title: Directed evolution for thermostabilization of a hygromycin B phosphotransferase from Streptomyces hygroscopicus.
abstract: To obtain a selection marker gene functional in a thermophilic bacterium, Thermus thermophilus, an in vivo-directed evolutionary strategy was conducted on a hygromycin B phosphotransferase gene (hyg) from Streptomyces hygroscopicus. The expression of wild-type hyg in T. thermophilus provided hygromycin B (HygB) resistance up to 60 °C. Through selection of mutants showing HygB resistance at higher temperatures, eight amino acid substitutions and the duplication of three amino acids were identified. A variant containing seven substitutions and the duplication (HYG10) showed HygB resistance at a highest temperature of 74 °C. Biochemical and biophysical analyses of recombinant HYG and HYG10 revealed that HYG10 was in fact thermostabilized. Modeling of the three-dimensional structure of HYG10 suggests the possible roles of the various substitutions and the duplication on thermostabilization, of which three substitutions and the duplication located at the enzyme surface suggested that these mutations made the enzyme more hydrophilic and provided increased stability in aqueous solution.
authors.affiliation: Faculty of Life and Environmental Sciences, University of Tsukuba.
authors.forename: Naohisa
authors.initials: N
authors.lastname: Sugimoto
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Takakura
authors.forename: Kentaro
authors.initials: K
authors.lastname: Shiraki
authors.forename: Shinya
authors.initials: S
authors.lastname: Honda
authors.forename: Naoki
authors.initials: N
authors.lastname: Takaya
authors.forename: Takayuki
authors.initials: T
authors.lastname: Hoshino
authors.forename: Akira
authors.initials: A
authors.lastname: Nakamura
mesh_terms: D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019020:Directed Molecular Evolution / Q000379:methods*; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005819:Genetic Markers; D006358:Hot Temperature; D057927:Hydrophobic and Hydrophilic Interactions; D006921:Hygromycin B / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013302:Streptomyces / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D013816:Thermodynamics; D016963:Thermus thermophilus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D005819:Genetic Markers; D011994:Recombinant Proteins; D006921:Hygromycin B; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase