Reconstruction of arginine deiminase pathway sustains a higher-energy state in mammalian cells.

ATP is the universal "energy currency" of the cell, and its supply may represent one of several limiting factors influencing the productivity of mammalian cell factories. Here, we present a novel, mitochondria-independent approach to enhance cellular energy metabolism. We engineered Chinese hamster ovary (CHO) cells to express the bacterial arginine deiminase (ADI) pathway along with two arginine transporters. This system enables the direct, cytosolic conversion of arginine to ATP, effectively generating energy without relying solely on the cell's native metabolic machinery. ADI pathway expression was associated with intracellular ATP and concurrent improvements in culture performance across different CHO cell backgrounds. In contrast, cell lines engineered only for enhanced arginine uptake showed no performance gain, consistent with the hypothesis that de-novo ATP generation may contribute to improve productivity. Metabolic profiling revealed that the ADI pathway affects cellular metabolism. We observed a downshift in glycolysis, characterized by decreased glucose consumption and reduced lactate and alanine production, while amino acid and TCA cycle intermediaries remained broadly unchanged. Adenylate measurements and AMPK signalling analysis confirmed a higher energy state (ATP↑, ADP/ATP↓, p-AMPK/AMPK↓) in engineered cells. Supplementing the cell culture medium with arginine or citrulline was associated with further increases in growth and mAb titres in ADI-expressing cells. These results establish the ADI pathway as a powerful and distinct method for enhancing cellular energy. This mitochondria-independent approach highlights a new paradigm for improving the efficiency of industrial bioprocesses.