title: An N-terminal protein degradation tag enables robust selection of highly active enzymes.
abstract: Degradation tags are short peptide sequences that target proteins for destruction by housekeeping proteases. We previously utilized the C-terminal SsrA tag in directed evolution experiments to decrease the intracellular lifetime of a growth-limiting enzyme and thereby facilitate selection of highly active variants. In this study, we examine the N-terminal RepA tag as an alternative degradation signal for laboratory evolution. Although RepA proved to be less effective than SsrA at lowering protein concentrations in the cell, its N-terminal location dramatically reduced the occurrence of truncation and frameshift artifacts in selection experiments. We exploited this improvement to evolve a topologically redesigned chorismate mutase that is intrinsically disordered but already highly active for the conversion of chorismate to prephenate. After three rounds of mutagenesis and high-stringency selection, a robust and more nativelike variant was obtained that exhibited a catalytic efficiency (k(cat)/K(M) = 84000 M(-1) s(-1)) comparable to that of a natural dimeric chorismate mutase. Because of concomitant increases in catalyst yield, the level of intracellular prephenate production increased approximately 30-fold overall over the course of evolution.
authors.affiliation: Laboratory of Organic Chemistry, ETH Zurich, CH-8093 Zurich, Switzerland.
authors.forename: Maren
authors.initials: M
authors.lastname: Butz
authors.forename: Martin
authors.initials: M
authors.lastname: Neuenschwander
authors.forename: Peter
authors.initials: P
authors.lastname: Kast
authors.forename: Donald
authors.initials: D
authors.lastname: Hilvert
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D002826:Chorismate Mutase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003509:Cyclohexanecarboxylic Acids / Q000378:metabolism; D053138:Cyclohexenes / Q000378:metabolism; D019020:Directed Molecular Evolution / Q000379:methods*; D007700:Kinetics; D017015:Methanococcales / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering
chemical_list: D019843:Archaeal Proteins; D003509:Cyclohexanecarboxylic Acids; D053138:Cyclohexenes; D002826:Chorismate Mutase; C005550:prephenic acid