{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T04:45:33Z","timestamp":1785732333824,"version":"3.56.0"},"reference-count":47,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,8,1]],"date-time":"2026-08-01T00:00:00Z","timestamp":1785542400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"crossref","award":["82304838"],"award-info":[{"award-number":["82304838"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]},{"award":["82304838"],"award-info":[{"award-number":["82304838"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100007129","name":"Natural Science Foundation of Shandong Province","doi-asserted-by":"publisher","award":["ZR2022MC159"],"award-info":[{"award-number":["ZR2022MC159"]}],"id":[{"id":"10.13039\/501100007129","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>Carcinine is an imidazole dipeptide with potent antioxidant and antiglycation properties, although its chemical synthesis currently relies on severely environmentally harmful processes. In this work, a multi-enzyme cascade biotransformation system comprising 4\u2032-phosphopantetheinyl transferase and non-ribosomal peptide synthetase was constructed. To overcome the limitations arising from stochastic spatial distribution and suboptimal mass transfer associated with independent enzymes, a fusion protein strategy was adopted. The two enzymes were fused via a flexible genetic linker within plasmid pET28a-SFP-L-Ebony, which enabled robust soluble expression in Escherichia coli. Concurrently, the endogenous peptidase genes (pepA, pepB, pepD, and pepN) were systematically knocked out using CRISPR\/Cas9-mediated gene editing. This quadruple protease-deficient strain (designated SFP-L-Ebony-\u0394pepABDN) effectively suppressed product degradation. Subsequent optimization revealed that optimal catalytic performance occurred at 25 \u00b0C and pH 7.0. The highest biotransformation efficiency was achieved using 15 g\/L crude enzymes, in the presence of 2 mM ATP and 10 mM MgCl2. Through a fed-batch substrate feeding strategy in a 50 mL reaction system, the final carcinine titer reached 7.0 g\/L after 48 h. This study, therefore, provides an efficient and sustainable technological pathway for the green biomanufacturing of carcinine as well as other high-value dipeptides.<\/jats:p>","DOI":"10.3390\/biom16081124","type":"journal-article","created":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T01:43:27Z","timestamp":1785721407000},"page":"1124","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Construction of Engineered Escherichia coli and Optimization of Conditions for Carcinine Synthesis via Multi-Enzyme Cascade Catalysis"],"prefix":"10.3390","volume":"16","author":[{"given":"Haoni","family":"Luan","sequence":"first","affiliation":[{"name":"School of Pharmaceutical Sciences and Food Engineering, Liaocheng University, Liaocheng 252000, China"},{"name":"Shandong Key Laboratory of Applied Technology for Protein and Peptide Drugs, Liaocheng 252000, China"},{"name":"State Key Laboratory of Macromolecular Drugs and Large-Scale Preparation, Liaocheng 252000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rui","family":"Yang","sequence":"additional","affiliation":[{"name":"School of Pharmaceutical Sciences and Food Engineering, Liaocheng University, Liaocheng 252000, China"},{"name":"Shandong Key Laboratory of Applied Technology for Protein and Peptide Drugs, Liaocheng 252000, China"},{"name":"State Key Laboratory of Macromolecular Drugs and Large-Scale Preparation, Liaocheng 252000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenhan","family":"Qiu","sequence":"additional","affiliation":[{"name":"School of Pharmaceutical Sciences and Food Engineering, Liaocheng University, Liaocheng 252000, China"},{"name":"Shandong Key Laboratory of Applied Technology for Protein and Peptide Drugs, Liaocheng 252000, China"},{"name":"State Key Laboratory of Macromolecular Drugs and Large-Scale Preparation, Liaocheng 252000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kaiyue","family":"Feng","sequence":"additional","affiliation":[{"name":"College of Agriculture and Biology, Liaocheng University, Liaocheng 252000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wei","family":"Xu","sequence":"additional","affiliation":[{"name":"School of Pharmaceutical Sciences and Food Engineering, Liaocheng University, Liaocheng 252000, China"},{"name":"Shandong Key Laboratory of Applied Technology for Protein and Peptide Drugs, Liaocheng 252000, China"},{"name":"State Key Laboratory of Macromolecular Drugs and Large-Scale Preparation, Liaocheng 252000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fei","family":"Wang","sequence":"additional","affiliation":[{"name":"School of Pharmaceutical Sciences and Food Engineering, Liaocheng University, Liaocheng 252000, China"},{"name":"Shandong Key Laboratory of Applied Technology for Protein and Peptide Drugs, Liaocheng 252000, China"},{"name":"State Key Laboratory of Macromolecular Drugs and Large-Scale Preparation, Liaocheng 252000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wei","family":"Feng","sequence":"additional","affiliation":[{"name":"School of Pharmaceutical Sciences and Food Engineering, Liaocheng University, Liaocheng 252000, China"},{"name":"Shandong Key Laboratory of Applied Technology for Protein and Peptide Drugs, Liaocheng 252000, China"},{"name":"State Key Laboratory of Macromolecular Drugs and Large-Scale Preparation, Liaocheng 252000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9195-7769","authenticated-orcid":false,"given":"Peng","family":"Song","sequence":"additional","affiliation":[{"name":"School of Pharmaceutical Sciences and Food Engineering, Liaocheng University, Liaocheng 252000, China"},{"name":"Shandong Key Laboratory of Applied Technology for Protein and Peptide Drugs, Liaocheng 252000, China"},{"name":"State Key Laboratory of Macromolecular Drugs and Large-Scale Preparation, Liaocheng 252000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,1]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"301","DOI":"10.1016\/0306-4492(75)90077-5","article-title":"La carcinine (\u03b2-alanyl-histamine), substance caracteristique du c\u0229ur de Carcinus maenas (L.): Synthese chimique et recherche d\u2019un role eventuel dans la regulation cardiaque du crustace","volume":"51","author":"Arnould","year":"1975","journal-title":"Comp. 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