title: Thermoadaptation-directed enzyme evolution in an error-prone thermophile derived from Geobacillus kaustophilus HTA426.
abstract: Thermostability is an important property of enzymes utilized for practical applications because it allows long-term storage and use as catalysts. In this study, we constructed an error-prone strain of the thermophile Geobacillus kaustophilus HTA426 and investigated thermoadaptation-directed enzyme evolution using the strain. A mutation frequency assay using the antibiotics rifampin and streptomycin revealed that G. kaustophilus had substantially higher mutability than Escherichia coli and Bacillus subtilis. The predominant mutations in G. kaustophilus were A · T→G · C and C · G→T · A transitions, implying that the high mutability of G. kaustophilus was attributable in part to high-temperature-associated DNA damage during growth. Among the genes that may be involved in DNA repair in G. kaustophilus, deletions of the mutSL, mutY, ung, and mfd genes markedly enhanced mutability. These genes were subsequently deleted to construct an error-prone thermophile that showed much higher (700- to 9,000-fold) mutability than the parent strain. The error-prone strain was auxotrophic for uracil owing to the fact that the strain was deficient in the intrinsic pyrF gene. Although the strain harboring Bacillus subtilis pyrF was also essentially auxotrophic, cells became prototrophic after 2 days of culture under uracil starvation, generating B. subtilis PyrF variants with an enhanced half-denaturation temperature of >10°C. These data suggest that this error-prone strain is a promising host for thermoadaptation-directed evolution to generate thermostable variants from thermolabile enzymes.
authors.affiliation: Functional Genomics of Extremophiles, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, Tottori, Japan hirokap@xpost.plala.or.jp.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Suzuki
authors.affiliation: Functional Genomics of Extremophiles, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, Tottori, Japan.
authors.forename: Jyumpei
authors.initials: J
authors.lastname: Kobayashi
authors.affiliation: Applied Molecular Microbiology and Biomass Chemistry, Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Wada
authors.affiliation: Functional Genomics of Extremophiles, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan.
authors.forename: Megumi
authors.initials: M
authors.lastname: Furukawa
authors.affiliation: Microbial Genetic Division, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Fukuoka, Japan.
authors.forename: Katsumi
authors.initials: K
authors.lastname: Doi
mesh_terms: D045643:DNA Repair Enzymes / Q000235:genetics; D004795:Enzyme Stability / Q000528:radiation effects; D004798:Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D005827:Genetics, Microbial; D056506:Geobacillus / Q000201:enzymology* / Q000235:genetics / Q000528:radiation effects*; D008967:Molecular Biology; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D059645:Mutation Rate; D055550:Protein Stability / Q000528:radiation effects; D012641:Selection, Genetic; D013696:Temperature
chemical_list: D004798:Enzymes; D050505:Mutant Proteins; D045643:DNA Repair Enzymes