Metabolite retention times were identified by injecting unlabeled standards.
"],"repository":["MetaboLights"],"study_status":["Public"],"ptm_modification":[""],"instrument_platform":["Liquid Chromatography MS - negative - hilic"],"chromatography_protocol":["Quantification of 13C-incorporation was performed by coupling liquid chromatography (Vanquish™, Thermo Fisher Scientific, Waltham, MA, USA) with a MS (Q Exactive™ Focus orbitrap, Thermo Fisher
Scientific, Waltham, MA, USA) controlled by the Xcalibur software (version 7.3, Thermo Fisher Scientific, Waltham, MA, USA). The separation was performed by liquid chromatography with a SeQuant® ZIC®-pHILIC Column (5 μm polymer 150 × 2.1 mm, Merck KGaA, Germany) at a flow rate of 0.15 mL/min. For an optimal efficiency of separation, the mobile phase was composed of a changing gradient of A (5% ACN, 10 mM ammonium acetate, pH = 9.2 adjusted by NH4OH) and B (90% acetonitrile, 10 mM ammonium acetate, pH = 9.2 by NH4OH) following the program (0 min, 95% B; 2 min, 95% B; 3 min, 89.4% B; 5 min, 89.4% B; 6 min, 83.8% B; 7 min, 83.8% B; 8 min, 78.2% B; 9 min, 78.2% B; 10 min, 55.9% B; 12 min, 55.9% B; 13 min, 27.9% B; 16 min, 27.9% B; 18 min, 0% B; 23 min, 0% B; 24 min, 95% B; 30 min, 95% B). The temperature of the autosampler and oven was kept at 6 °C and 25 °C, respectively. The injection volume was 5 μL.
"],"publication":["Enzyme engineering for optimizing biosynthesis of 2,4-dihydroxybutyric acid via the synthetic threose-dependent glycolaldehyde assimilation (STEGA) pathway. 10.1016/j.ymben.2026.04.005. PMID:41991054"],"submitter_affiliation":["Dresden University of Technology"],"submitter_name":["Alrik Titze"],"organism_part":["metabolite","Analytical standard"],"technology_type":["mass spectrometry assay"],"disease":[""],"extraction_protocol":["For each sample, cells from 1.5 mL culture were captured by a polyamide membrane filter with the pore size of 0.2 μm (Sartorius, Germany) and immediately washed with 20 g/L mL glass tube which contained 5 mL of 75% ethanol pre-heated to 80 °C. The glass tubes were vortexed for 10 s and incubated at 80 °C for 3 min before being chilled on ice for10 min. The supernatant containing intracellular metabolites was separated from the cell debris by centrifuging the suspension without the polyamide filters at 13,000 × g for 5 min and kept at -20°C. Prior to LC-MS measurement, the liquid residue was removed by vacuum drying at 45 °C for 4 h using a benchtop vacuum concentrator CentriVap Concentrator System (Labconco, USA). The obtained metabolites were collected by re-suspension in 250 μL
deionized water. All the samples to be measured using LC-MS were prepared in a matrix of 60% acetonitrile and 40% water (v/v) which contained 10 mM ammonium acetate and was adjusted to pH = 9.2.
"],"organism":["Escherichia coli str. K-12 substr. MG1655","Analytical standard"],"full_dataset_link":["https://www.ebi.ac.uk/metabolights/MTBLS15050"],"author":["Thomas Walther. Technische Universität Dresden. thomas_walther@tu-dresden.de.","Alrik Titze. Technische Universität Dresden. alrik.titze@tu-dresden.de."],"data_transformation_protocol":["Peak areas were extracted from the raw files using the software TraceFinder (Version 5.1, Thermo Fisher Scientific, Waltham, MA, USA) and corrected for the contribution of all naturally abundant isotopes using the software IsoCor version 2.2.3
"],"study_factor":["Gene mutations","Biological replicate"],"submitter_email":["alrik.titze@tu-dresden.de"],"sample_collection_protocol":["E. coli MG1655 ΔyqhD ΔaldA ΔlldD cells containing the plasmids pEXT22-Ec.mdh5Q-Hh.araD, pEXT21-Re.kdgT and pACT3-Ec.fsaATA-Pc.tadH-Tt.lac11v1 was grown alongside adapted strains where the Ec.FsaA-TA was replaced by Ec.FsaA-TA additionally containing the mutations R134I, R134M or R134V. Cells were grown from an intial OD600 of 0.2 in 10 mL M9 medium supplemented with 20 g/L glucose in 100 mL, non-baffled shake flasks. When OD600 reached 0.6, IPTG (0.5 mM) was added to induce gene expression and when OD600 reached 2.0, 10 mM of uniformely 13C labelled Glycolaldehyde were added. 1.5 mL of samples were taken from the cultivation 2h after feeding the labelled Glycolaldehyde. They were quenched directly as described in the section 'extraction'
"],"omics_type":["Metabolomics"],"study_design":["Technische Universität Dresden","Metabolomics","Standard for Seduheptulose 7P","targeted analysis","Standard for Fructose 6P and Pentose 5P","Analytical standard","targeted metabolite profiling","13c label","experimental sample","glycolaldehyde","Vanquish","metabolite","Escherichia coli str. K-12 substr. MG1655","Thermo Scientific Q Exactive Focus"],"curator_keywords":["Technische Universität Dresden","Metabolomics","Standard for Seduheptulose 7P","targeted analysis","Standard for Fructose 6P and Pentose 5P","Analytical standard","targeted metabolite profiling","13c label","experimental sample","glycolaldehyde","Vanquish","metabolite","Escherichia coli str. K-12 substr. MG1655","Thermo Scientific Q Exactive Focus"],"mass_spectrometry_protocol":["The LC was coupled to an MS (Q Exactive™ Focus orbitrap, Thermo Fisher Scientific, Waltham, MA, USA). The instrumental settings according to the electrospray ionization were optimised for a flow rate of 0.15 mL/min. Final parameters were adjusted as follows: sheath gas flow rate 32 (arbitrary units), auxiliary gas flow rate 8 (arbitrary units), sweep gas flow rate 0 (arbitrary units), spray voltage −3.5 kV, capillary temperature at 250 °C and auxiliary gas temperature at 200 °C. The metabolites were identified according to the monoisotopic mass and retention time of the unlabelled standards.
