilized as 'plug-and-play' module for the metabolic engineering of two important microbial platform organisms, paving the way for multiple applications for a more efficient and carbon-conserving upcycling of EG in the future.
Assuntos
Pseudomonas putida , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Plásticos/metabolismo , Etilenoglicol/metabolismo , Polietilenotereftalatos/metabolismo , Carbono/metabolismoRESUMO
In the biosynthesis of the iron-guanylylpyridinol (FeGP) cofactor, 6-carboxymethyl-5-methyl-4-hydroxy-2-pyridinol (1) is 3-methylated to form 2, then 4-guanylylated to form 3, and converted into the full cofactor. HcgA-G proteins catalyze the biosynthetic reactions. Herein, we report the function of two radical S-adenosyl methionine enzymes, HcgA and HcgG, as uncovered by in vitro complementation experiments and the use of purified enzymes. In vitro biosynthesis using the cell extract from the Methanococcus maripaludis ΔhcgA strain was complemented with HcgA or precursors 1, 2 or 3. The results suggested that HcgA catalyzes the biosynthetic reaction that forms 1. We demonstrated the formation of 1 by HcgA using the 3â kDa cell extract filtrate as the substrate. Biosynthesis in the ΔhcgG system was recovered by HcgG but not by 3, which indicated that HcgG catalyzes the reactions after the biosynthesis of 3. The data indicated that HcgG contributes to the formation of CO and completes biosynthesis of the FeGP cofactor.
Assuntos
Hidrogenase , Proteínas Ferro-Enxofre , Hidrogenase/metabolismo , Extratos Celulares , Proteínas Ferro-Enxofre/metabolismo , S-Adenosilmetionina/metabolismo , Ferro/metabolismoRESUMO
The promiscuous activities of enzymes provide fertile ground for the evolution of new metabolic pathways. Here, we systematically explore the ability of E. coli to harness underground metabolism to compensate for the deletion of an essential biosynthetic pathway. By deleting all threonine deaminases, we generated a strain in which isoleucine biosynthesis was interrupted at the level of 2-ketobutyrate. Incubation of this strain under aerobic conditions resulted in the emergence of a novel 2-ketobutyrate biosynthesis pathway based upon the promiscuous cleavage of O-succinyl-L-homoserine by cystathionine γ-synthase (MetB). Under anaerobic conditions, pyruvate formate-lyase enabled 2-ketobutyrate biosynthesis from propionyl-CoA and formate. Surprisingly, we found this anaerobic route to provide a substantial fraction of isoleucine in a wild-type strain when propionate is available in the medium. This study demonstrates the selective advantage underground metabolism offers, providing metabolic redundancy and flexibility which allow for the best use of environmental carbon sources.
Assuntos
Butiratos/metabolismo , Carbono-Oxigênio Liases/metabolismo , Escherichia coli/metabolismo , Deleção de Genes , Homosserina/análogos & derivados , Isoleucina/metabolismo , Escherichia coli/genética , Homosserina/metabolismo , Redes e Vias MetabólicasRESUMO
The mammalian gene Nit1 (nitrilase-like protein 1) encodes a protein that is highly conserved in eukaryotes and is thought to act as a tumor suppressor. Despite being â¼35% sequence identical to ω-amidase (Nit2), the Nit1 protein does not hydrolyze efficiently α-ketoglutaramate (a known physiological substrate of Nit2), and its actual enzymatic function has so far remained a puzzle. In the present study, we demonstrate that both the mammalian Nit1 and its yeast ortholog are amidases highly active toward deaminated glutathione (dGSH; i.e., a form of glutathione in which the free amino group has been replaced by a carbonyl group). We further show that Nit1-KO mutants of both human and yeast cells accumulate dGSH and the same compound is excreted in large amounts in the urine of Nit1-KO mice. Finally, we show that several mammalian aminotransferases (transaminases), both cytosolic and mitochondrial, can form dGSH via a common (if slow) side-reaction and provide indirect evidence that transaminases are mainly responsible for dGSH formation in cultured mammalian cells. Altogether, these findings delineate a typical instance of metabolite repair, whereby the promiscuous activity of some abundant enzymes of primary metabolism leads to the formation of a useless and potentially harmful compound, which needs a suitable "repair enzyme" to be destroyed or reconverted into a useful metabolite. The need for a dGSH repair reaction does not appear to be limited to eukaryotes: We demonstrate that Nit1 homologs acting as excellent dGSH amidases also occur in Escherichia coli and other glutathione-producing bacteria.
Assuntos
Aminoidrolases/metabolismo , Glutationa/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transaminases/metabolismo , Aminoidrolases/fisiologia , Animais , Desaminação , Humanos , Hidrólise , Camundongos , Camundongos Knockout , Especificidade por SubstratoRESUMO
The D or L form of 2-hydroxyglutarate (2HG) accumulates in certain rare neurometabolic disorders, and high D-2-hydroxyglutarate (D-2HG) levels are also found in several types of cancer. Although 2HG has been detected in Saccharomyces cerevisiae, its metabolism in yeast has remained largely unexplored. Here, we show that S. cerevisiae actively forms the D enantiomer of 2HG. Accordingly, the S. cerevisiae genome encodes two homologs of the human D-2HG dehydrogenase: Dld2, which, as its human homolog, is a mitochondrial protein, and the cytosolic protein Dld3. Intriguingly, we found that a dld3Δ knock-out strain accumulates millimolar levels of D-2HG, whereas a dld2Δ knock-out strain displayed only very moderate increases in D-2HG. Recombinant Dld2 and Dld3, both currently annotated as D-lactate dehydrogenases, efficiently oxidized D-2HG to α-ketoglutarate. Depletion of D-lactate levels in the dld3Δ, but not in the dld2Δ mutant, led to the discovery of a new type of enzymatic activity, carried by Dld3, to convert D-2HG to α-ketoglutarate, namely an FAD-dependent transhydrogenase activity using pyruvate as a hydrogen acceptor. We also provide evidence that Ser3 and Ser33, which are primarily known for oxidizing 3-phosphoglycerate in the main serine biosynthesis pathway, in addition reduce α-ketoglutarate to D-2HG using NADH and represent major intracellular sources of D-2HG in yeast. Based on our observations, we propose that D-2HG is mainly formed and degraded in the cytosol of S. cerevisiae cells in a process that couples D-2HG metabolism to the shuttling of reducing equivalents from cytosolic NADH to the mitochondrial respiratory chain via the D-lactate dehydrogenase Dld1.