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Repurposing a bacterial quality control mechanism to enhance enzyme production in living cells.

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.

SUBMITTER: Boock JT

PROVIDER: S-EPMC4576832 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Repurposing a bacterial quality control mechanism to enhance enzyme production in living cells.

Boock Jason T JT King Brian C BC Taw May N MN Conrado Robert J RJ Siu Ka-Hei KH Stark Jessica C JC Walker Larry P LP Gibson Donna M DM DeLisa Matthew P MP

Journal of molecular biology 20150112 6 Pt B

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 ...[more]

PMID: 25591491

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Repurposing a bacterial quality control mechanism to enhance enzyme production in living cells.

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Repurposing a bacterial quality control mechanism to enhance enzyme production in living cells.

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