Versatile peroxidases (VP) are promiscuous biocatalysts with the highest fragility to hydroperoxides yet reported due to a complex molecular architecture, with three catalytic sites and several oxidation pathways. To improve the VP resistance to H₂O_2, an evolved version of this enzyme was subjected to a range of directed evolution and hybrid strategies in Saccharomyces cerevisiae. After five generations of random, saturation, and domain mutagenesis, together with in vivo DNA recombination, several structural determinants behind the oxidative destabilization of the enzyme were unmasked. To establish a balance between activity and stability, selected beneficial mutations were introduced into novel mutational environments by the in vivo exchange of sequence blocks, promoting epistatic interactions. The best variant of this process accumulated 8 mutations that increased the half-life of the protein from 3 (parental type) to 35 min in the presence of 3000 equiv of H₂O₂ and with a 6 °C upward shift in thermostability. Multiple structural alignment with other H₂O₂-tolerant heme peroxidases help to understand the possible roles played by the new mutations in the overall oxidative stabilization of these enzymes.

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