Thermoadaptation-directed enzyme evolution in an error-prone thermophile derived from Geobacillus kaustophilus HTA426

Hirokazu Suzuki, Jyumpei Kobayashi, Keisuke Wada, Megumi Furukawa, Katsumi Doi

研究成果: ジャーナルへの寄稿記事

9 引用 (Scopus)

抄録

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. kaustophiluswere 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.

元の言語英語
ページ(範囲)149-158
ページ数10
ジャーナルApplied and environmental microbiology
81
発行部数1
DOI
出版物ステータス出版済み - 1 1 2015

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Bacillus kaustophilus
thermophilic microorganisms
Geobacillus
Bacillus subtilis
enzyme
Uracil
gene
Enzymes
enzymes
Genes
mutation
uracil
DNA
Temperature
Streptomycin
Mutation Rate
Rifampin
Starvation
starvation
DNA Repair

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Food Science
  • Applied Microbiology and Biotechnology
  • Ecology

これを引用

Thermoadaptation-directed enzyme evolution in an error-prone thermophile derived from Geobacillus kaustophilus HTA426. / Suzuki, Hirokazu; Kobayashi, Jyumpei; Wada, Keisuke; Furukawa, Megumi; Doi, Katsumi.

:: Applied and environmental microbiology, 巻 81, 番号 1, 01.01.2015, p. 149-158.

研究成果: ジャーナルへの寄稿記事

Suzuki, Hirokazu ; Kobayashi, Jyumpei ; Wada, Keisuke ; Furukawa, Megumi ; Doi, Katsumi. / Thermoadaptation-directed enzyme evolution in an error-prone thermophile derived from Geobacillus kaustophilus HTA426. :: Applied and environmental microbiology. 2015 ; 巻 81, 番号 1. pp. 149-158.
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