{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T14:38:48Z","timestamp":1786545528799,"version":"build-2736575974"},"reference-count":42,"publisher":"Wiley","issue":"15","license":[{"start":{"date-parts":[[2026,7,24]],"date-time":"2026-07-24T00:00:00Z","timestamp":1784851200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,7,24]],"date-time":"2026-07-24T00:00:00Z","timestamp":1784851200000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["chemistry-europe.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["ChemSusChem"],"published-print":{"date-parts":[[2026,8,14]]},"abstract":"<jats:p>\n                    Multienzyme cascades are emerging as alternatives to fermentation for converting biogenic feedstocks into value\u2010added chemicals and fuels. A major bottleneck in redox transformations is the loss of costly and unstable cofactors in continuous\u2010flow systems, typically necessitating co\u2010immobilization and complex regeneration schemes. Self\u2010sufficient biocatalytic nanomachines, created by fusing enzymes with tethered cofactors, offer a streamlined solution by enabling intramolecular cofactor retention and recycling. Here, we demonstrate that such nanomachines can be integrated into a complete redox\u2010dependent cascade, exemplified by a 10\u2010step glucose\u2010to\u2010isobutanol pathway. Oxidoreductases were fused into redox pairs using peptide linkers containing a single cysteine for covalent attachment of polyethylene glycol (PEG)\u2010modified nicotinamide cofactors, with rational selection of monomeric enzymes ensuring structural compatibility and soluble expression. The resulting nanomachines remained catalytically competent and produced isobutanol in both batch and continuous\u2010flow operation without the addition of free NAD\n                    <jats:sup>+<\/jats:sup>\n                    . A total turnover number of \u223c1.4 \u00d7 10\n                    <jats:sup>4<\/jats:sup>\n                    was achieved for tethered PEG\u2013NAD\n                    <jats:sup>+<\/jats:sup>\n                    , among the highest reported for immobilized nicotinamide cofactors. Use of the ultrastable analogue PEG\u2013cNAD\n                    <jats:sup>+<\/jats:sup>\n                    further demonstrates the modularity of the platform. Together, these results establish a proof\u2010of\u2010concept for cofactor\u2010autonomous multienzyme redox cascades in continuous flow and define design principles for future cell\u2010free synthesis systems.\n                  <\/jats:p>","DOI":"10.1002\/cssc.70914","type":"journal-article","created":{"date-parts":[[2026,7,25]],"date-time":"2026-07-25T05:07:11Z","timestamp":1784956031000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Extending Cofactor\u2010Tethered Nanomachines to Complex Multienzyme Redox Cascades in Continuous Flow"],"prefix":"10.1002","volume":"19","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3574-6358","authenticated-orcid":false,"given":"Zinnia","family":"Dsouza","sequence":"first","affiliation":[{"name":"Chair of Chemistry of Biogenic Resources Campus Straubing for Biotechnology and Sustainability Technical University of Munich  Straubing Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-4865-6115","authenticated-orcid":false,"given":"Jan\u2010Simon Jeshua","family":"Friedrichs","sequence":"additional","affiliation":[{"name":"Chair of Chemistry of Biogenic Resources Campus Straubing for Biotechnology and Sustainability Technical University of Munich  Straubing Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0021-3466","authenticated-orcid":false,"given":"Okke","family":"Melse","sequence":"additional","affiliation":[{"name":"Chair of Chemistry of Biogenic Resources Campus Straubing for Biotechnology and Sustainability Technical University of Munich  Straubing Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8619-0631","authenticated-orcid":false,"given":"Gerhard","family":"Schenk","sequence":"additional","affiliation":[{"name":"School of Chemistry and Molecular Biosciences The University of Queensland  St. Lucia Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5458-9330","authenticated-orcid":false,"given":"Volker","family":"Sieber","sequence":"additional","affiliation":[{"name":"Chair of Chemistry of Biogenic Resources Campus Straubing for Biotechnology and Sustainability Technical University of Munich  Straubing Germany"},{"name":"School of Chemistry and Molecular Biosciences The University of Queensland  