{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,17]],"date-time":"2026-06-17T23:30:43Z","timestamp":1781739043313,"version":"3.54.5"},"reference-count":49,"publisher":"Wiley","issue":"6","license":[{"start":{"date-parts":[[2026,3,9]],"date-time":"2026-03-09T00:00:00Z","timestamp":1773014400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2026,3,9]],"date-time":"2026-03-09T00:00:00Z","timestamp":1773014400000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["analyticalsciencejournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Biotech &amp; Bioengineering"],"published-print":{"date-parts":[[2026,6]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>The production of complex biologics in Chinese hamster ovary (CHO) cells is constrained by the lack of selection systems capable of coordinating multiple transgenes. Conventional single\u2010marker systems have low saturable thresholds that limit enrichment efficiency, while multi\u2010auxotrophic platforms often impose metabolic burdens. Here, we present a rationally designed tyrosine\u2010auxotrophic system that overcomes these limitations by establishing a high\u2010threshold cooperative selection mechanism. This is achieved through the reconstruction of an essential pathway comprising pterin\u20104\u03b1 carbinolamine dehydratase 1 (PCBD1), phenylalanine hydroxylase (PAH), and quinoid dihydropteridine reductase (QDPR). We generated a triple\u2010knockout CHO host via CRISPR\/Cas9, wherein survival under tyrosine deprivation became strictly dependent on the balanced co\u2010expression of all three rescue genes. This architecture creates a selection pressure that is not saturable by any single gene, enabling efficient co\u2010enrichment. Applied to monoclonal antibody (mAb) production, the system enriched triple\u2010positive populations to 97.49%, resulting in significantly enhanced homogeneity and coordinated upregulation of antibody chain expression. Optimized pools achieved titers of 0.35\u2009g\/L in fed\u2010batch and 1.60\u2009g\/L in perfusion cultures without tyrosine feeding. Consequently, pathway reconstitution rewired central metabolism, reducing byproducts and enhancing biosynthesis. This antibiotic\u2010free multi\u2010marker platform establishes a new paradigm for stringent multigene co\u2010expression, advancing CHO cell engineering for next\u2010generation biologics.<\/jats:p>","DOI":"10.1002\/bit.70185","type":"journal-article","created":{"date-parts":[[2026,3,10]],"date-time":"2026-03-10T03:14:32Z","timestamp":1773112472000},"page":"1503-1517","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["The Innovative Multi\u2010Marker Selection System Based on Tyrosine Synthesis Pathway for Monoclonal Antibody Expression in CHO Cells"],"prefix":"10.1002","volume":"123","author":[{"given":"Lei","family":"Cao","sequence":"first","affiliation":[{"name":"State Key Laboratory of Bioreactor Engineering East China University of Science and Technology Shanghai China"},{"name":"Shanghai Collaborative Innovation Center for Biomanufacturing Technology (SCIBT) Shanghai China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daoyuan","family":"Na","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Bioreactor Engineering East China University of Science and Technology Shanghai China"},{"name":"Shanghai Collaborative Innovation Center for Biomanufacturing Technology (SCIBT) Shanghai China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jun","family":"Cheng","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Bioreactor Engineering East China University of Science and Technology Shanghai China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liang","family":"Zhao","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Bioreactor Engineering East China University of Science and Technology Shanghai China"},{"name":"Shanghai Collaborative Innovation Center for Biomanufacturing Technology (SCIBT) Shanghai China"},{"name":"Shanghai BioEngine Sci\u2010Tech Co., LTD Shanghai P. 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