Abstract

Chinese Hamster Ovary cells (CHO cells) are recombinant protein-producing cells widely used to produce biopharmaceutical products. Improving the recombinant protein production efficiency of CHO cells has been the subject of many studies over the last decade, due to its capacity to produce complex recombinant proteins of interest with human-like post-translational modifications. However, CHO cells are less efficient and more costly than bacteria and yeast-based expression systems. While many engineering methods have been previously described, the delicate balance between oxidative metabolism and glycolytic metabolism, which promotes protein production and cell proliferation respectively, remains a challenge. Recent sequencing studies performed on CHO cells mitochondria, the powerhouse of the cell, and the center of oxidative metabolism, have provided a deeper understanding of the mechanism underlying CHO cell metabolism. These studies have shown the existence of heteroplasmic mutations within CHO cell mitochondrial DNA, which have a potential impact on cell metabolism. In this study, the engineering of CHO cell metabolism was attempted by focusing the efforts on the mitochondria and oxidative metabolism, aiming to increase recombinant protein production by CHO cells. The potential influence of heteroplasmy variants previously found in CHO cells was evaluated to establish the future potential of creating ``de novo´´ heteroplasmic variants that will improve the recombinant protein production of the cell. The evolution of heteroplasmy over time in CHO cells was also assessed by performing deep sequencing on 12 different newly generated CHO clones at different time points. Additionally, genes closely related to mitochondrial metabolism, biosynthesis, and fission, including PGC1