Chinese hamster ovary (CHO) cells serve as the predominant host for monoclonal antibody (mAb) production in biopharmaceutical manufacturing (Walsh and Walsh, 2022, O’Flaherty et al., 2020). In fed-batch cultures, where viable cell densities can exceed 30×106 cells/ml and product titers reach gram-per-liter levels, the escalating demand for essential nutrients necessitates precisely controlled feeding strategies to maintain process efficiency and product quality (Mulukutla et al., 2017, Yu et al., 2011).

High-density cell cultures typically rely on periodic supplementation with balanced, concentrated media formulations, requiring precise compositional adjustments and feeding schedules (Kavoni et al., 2025). Recent advances in metabolic pathway engineering now enable the revitalization of endogenous biosynthetic pathways in CHO cells, reducing dependence on exogenous nutrients. Synthetic genomics techniques have successfully reconstructed valine biosynthesis, creating CHO cell lines capable of valine-free cultivation (Trolle et al., 2022). Similar approaches with pyrroline-5-carboxylase synthetase (P5CS) and arginase overexpression have remodeled proline and polyamine pathways (Capella Roca et al., 2019, Sun et al., 2020, Budge et al., 2021). While previous studies primarily regarded pathway engineering as a tool for cell line selection to enhance growth and production (Capella Roca et al., 2019, Sun et al., 2020, Budge et al., 2021, Dumontet et al., 2023, Zhang et al., 2020a, Zhang et al., 2022, Cacciatore et al., 2010), its potential to reduce byproducts via metabolic reprogramming—specifically of tyrosine and glutamine metabolic pathways—has been demonstrated (Mulukutla et al., 2019, Noh et al., 2018, Ley et al., 2019). These findings collectively establish metabolic reprogramming of nutrient pathways as a promising alternative feeding strategy.

Tyrosine is in high demand for high-density cell cultures yet exhibits the lowest solubility among all amino acids in neutral media, presenting a major challenge for meeting its nutritional requirements (Carta and Tola, 1996, Tang et al., 2019, Zhang et al., 2020b). Current strategies to address tyrosine solubility include developing soluble derivatives and using alkaline feed solutions (Zimmer et al., 2014, Kang et al., 2012). While derivatives suffer from poor cost-effectiveness, alkaline feeds introduce operational complexity and pH fluctuations (Zimmer et al., 2014). Advances in sequencing have clarified the tetrahydrobiopterin (BH4)-dependent tyrosine biosynthesis pathway in mammalian cells. In this pathway, phenylalanine hydroxylase (PAH) catalyzes the hydroxylation phenylalanine to tyrosine, a reaction that strictly requires BH4 as an essential cofactor. To sustain PAH activity, the BH4 regeneration cycle — mediated by pterin-4α-carbinolamine dehydratase 1 (PCBD1) and quinoid dihydropteridine reductase (QDPR) — ensures continuous BH4 bioavailability (KEGG pathways: cge00400, cge00790) (Wang et al., 2013, Flydal and Martinez, 2013, Himmelreich et al., 2021, Eichwald et al., 2023, Nezhad et al., 2023). Despite this mechanistic clarity, CHO cells still face inherent limitations in endogenous tyrosine synthesis.

Previous studies have confirmed that the downregulated expression of pterin-4α-carbinolamine dehydratase 1 (PCBD1) and phenylalanine hydroxylase (PAH)—key enzymes for tyrosine synthesis in Chinese hamster ovary (CHO) cells—results in limited tyrosine biosynthetic capacity and exogenous tyrosine-dependent proliferation (Mulukutla et al., 2019, Cheng et al., 2024, Román et al., 2019). Furthermore, overexpressing PCBD1 and PAH can restore de novo tyrosine synthesis, and this property can be utilized to reduce the accumulation of phenylalanine and its inhibitory derivatives during cell culture (Mulukutla et al., 2019, Pereira et al., 2018); additionally, a PCBD1-based selection platform has been established, enabling antibiotic-free enrichment of PAH-overexpressing CHO cells (Cheng et al., 2024). However, two critical limitations persist in existing research. First, most studies focus exclusively on PCBD1/PAH expression regulation, without systematically investigating the role of other key pathway enzymes (e.g., QDPR) in the tyrosine synthesis pathway—this gap severely limits the development and industrialization of tyrosine-free culture strategies. Second, CHO cell clones engineered to overexpress PCBD1/PAH show poor adaptability in high-density cultures (Cheng et al., 2024), which hampers the translational potential of the tyrosine autotrophy-based selection system.

To address these limitations, this study first evaluated the growth and mAb production performance of recombinant CHO (rCHO) cell lines across passage, batch, and fed-batch cultures under varying tyrosine concentrations. Subsequently, the transcriptional and protein expression levels of tyrosine biosynthesis-related genes in rCHO cells were characterized. Based on these findings, tyrosine metabolic engineering was applied to enhance the tyrosine biosynthetic capacity of rCHO cells. Furthermore, the effects of key enzyme expression and substrate concentration on tyrosine biosynthesis in rCHO cells were investigated. This approach yields deeper insights into the differential tyrosine requirements across bioprocesses, laying the foundation for advancing tyrosine metabolic engineering in high-density cell cultures and facilitating the industrialization of tyrosine-free feeding strategies — an important step toward simplifying fed-batch operations and reducing biopharmaceutical production costs.