Beyond tyrosine feeding: A novel fed-batch cultivation strategy based on tyrosine metabolic engineering in recombinant CHO cells.

CHO cells dominate monoclonal antibody (mAb) production in fed-batch biomanufacturing, where tyrosine supply is limited by low solubility in neutral media and the complexities of alkaline tyrosine feeds. Existing studies confirm overexpressing pterin-4α-carbinolamine dehydratase 1 (PCBD1) and phenylalanine hydroxylase (PAH) restores tyrosine prototrophy and matches non-engineered cells' production in tyrosine-supplemented cultures. However, these studies focus on enzyme regulation without resolving feeding-phase supply challenges in high-density scenarios. To address this, this study verified that recombinant CHO (rCHO) cells rely strongly on exogenous tyrosine for growth and production. Multi-level expression analysis further confirmed low PCBD1/PAH levels restrict endogenous tyrosine synthesis, and identified quinoid dihydropteridine reductase (QDPR)-a key tetrahydrobiopterin (BH4) regeneration enzyme-as a previously unrecognized bottleneck, particularly in tyrosine-limited conditions. By co-overexpressing QDPR in high PCBD1/PAHexpressing cells to remodel the tyrosine biosynthesis pathway, a novel fed-batch strategy was established: basal medium with 3.0 mM tyrosine and tyrosine-free singlefeeding medium. Results showed this strategy, effective in high-density fed-batch settings, enabled rCHO cells to reach a final mAb titer of 4.24 g/L, representing a 32.50% increase compared to cells overexpressing only PCBD1 and PAH, and a 10.70 % increase compared to the conventional strategy. In summary, the strategy offers a simplified nutrient alternative by eliminating alkaline tyrosine feeds, highlighting holistic metabolic pathway optimization's importance in biomanufacturing and targeted value for high-density, tyrosine-limited CHO cell-based mAb production.