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ARS Home » Northeast Area » Ithaca, New York » Robert W. Holley Center for Agriculture & Health » Emerging Pests and Pathogens Research » Research » Publications at this Location » Publication #316255
Title: Repurposing a bacterial quality control mechanism to enhance enzyme production in living cells

Author
item BOOCK, JASON - Cornell University
item KING, BRIAN - Cornell University
item TAW, MAY - Cornell University
item CONRADO, ROBERT - Cornell University
item SIU, KA-HEI - Cornell University
item STARK, JESSICA - Cornell University
item WALKER, LARRY - Cornell University
item Gibson, Donna
item DELISA, MATTHEW - Cornell University

Submitted to: Journal of Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/2/2015
Publication Date: 2/1/2015
Citation: Boock, J.T., King, B.C., Taw, M.N., Conrado, R.J., Siu, K., Stark, J.C., Walker, L.P., Gibson, D.M., Delisa, M.P. 2015. Repurposing a bacterial quality control mechanism to enhance enzyme production in living cells. Journal of Molecular Biology. 427:1451-63.

Interpretive Summary: The quantity and selectivity of enzymes used for lignocellulosic digestion is a major barrier, creating the need for a select suite of enzymes with enhanced activity and functionality. Using a bacterium, such as Escherichia coli, would be ideal for producing large quantities of enzyme, but enzymes such as those needed for lignocellulose digestion, are typically nonfunctional due to incorrect folding. In this study, we developed a strategy for rapid molecular reengineering of a fungal enzyme, by use of a robust genetic selection strategy and high throughput screening for engineered enzyme evolution. We were able to demonstrate a 30 fold increase in production of a highly soluble functional form of the enzyme with only two amino acid substitutions without compromising enzyme catalytic efficiency. This selection strategy may be useful for other fungal enzymes where increased efficient production is needed.

Technical Abstract: 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.