{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,11]],"date-time":"2026-08-11T23:34:43Z","timestamp":1786491283131,"version":"build-2736575974"},"reference-count":47,"publisher":"Wiley","issue":"1","license":[{"start":{"date-parts":[[2021,11,14]],"date-time":"2021-11-14T00:00:00Z","timestamp":1636848000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["analyticalsciencejournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Engineering in Life Sciences"],"published-print":{"date-parts":[[2022,1]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Glycine cleavage system (GCS) plays a central role in one\u2010carbon (C1) metabolism and receives increasing interest as a core part of the recently proposed reductive glycine pathway (rGlyP) for assimilation of CO<jats:sub>2<\/jats:sub> and formate. Despite decades of research, GCS has not yet been well understood and kinetic data are barely available. This is to a large degree because of the complexity of GCS, which is composed of four proteins (H, T, P, and L) and catalyzes reactions involving different substrates and cofactors. In vitro kinetics of reconstructed microbial multi\u2010enzyme glycine cleavage\/synthase system is desired to better implement rGlyP in microorganisms like <jats:italic>Escherichia coli<\/jats:italic> for the use of C1 resources. Here, we examined in vitro several factors that may affect the rate of glycine synthesis via the reverse GCS reaction. We found that the ratio of GCS component proteins has a direct influence on the rate of glycine synthesis, namely higher ratios of P protein and especially H protein to T and L proteins are favorable, and the carboxylation reaction catalyzed by P protein is a key step determining the glycine synthesis rate, whereas increasing the ratio of L protein to other GCS proteins does not have significant effect and the ratio of T protein to other GCS proteins should be kept low. The effect of substrate concentrations on glycine synthesis is quite complex, showing interdependence with the ratios of GCS component proteins. Furthermore, adding the reducing agent dithiothreitol to the reaction mixture not only results in great tolerance to high concentration of formaldehyde, but also increases the rate of glycine synthesis, probably due to its functions in activating P protein and taking up the role of L protein in the non\u2010enzymatic reduction of H<jats:sub>ox<\/jats:sub> to H<jats:sub>red<\/jats:sub>. Moreover, the presence of some monovalent and divalent metal ions can have either positive or negative effect on the rate of glycine synthesis, depending on their type and their concentration.<\/jats:p>","DOI":"10.1002\/elsc.202100047","type":"journal-article","created":{"date-parts":[[2021,11,15]],"date-time":"2021-11-15T00:38:24Z","timestamp":1636936704000},"page":"40-53","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":30,"title":["Improvement of glycine biosynthesis from one\u2010carbon compounds and ammonia catalyzed by the glycine cleavage system in vitro"],"prefix":"10.1002","volume":"22","author":[{"given":"Yingying","family":"Xu","sequence":"first","affiliation":[{"name":"Beijing Advanced Innovation Center for Soft Matter Science and Engineering Beijing University of Chemical Technology  Beijing P. R. China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jie","family":"Ren","sequence":"additional","affiliation":[{"name":"Beijing Advanced Innovation Center for Soft Matter Science and Engineering Beijing University of Chemical Technology  Beijing P. R. China"},{"name":"State Key Laboratory for Biology of Plant Diseases and Insect Pests\/Key Laboratory of Control of Biological Hazard Factors (Plant Origin) for Agri\u2010product Quality and Safety Ministry of Agriculture Institute of Plant Protection Chinese Academy of Agricultural Sciences  Beijing P. R. China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wei","family":"Wang","sequence":"additional","affiliation":[{"name":"Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology  Hamburg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9768-7096","authenticated-orcid":false,"given":"An\u2010Ping","family":"Zeng","sequence":"additional","affiliation":[{"name":"Beijing Advanced Innovation Center for Soft Matter Science