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In-vivo directed evolution of galactose oxidase by stationary phase adaptive mutations and phylogenetic analysis of error-prone polymerases

Öreroğlu, Ayla
In this study, the novel idea of in-vivo directed evolution was applied in order to achieve variants of the enzyme galactose oxidase with increased activity. This procedure was done under starvation conditions in Escherichia coli BL21 Star (DE3). Previous studies have been carried out in order to improve the activity of this enzyme using directed evolution methods. In this study, the same idea was used in-vivo, during stationary phase adaptive mutations inside the host organism, hence called in-vivo directed evolution. This method gave variants with improved enzyme activity as compared with the wild-type enzyme, and some variants showed activities that were even higher than the variants of previous directed evolution studies, hence making this method a promising approach for the random mutagenesis of genes of interest. The above mentioned mutations are carried out by a special group of polymerases, the error-prone polymerases. Phylogenetic analysis of these error-prone polymerases was also carried out in order to investigate the relationship between the number of error-prone polymerases and the level of complexity of organisms, and both the number of error-prone polymerases and the ratio of error-prone polymerases to total DNA polymerases of six organisms were studied. It was found that as the organism gets more complex, the number of error-prone polymerases and their ratio to the total polymerases increase.