A novel random mutagenesis approach using human mutagenic DNA polymerases to generate enzyme variant libraries

Abstract

The in vitro MutaGen™ procedure is a new random mutagenesis method based on the use of low-fidelity DNA polymerases. In the present study, this technique was applied on a 2 kb gene encoding amylosucrase, an attractive enzyme for the industrial synthesis of amylose-like polymers. Mutations were first introduced during a single replicating step performed by mutagenic polymerases pol β and pol η. Three large libraries (>105 independent clones) were generated (one with pol β and two with pol η). The sequence analysis of randomly chosen clones confirmed the potential of this strategy for the generation of diversity. Variants generated by pol β were 4-7-fold less mutated than those created with pol η, indicating that our approach enables mutation rate control following the DNA polymerase employed for mutagenesis. Moreover, pol β and pol η provide different and complementary mutation spectra, allowing a wider sequence space exploration than error-prone PCR protocols employing Taq polymerase. Interestingly, some of the variants generated by pol η displayed unusual modifications, including combinations of base substitutions and codon deletions which are rarely generated using other methods. By taking advantage of the mutation bias of naturally highly error-prone DNA polymerases, MutaGen™ thus appears as a very useful tool for gene and protein randomisation. © The Author 2008. Published by Oxford University Press. All rights reserved.