(188i) Expanding the N-End Rule Pathway of Protein Degradation in Escherichia coli

Protein half-life is an important variable for biomolecular engineers to tune and control for a variety of protein containing products, such as biologics or engineered microbial factories. The N-end rule pathway of protein degradation dictates the half-life of proteins based on their amino-terminal residue. The natural N-end rule in Escherichia coli recognizes 6 out of the 20 standard amino acids as those that confer rapid destabilization of proteins if at their N-terminus. Conditional expansion of the spectrum of N-end destabilizing amino acids for targeted proteolysis would add to the toolkit for controlling genetic circuits and aid in achieving discrimination of non-standard amino acids. Here, we demonstrate successful hierarchical expansion of N-end destabilizing residues in E. coli primarily by expression of heterologous N-recognins. Under these conditions, several additional amino acids become destabilizing at the N-terminus, including isoleucine, valine, aspartate, glutamate, and cysteine (11 total). By nearly doubling the number of conditional substrates, this work increases the relevance of the N-end rule for engineering and accelerates fundamental characterization of heterologous N-recognins to eventually reveal preferences for positions downstream of the amino-terminal residue and protein degradation kinetics.