CHO genome mining for synthetic promoter design

Area of Science:

• Synthetic biology
• Mammalian cell engineering
• Biotechnology

Background:

• Synthetic promoters offer customizable functionalities for mammalian hosts like CHO cells.
• Designing synthetic promoters requires integrating host genomic information and bioprocess needs.

Purpose of the Study:

• To develop and validate a bioprocess-directed method for designing synthetic promoters using CHO genomic data.
• To create synthetic promoters with constitutive high-level transcription and upregulation under specific bioprocess conditions (hypothermia, late-stage culture).

Main Methods:

• Analyzed CHO gene expression (RNA-Seq) and mRNA half-life to select genes with varying transcriptional activity.
• Identified and screened transcription factor regulatory elements (TFREs) for high activity in biphasic cultures.
• Designed synthetic promoters in silico using TFRE combinations and validated them via transient transfection and stable cell line development.

Main Results:

• Synthetic promoters achieved up to a 2.5-fold increase in transcriptional strength over CMV-IE promoter in transient transfections.
• Stable transfectant pools using synthetic promoters showed up to 1.6-fold increased reporter production compared to CMV promoter pools.
• Enhanced productivity was maintained in fed-batch cultures, even with mild hypothermia during stationary phase.

Conclusions:

• Bioprocess context and host cell characteristics are crucial for de novo design of synthetic genetic parts.
• The developed method enables the creation of synthetic promoters tailored for improved recombinant protein production in mammalian cell engineering.
• Synthetic promoters represent a promising tool for optimizing biomanufacturing processes in CHO cells.