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Here, an isozyme co\u2010expression strategy was proposed and evaluated in the (\n                    <jats:italic>R<\/jats:italic>\n                    )\u20101,3\u2010BDO biosynthetic pathway of\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    . First, nine strains harboring different combinations of three candidate thiolases (AtoB, PhaA, Thl) and three candidate aldehyde dehydrogenases (Bld, Ald, AdhE2) were constructed and evaluated. Among them, the Thl\u2010PhaB\u2010Bld strain (B3\u20107) exhibited the highest (\n                    <jats:italic>R<\/jats:italic>\n                    )\u20101,3\u2010BDO production (1.4\u00a0g\/L). The remaining isozymes (AtoB, PhaA, Ald, and AdhE2) were then overexpressed individually in strain B3\u20107, with PhaA and AdhE2 showing the most significant improvement in (\n                    <jats:italic>R<\/jats:italic>\n                    )\u20101,3\u2010BDO production. Co\u2010expression of PhaA and AdhE2 further increased the (\n                    <jats:italic>R<\/jats:italic>\n                    )\u20101,3\u2010BDO titer to 4.2\u00a0g\/L, representing a 200% increase relative to strain B3\u20107. In addition, several metabolic effector molecules were screened as medium supplements, among which citric acid was identified as the most effective for improving (\n                    <jats:italic>R<\/jats:italic>\n                    )\u20101,3\u2010BDO production. Finally, in glucose\u2010feeding shake\u2010flask fermentation, the isozyme co\u2010expression strain produced 16.2\u00a0g\/L (\n                    <jats:italic>R<\/jats:italic>\n                    )\u20101,3\u2010BDO. Collectively, the isozyme co\u2010expression strategy effectively enhanced (\n                    <jats:italic>R<\/jats:italic>\n                    )\u20101,3\u2010BDO production and represents a promising approach for optimizing multi\u2010step biosynthetic pathways.\n                  <\/jats:p>","DOI":"10.1002\/biot.70224","type":"journal-article","created":{"date-parts":[[2026,4,8]],"date-time":"2026-04-08T10:38:00Z","timestamp":1775644680000},"source":"Crossref","is-referenced-by-count":0,"title":["Production of (\n                    <i>R<\/i>\n                    )\u20101,3\u2010butanediol via an Isozyme co\u2010expression Strategy in\n                    <i>Escherichia coli<\/i>"],"prefix":"10.1002","volume":"21","author":[{"given":"Huifang","family":"Zhang","sequence":"first","affiliation":[{"name":"College of Biotechnology and Pharmaceutical Engineering Nanjing Tech University  Nanjing 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