title: 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.
authors.affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Jason T
authors.initials: JT
authors.lastname: Boock
authors.affiliation: Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Brian C
authors.initials: BC
authors.lastname: King
authors.affiliation: Department of Microbiology, Cornell University, Ithaca, NY 14853, USA.
authors.forename: May N
authors.initials: MN
authors.lastname: Taw
authors.affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Conrado
authors.affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Ka-Hei
authors.initials: KH
authors.lastname: Siu
authors.affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Jessica C
authors.initials: JC
authors.lastname: Stark
authors.affiliation: Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Larry P
authors.initials: LP
authors.lastname: Walker
authors.affiliation: 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.
authors.forename: Donna M
authors.initials: DM
authors.lastname: Gibson
authors.affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA. Electronic address: md255@cornell.edu.
authors.forename: Matthew P
authors.initials: MP
authors.lastname: DeLisa
mesh_terms: D001120:Arginine / Q000737:chemistry; D002480:Cellulase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins; D060585:Fusariosis / Q000378:metabolism / Q000382:microbiology; D005670:Fusarium / Q000201:enzymology* / Q000254:growth & development; D026901:Membrane Transport Proteins; D009154:Mutation / Q000235:genetics; D015202:Protein Engineering; D017510:Protein Folding; D055550:Protein Stability; D011786:Quality Control; D012995:Solubility
chemical_list: D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C408585:twin-arginine translocase complex, E coli; D001120:Arginine; C407024:endoglucanase Cel5A; D002480:Cellulase
keywords: cellulase; directed evolution; enzyme engineering; protein folding quality control; twin-arginine translocation