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This study explores the potential of bacterially derived glutamine synthetases (GS) as a novel generation of metabolic selection markers to further enhance CHO cell culture performance. GS\u2010I proteins were extracted from the genomes of enterobacterial and actinomycetes species. Three of these enzymes demonstrated functionality when stably transfected into GS\u2010deficient CHO cells, leading to a 3\u2010 to 4\u2010fold increase in antibody titer compared to endogenous GS from <jats:italic>Cricetulus griseus<\/jats:italic>. This study indicates that the functionality of bacterial GS enzymes in mammalian cells is determined by solvent accessibility and the geometry of the catalytic binding pocket. Dysfunctional variants showed a less accessible bifunnel. Bacterial GS were evaluated for their bioprocess performance leading to superior stable pool and clone performance. Transcriptome analysis further revealed that regulatory cellular mechanisms were decoupled in a cross\u2010species set\u2010up, reinforcing the suitability of repurposing bacterial enzymes as selection markers in mammalian cell lines. By modulating the selection stringency, an increase in expression performance was achieved without impairing the bioprocess behavior or long\u2010term cell line stability.<\/jats:p>","DOI":"10.1002\/biot.70119","type":"journal-article","created":{"date-parts":[[2025,9,8]],"date-time":"2025-09-08T04:29:13Z","timestamp":1757305753000},"source":"Crossref","is-referenced-by-count":4,"title":["Bacterial Glutamine Synthetases as a Novel Metabolic Selection Marker to Improve CHO Cell Culture Performance Through Selection Stringency Modulation"],"prefix":"10.1002","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5431-1398","authenticated-orcid":false,"given":"Daniel","family":"Heinzelmann","sequence":"first","affiliation":[{"name":"Bioprocess Development Biologicals Cell Line Development Boehringer Ingelheim GmbH &amp; Co. 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