Generation of thermostable enzyme genes using spontaneous mutations in thermophile Geobacillus kaustophilus HTA426

摘要

While enzymes catalysts have been widely used in industry fields, many enzymes are not practical in spite of their catalytic activities that meet industry demand. A major reason for this is enzyme instability. Because enzyme stability can be commonly improved by enhancing enzyme thermostability, techniques to confer thermostability on enzymes could expand opportunities for enzyme utilization as industrial catalysts. Geobacillus kaustophilus HTA426 is an aerobic, high growth, Gram-positive thermophile that grows between 42 °C and 74 °C. In a previous study, we found that strain HTA426 exhibits frequent spontaneous mutations under certain conditions. In this study, we constructed two G. kaustophilus strains, MK242 and MK480. Strain MK242 lacked pyrF gene involved in uracil biosynthesis and thus was auxotrophic for uracil. Strain MK480 was constructed from strain MK242 by deleting five genes for DNA repair. Mutation assay confirmed frequent and various mutations in strain MK480. Two mesophile genes, Bacillus subtilis pyrF (BSpyrF) and Streptomyces azureus tsrR (SAtsrR) encoding thiostrepton resistance protein, were introduced in strain MK480 and propagated under uracil-starving and thiostrepton-containing conditions, respectively. After successive cultivations, the gene sequences in grown cells were analyzed to obtain two mutant genes, BSpyrFel (with C497T mutation) and SAtsrRel (with C772T mutation). BSpyrFe1 conferred prototrophy for uracil on strain MK242 at 70 °C, whereas BSpyrF did not. Although SAtsrR was not functional over 50 °C, SAtsrRe1 conferred thiostrepton resistivity on strain MK242 even at 55 °C. These results suggest that spontaneous mutation in G. kaustophilus MK480 provides a new approach to generate thermostable enzymes.