and Engineering Beijing University of Chemical Technology  Beijing P. R. China"},{"name":"Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology  Hamburg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2021,11,14]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibtech.2017.08.011"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.copbio.2021.01.003"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2019.11.009"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.biochem.6b00495"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cbpa.2016.07.005"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymben.2016.10.015"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.copbio.2017.11.010"},{"key":"e_1_2_9_9_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.aav3506"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymben.2018.03.015"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbabio.2012.10.013"},{"key":"e_1_2_9_12_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-020-18906-7"},{"key":"e_1_2_9_13_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1715549114"},{"key":"e_1_2_9_14_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1912289117"},{"key":"e_1_2_9_15_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1810386115"},{"key":"e_1_2_9_16_1","doi-asserted-by":"publisher","DOI":"10.1021\/acssynbio.8b00131"},{"key":"e_1_2_9_17_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41589-020-0473-5"},{"key":"e_1_2_9_18_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41564-020-00793-9"},{"key":"e_1_2_9_19_1","doi-asserted-by":"publisher","DOI":"10.1002\/bit.27658"},{"key":"e_1_2_9_20_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymben.2020.08.004"},{"key":"e_1_2_9_21_1","doi-asserted-by":"publisher","DOI":"10.1021\/acssynbio.8b00464"},{"key":"e_1_2_9_22_1","doi-asserted-by":"publisher","DOI":"10.1128\/JB.140.2.468-478.1979"},{"key":"e_1_2_9_23_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1574-6968.1999.tb13705.x"},{"key":"e_1_2_9_24_1","doi-asserted-by":"publisher","DOI":"10.1016\/0003-9861(69)90377-4"},{"key":"e_1_2_9_25_1","doi-asserted-by":"publisher","DOI":"10.1093\/oxfordjournals.jbchem.a130483"},{"key":"e_1_2_9_26_1","doi-asserted-by":"publisher","DOI":"10.1016\/0003-9861(76)90236-8"},{"key":"e_1_2_9_27_1","doi-asserted-by":"publisher","DOI":"10.1016\/S1360-1385(01)01892-1"},{"key":"e_1_2_9_28_1","doi-asserted-by":"publisher","DOI":"10.1042\/bj2550169"},{"key":"e_1_2_9_29_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(17)35920-3"},{"key":"e_1_2_9_30_1","doi-asserted-by":"publisher","DOI":"10.1016\/0014-5793(86)80005-9"},{"key":"e_1_2_9_31_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(19)68336-5"},{"key":"e_1_2_9_32_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(20)82108-5"},{"key":"e_1_2_9_33_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF00407945"},{"key":"e_1_2_9_34_1","doi-asserted-by":"publisher","DOI":"10.1038\/s42003-020-01401-6"},{"key":"e_1_2_9_35_1","doi-asserted-by":"publisher","DOI":"10.1002\/bit.27526"},{"key":"e_1_2_9_36_1","doi-asserted-by":"publisher","DOI":"10.1186\/s13036-019-0164-5"},{"key":"e_1_2_9_37_1","doi-asserted-by":"publisher","DOI":"10.1104\/pp.94.2.833"},{"key":"e_1_2_9_38_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi00208a010"},{"key":"e_1_2_9_39_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi00014a028"},{"key":"e_1_2_9_40_1","doi-asserted-by":"publisher","DOI":"10.1186\/s13036-020-00237-2"},{"key":"e_1_2_9_41_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M113.509976"},{"key":"e_1_2_9_42_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.aao3407"},{"key":"e_1_2_9_43_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-021-03456-9"},{"key":"e_1_2_9_44_1","doi-asserted-by":"publisher","DOI":"10.1046\/j.1432-1327.2000.01301.x"},{"key":"e_1_2_9_45_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.274.37.26344"},{"key":"e_1_2_9_46_1","doi-asserted-by":"publisher","DOI":"10.1093\/oxfordjournals.jbchem.a134009"},{"key":"e_1_2_9_47_1","article-title":"Understanding and engineering glycine cleavage system and related metabolic pathways for C1\u2010based biosynthesis","author":"Ren J.","year":"2021","journal-title":"Adv. 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