St. Lucia Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,7,24]]},"reference":[{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biotechadv.2015.03.010"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.1021\/op1002159"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1002\/cbic.201700687"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.copbio.2022.102868"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.1021\/acscatal.7b03440"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/nchem.2459"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chempr.2017.04.009"},{"key":"e_1_2_8_9_1","doi-asserted-by":"publisher","DOI":"10.3390\/molecules26030573"},{"key":"e_1_2_8_10_1","doi-asserted-by":"publisher","DOI":"10.1002\/cctc.201300976"},{"key":"e_1_2_8_11_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(18)81208-X"},{"key":"e_1_2_8_12_1","doi-asserted-by":"publisher","DOI":"10.1006\/abio.1998.2656"},{"key":"e_1_2_8_13_1","doi-asserted-by":"publisher","DOI":"10.1210\/er.2009-0026"},{"key":"e_1_2_8_14_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymben.2016.02.005"},{"key":"e_1_2_8_15_1","doi-asserted-by":"publisher","DOI":"10.1039\/D0NP00004C"},{"key":"e_1_2_8_16_1","doi-asserted-by":"publisher","DOI":"10.3390\/catal12111454"},{"key":"e_1_2_8_17_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbiotec.2014.07.449"},{"key":"e_1_2_8_18_1","doi-asserted-by":"publisher","DOI":"10.1007\/s41981-024-00315-2"},{"key":"e_1_2_8_19_1","doi-asserted-by":"publisher","DOI":"10.1002\/anie.201609758"},{"key":"e_1_2_8_20_1","doi-asserted-by":"publisher","DOI":"10.1021\/acsanm.5c01612"},{"key":"e_1_2_8_21_1","doi-asserted-by":"publisher","DOI":"10.3390\/catal11040409"},{"key":"e_1_2_8_22_1","doi-asserted-by":"publisher","DOI":"10.1039\/D0RE00483A"},{"key":"e_1_2_8_23_1","doi-asserted-by":"publisher","DOI":"10.1016\/0385-6380(88)90103-3"},{"key":"e_1_2_8_24_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.bioconjchem.8b00766"},{"key":"e_1_2_8_25_1","doi-asserted-by":"publisher","DOI":"10.1002\/cssc.202200888"},{"key":"e_1_2_8_26_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cej.2022.135400"},{"key":"e_1_2_8_27_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41929-019-0353-0"},{"key":"e_1_2_8_28_1","doi-asserted-by":"publisher","DOI":"10.1002\/cssc.201200365"},{"key":"e_1_2_8_29_1","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202017027"},{"key":"e_1_2_8_30_1","doi-asserted-by":"publisher","DOI":"10.1039\/D6CB00036C"},{"key":"e_1_2_8_31_1","doi-asserted-by":"publisher","DOI":"10.1002\/cbic.200700500"},{"key":"e_1_2_8_32_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-019-48151-y"},{"key":"e_1_2_8_33_1","doi-asserted-by":"publisher","DOI":"10.1002\/cssc.202301132"},{"key":"e_1_2_8_34_1","doi-asserted-by":"publisher","DOI":"10.1021\/acscatal.4c00958"},{"key":"e_1_2_8_35_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biotechadv.2022.108016"},{"key":"e_1_2_8_36_1","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.03873-12"},{"key":"e_1_2_8_37_1","doi-asserted-by":"publisher","DOI":"10.1002\/pro.2852"},{"key":"e_1_2_8_38_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi101163u"},{"key":"e_1_2_8_39_1","doi-asserted-by":"publisher","DOI":"10.1002\/pro.3015"},{"key":"e_1_2_8_40_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF00928468"},{"key":"e_1_2_8_41_1","doi-asserted-by":"publisher","DOI":"10.1002\/cssc.202501120"},{"key":"e_1_2_8_42_1","doi-asserted-by":"publisher","DOI":"10.1002\/cbic.202200549"},{"key":"e_1_2_8_43_1","doi-asserted-by":"publisher","DOI":"10.3390\/molecules25051223"}],"container-title":["ChemSusChem"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/chemistry-europe.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cssc.70914","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/chemistry-europe.onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/cssc.70914","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/chemistry-europe.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cssc.70914","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T13:42:50Z","timestamp":1786542170000},"score":1,"resource":{"primary":{"URL":"https:\/\/chemistry-europe.onlinelibrary.wiley.com\/doi\/10.1002\/cssc.70914"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,24]]},"references-count":42,"journal-issue":{"issue":"15","published-print":{"date-parts":[[2026,8,14]]}},"alternative-id":["10.1002\/cssc.70914"],"URL":"https:\/\/doi.org\/10.1002\/cssc.70914","archive":["Portico"],"relation":{},"ISSN":["1864-5631","1864-564X"],"issn-type":[{"value":"1864-5631","type":"print"},{"value":"1864-564X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,24]]},"assertion":[{"value":"2026-06-05","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-07-09","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-07-24","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e70914"}}