Engineering of small sized DNAs by error-prone multiply-primed rolling circle amplification for introduction of random point mutations

Small sized DNAs per se or their encoding peptides play various roles in biological systems and for biocatalyst development thus, engineering of those small sized DNAs/peptides is of great interest. By self-ligation of small sized DNAs, circular small sized DNA templates were prepared for error-prone rolling circle amplification using multiply-primed random hexamers to create tandem repeats of small sized DNAs and simultaneous introduction of random point mutations into those tandem repeats of small sized DNAs. We applied this method to randomize the signal peptide of a glucoamylase in recombinant Saccharomyces cerevisiae. Random point mutations were efficiently introduced into small sized DNA encoding the signal peptide of glucoamylase and the resulting recombinant S. cerevisiae with beneficial point mutations in its signal peptide was able to secrete ca. 30% more glucoamylase than that with native signal peptide.