"],"metabolite_name":["Sedoheptulose 7-Phosphate","Fructose 6-Phosphate","Ribose 5-Phosphate"],"pubmed_abstract":["Ethylene glycol is a potential feedstock for next-generation biorefineries, as it can be derived from both plastic waste and carbon dioxide. The synthetic D-threose-dependent glycolaldehyde assimilation (STEGA) pathway, which is orthogonal to central metabolism of Escherichia coli, was recently constructed for the biosynthesis of the platform chemical 2,4-dihydroxybutyric acid (DHB) from ethylene glycol (EG). However, the performance of this pathway was limited by the low catalytic efficiency of key enzymes. Therefore, in the present study, structure-guided semi-rational engineering was employed to improve the activities of D-threose aldolase and D-threose dehydrogenase, originally provided by the fructose-6-phosphate aldolase L107Y:A129G mutant (Ec.FsaATA) from E. coli and the promiscuous D-threo-1-aldose dehydrogenase Pc.TadH from Paraburkholderia caryophylli, respectively. The substrate specificity of Ec.FsaATA was improved by replacing Arg134 by either isoleucine, methionine or valine which effectively eliminated activity of the mutant enzyme toward phosphorylated substrates while retaining homo-aldol condensation activity toward glycolaldehyde for D-threose formation. In-vivo implementation of the improved aldolase within the STEGA pathway reduced off-target glycolaldehyde flux, as demonstrated by 13C-carbon tracing experiments. Furthermore, the engineered Pc.TadH A24G:F58L double mutant exhibited an 11-fold increase in specificity constant (kcat/Km) on D-threose compared to the wild-type enzyme. As a consequence of these improvements, co-expression of both engineered enzymes in the STEGA pathway significantly enhanced EG-to-DHB bioconversion, achieving a 68% increase in final DHB titer (5.2 ± 0.11 mM) and a 23% improvement in carbon yield (0.16 ± 0.003 Cmol/Cmol)."],"pubmed_title":["Enzyme engineering for optimizing biosynthesis of 2,4-dihydroxybutyric acid via the synthetic threose-dependent glycolaldehyde assimilation (STEGA) pathway."],"pubmed_authors":["Wen Linxuan L, Titze Alrik A, Topham Christopher M CM, Radde Johannes J, Frazão Cláudio J R CJR, Walther Thomas T"],"additional_accession":[]},"is_claimable":false,"name":"Enzyme engineering for optimizing biosynthesis of 2,4-dihydroxybutyric acid via the synthetic threose-dependent glycolaldehyde assimilation (STEGA) pathway","description":"Ethylene glycol is a potential feedstock for next-generation biorefineries, as it can be derived from both plastic waste and carbon dioxide. The synthetic D-threose-dependent glycolaldehyde assimilation (STEGA) pathway, which is orthogonal to central metabolism of Escherichia coli, was recently constructed for the biosynthesis of the platform chemical 2,4-dihydroxybutyric acid (DHB) from ethylene glycol (EG). However, the performance of this pathway was limited by the low catalytic efficiency of key enzymes. Therefore, in the present study, structureguided semi-rational engineering was employed to improve the activities of D-threose aldolase and D-threose dehydrogenase, originally provided by the fructose-6-phosphate aldolase L107Y:A129G mutant (Ec.FsaATA) fromE. coli and the promiscuous D-threo-1-aldose dehydrogenase Pc.TadH from Paraburkholderia caryophylli, respectively. The substrate specificity of Ec.FsaATA was improved by replacing Arg134 by either isoleucine, methionine or valine which effectively eliminated activity of the mutant enzyme toward phosphorylated sub-strates while retaining homo-aldol condensation activity toward glycolaldehyde for D-threose formation. In-vivoimplementation of the improved aldolase within the STEGA pathway reduced off-target glycolaldehyde flux, as demonstrated by 13C-carbon tracing experiments. Furthermore, the engineered Pc.TadH A24G:F58L double mutant exhibited an 11-fold increase in specificity constant (kcat/Km) on D-threose compared to the wild-type enzyme. As a consequence of these improvements, co-expression of both engineered enzymes in the STEGA pathway significantly enhanced EG-to-DHB bioconversion, achieving a 68% increase in final DHB titer (5.2 ±0.11 mM) and a 23% improvement in carbon yield (0.16 ± 0.003 Cmol/Cmol).","dates":{"publication":"2026-07-15","submission":"2026-07-15"},"accession":"MTBLS15050","cross_references":{"MetaboLights":["MTBLC57634","MTBLC58273","MTBLC57483"],"pubmed":["41991054"],"ChEBI":["CHEBI:57634","CHEBI:58273","CHEBI:57483"]}}