ULK1 knockout increases recombinant transcript abundance and improves recombinant membrane protein expression.

Abstract

Cell homeostasis depends on pathways balancing degradation and synthesis, including autophagy. Following a previous small-molecule screen, suggesting unc-51-like kinase 1 (ULK1) as a target affecting recombinant protein expression, we generated an ULK1 knockout (KO) Expi293 cell line. The KO cell line increased the expression of a secreted model protein by up to 1.7-fold, and transcriptomic analysis suggested downregulation of key macroautophagy-associated genes despite upregulated productivity-associated stress genes. Transcriptional upregulation of membrane organization and protein localization processes indicated a potential for membrane protein production. Production of three model membrane proteins in the KO cell line yielded up to a 2-fold increase, with the highest increase in the soluble fraction. Transcriptomic analyses suggested an increased recombinant transcript abundance, broader downregulation of autophagy, and weaker stress responses. These findings indicate that autophagy-associated pathways modulate cellular responses to recombinant protein production and point to ULK1-associated pathways for host-cell engineering, especially for enhanced membrane protein production.

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