Repurposing a Bacterial Quality Control Mechanism to Enhance Enzyme Production in Living Cells

作者：Boock Jason T; King Brian C; Taw May N; Conrado Robert J; Siu Ka Hei; Stark Jessica C; Walker Larry P; Gibson Donna M; DeLisa Matthew P^*

来源：Journal of Molecular Biology, 2015, 427(6): 1451-1463.

DOI：10.1016/j.jmb.2015.01.003

• directed evolution
• cellulase
• enzyme engineering
• protein folding quality control
• twin-arginine translocation

Heterologous expression of many proteins in bacteria, yeasts, and plants is often limited by low titers of functional protein. To address this problem, we have created a two-tiered directed evolution strategy in Escherichia coli that enables optimization of protein production while maintaining high biological activity. The first tier involves a genetic selection for intracellular protein stability that is based on the folding quality control mechanism inherent to the twin-arginine translocation pathway, while the second is a semi-high-throughput screen for protein function. To demonstrate the utility of this strategy, we isolated variants of the endoglucanase Cel5A, from the plant-pathogenic fungus Fusarium graminearum, whose production was increased by as much as 30-fold over the parental enzyme. This gain in production was attributed to just two amino acid substitutions, and it was isolated after two iterations through the two-tiered approach. There was no significant tradeoff in activity on soluble or insoluble cellulose substrates. Importantly, by combining the folding filter afforded by the twin-arginine translocation quality control mechanism with a function-based screen, we show enrichment for variants with increased protein abundance in a manner that does not compromise catalytic activity, providing a highly soluble parent for engineering of improved or new function.

• 出版日期2015-3-27