Genome Amplification and Altered Transcriptome Aid in Survival and Enhanced Protein Secretion in Tunicamycin‐Resistant CHOK1 Cells

Chinese Hamster Ovary (CHO) cells are the predominant host for the production of biotherapeutics; however, there remains considerable potential to further enhance their cellular productivity. Adaptive laboratory evolution (ALE) combined with omics-based analysis has emerged as a promising approach toward generating host cell lines with desirable characteristics. In this study, CHOK1 cells were gradually adapted to tunicamycin (TM), an endoplasmic reticulum (ER) stressor, resulting in an 8-fold increase in resistance compared to the non-adapted cells with a marked enlargement of the ER. Notably, the per-cell secretion rate of total protein was 3- to 4-fold higher in the TM-adapted cells. Transcriptomic analysis revealed upregulation of several genes in the protein processing pathway, such as Dpagt1, the TM target gene, and ER stress response genes. The protein transport, secretion and ubiquitination pathways were also altered, potentially contributing to the increased protein secretion. Furthermore, genes participating in signaling cascades of PI3K-AKT, MAPK, and Ras pathways were differentially expressed, thereby aiding in its survival and proliferation. Whole genome sequencing confirmed the amplification of a large genome segment of chromosome 4, which included several genes upregulated at the mRNA level, including Dpagt1. Thus, the survival and increased protein secretion of TM-adapted cells can be attributed to a combination of transcriptional level changes and amplification of a large genome segment. Further, transient expression of a recombinant protein, SEAP, in TM-adapted cells showed an improvement in specific productivity of ∼1.4 fold as compared to the non-adapted cells, underscoring the importance of ALE as a cell engineering strategy.