Thermoadaptation-Directed Enzyme Evolution in an Error-Prone Thermophile Derived from Geobacillus kaustophilus HTA426

Abstract

ABSTRACTThermostability 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 thermophileGeobacillus kaustophilusHTA426 and investigated thermoadaptation-directed enzyme evolution using the strain. A mutation frequency assay using the antibiotics rifampin and streptomycin revealed thatG. kaustophilushad substantially higher mutability thanEscherichia coliandBacillus subtilis. The predominant mutations inG. kaustophiluswere A · T→G · C and C · G→T · A transitions, implying that the high mutability ofG. kaustophiluswas attributable in part to high-temperature-associated DNA damage during growth. Among the genes that may be involved in DNA repair inG. kaustophilus, deletions of themutSL,mutY,ung, andmfdgenes 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 intrinsicpyrFgene. Although the strain harboringBacillus subtilispyrFwas also essentially auxotrophic, cells became prototrophic after 2 days of culture under uracil starvation, generatingB. subtilisPyrF 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.