CHO endogenous weak promoters facilitating the development of high-yield cell lines.

Efficient and stable transgene expression in Chinese hamster ovary (CHO) cells remains a critical challenge for biotherapeutic protein production. To advance the exploration and application of endogenous promoters in CHO cell line development (CLD), we established a comprehensive library of CHO-derived promoters with a wide range of expression strengths, spanning 0.5-629.3% relative to the SV40 promoter. This library enabled precise regulation of selection marker gene expression, revealing a strong negative correlation between marker promoter activity and target GFP expression (r = -0.93, P < 0.001). Ultra-weak promoters effectively eliminated low-copy-number cells during early selection, enriching high-expression phenotypes. Combining the four most effective promoters with either UCOE or the PiggyBac (PB) transposon system further enhanced transgene expression stability and productivity. Notably, cell lines engineered with the Rps3 UCOE-2P-6 and PB-P28 designs achieved maximum antibody titers of 1.91 g/L and 4.69 g/L, and specific productivities of 22.23 pg/cell/day and 37.75 pg/cell/day, respectively, in 14-day fed-batch cultures, while maintaining high productivity over 60 days of continuous passaging. Collectively, this study presents a scalable and robust strategy for constructing high-yield, stable mammalian expression platforms, providing valuable insights for next-generation biotherapeutic manufacturing.