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-marker systems have low saturable thresholds that limit enrichment efficiency, while multi-auxotrophic platforms often impose metabolic burdens. Here, we present a rationally designed tyrosine-auxotrophic system that overcomes these limitations by establishing a high-threshold cooperative selection mechanism. This is achieved through the reconstruction of an essential pathway comprising pterin-4α carbinolamine dehydratase 1 (PCBD1), phenylalanine hydroxylase (PAH), and quinoid dihydropteridine reductase (QDPR). We generated a triple-knockout CHO host via CRISPR/Cas9, wherein survival under tyrosine deprivation became strictly dependent on the balanced co-expression of all three rescue genes. This architecture creates a selection pressure that is not saturable by any single gene, enabling efficient co-enrichment. Applied to monoclonal antibody (mAb) production, the system enriched triple-positive populations to 97.49%, resulting in significantly enhanced homogeneity and coordinated upregulation of antibody chain expression. Optimized pools achieved titers of 0.35 g/L in fed-batch and 1.60 g/L in perfusion cultures without tyrosine feeding. Consequently, pathway reconstitution rewired central metabolism, reducing byproducts and enhancing biosynthesis. This antibiotic-free multi-marker platform establishes a new paradigm for stringent multigene co-expression, advancing CHO cell engineering for next-generation biologics.
Cao et al. (2026) studied this question.