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J Mol Biol. 2015 Mar 27;427(6 Pt B):1451-1463. doi: 10.1016/j.jmb.2015.01.003. Epub 2015 Jan 12.

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

Author information

1
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA.
2
Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca, NY 14853, USA.
3
Department of Microbiology, Cornell University, Ithaca, NY 14853, USA.
4
Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853, USA.
5
Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca, NY 14853, USA; Agricultural Research Service, Robert W. Holley Center for Agriculture and Health, United States Department of Agriculture, Ithaca, NY 14853, USA.
6
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA. Electronic address: md255@cornell.edu.

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.

KEYWORDS:

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

PMID:
25591491
PMCID:
PMC4576832
DOI:
10.1016/j.jmb.2015.01.003
[Indexed for MEDLINE]
Free PMC Article

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