A Non-Conventional Epigenetic Engineering Strategy: The ALKBH5/ITGB5 Axis Enhances Recombinant Protein Production in CHO Cells through FAK-Mediated Proliferation and Improved Redox Status

Abstract Chinese hamster ovary (CHO) cells are the predominant host for producing complex recombinant therapeutic proteins. N6-methyladenosine (m6A) regulates recombinant protein production in CHO cells by influencing RNA stability and translation; however, the role of the m6A demethylase ALKBH5 remains poorly characterized in this context. Here, ALKBH5 overexpression increased the titers of three recombinant products by up to 2.87-fold, enhanced CHO cell proliferation, and reduced oxidative stress. Co-immunoprecipitation supported an association between ALKBH5 and ITGB5, and structure-guided mutagenesis identified Tyr205 of ITGB5 as functionally important for this association and ITGB5 protein stability. ALKBH5 overexpression did not measurably alter ITGB5 mRNA stability or m6A enrichment, supporting a mechanism that does not involve detectable m6A changes on the ITGB5 transcript, while not excluding m6A-dependent effects on other targets. FAK inhibition attenuated but did not abolish the production advantage, indicating that FAK signaling contributes to, but may not fully account for, the ALKBH5−ITGB5-associated phenotype. Metabolic and redox measurements further linked ALKBH5 overexpression to increased antioxidant capacity and ATP abundance, together with altered nutrient consumption and by-product formation. These findings identify ALKBH5 as a candidate host-cell engineering target for improving recombinant protein production and support further validation under industry-relevant fed-batch conditions.