Mutagenic Organized Recombination Process by Homologous In Vivo Grouping (MORPHING) for Directed Enzyme Evolution

<div><p>Approaches that depend on directed evolution require reliable methods to generate DNA diversity so that mutant libraries can focus on specific target regions. We took advantage of the high frequency of homologous DNA recombination in <i>Saccharomyces cerevisiae</i> to develop a strategy for domain mutagenesis aimed at introducing and <i>in vivo</i> recombining random mutations in defined segments of DNA. <i><u>M</u></i>utagenic <i><u>O</u></i>rganized <i><u>R</u></i>ecombination <i><u>P</u></i>rocess by <i><u>H</u></i>omologous <i><u>IN</u></i> vivo <i><u>G</u></i>rouping (MORPHING) is a <i>one-pot</i> random mutagenic method for short protein regions that harnesses the <i>in vivo</i> recombination apparatus of yeast. Using this approach, libraries can be prepared with different mutational loads in DNA segments of less than 30 amino acids so that they can be assembled into the remaining unaltered DNA regions <i>in vivo</i> with high fidelity. As a proof of concept, we present two eukaryotic-ligninolytic enzyme case studies: i) the enhancement of the oxidative stability of a H₂O₂-sensitive versatile peroxidase by independent evolution of three distinct protein segments (Leu28-Gly57, Leu149-Ala174 and Ile199-Leu268); and ii) the heterologous functional expression of an unspecific peroxygenase by exclusive evolution of its native 43-residue signal sequence.</p>