Suzuki Hirokazu, Kobayashi Jyumpei, Wada Keisuke, Furukawa Megumi, Doi Katsumi
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
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.
Appl Environ Microbiol. 2015 Jan;81(1):149-58. doi: 10.1128/AEM.02577-14. Epub 2014 Oct 17.
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.
热稳定性是用于实际应用的酶的一项重要特性，因为它允许酶长期储存并用作催化剂。在本研究中，我们构建了嗜热栖热放线菌HTA426的易错菌株，并使用该菌株研究了热适应性导向的酶进化。使用抗生素利福平和链霉素进行的突变频率测定表明，嗜热栖热放线菌的突变率明显高于大肠杆菌和枯草芽孢杆菌。嗜热栖热放线菌中的主要突变是A·T→G·C和C·G→T·A转换，这意味着嗜热栖热放线菌的高突变率部分归因于生长过程中与高温相关的DNA损伤。在可能参与嗜热栖热放线菌DNA修复的基因中，mutSL、mutY、ung和mfd基因的缺失显著提高了突变率。随后删除这些基因以构建一个易错嗜热菌，该菌株的突变率比亲本菌株高得多（700至9000倍）。由于该菌株缺乏内在的pyrF基因，易错菌株对尿嘧啶营养缺陷。尽管携带枯草芽孢杆菌pyrF的菌株也基本营养缺陷，但在尿嘧啶饥饿条件下培养2天后细胞变为原养型，产生了半变性温度提高>10°C的枯草芽孢杆菌PyrF变体。这些数据表明，这种易错菌株是热适应性导向进化的一个有前途的宿主，可从热不稳定酶中产生热稳定变体。
