Stable and Promiscuous Galactose Oxidases Engineered by Directed Evolution, Atomistic Design, and Ancestral Sequence Reconstruction

Galactose oxidase (GOase) is a versatile biocatalyst with a wide range of potential applications, ranging from synthetic chemistry to bioelectrochemical devices. Previous GOase engineering by directed evolution generated the M-RQW mutant, with unprecedented new-to-nature oxidation activity at the C6-OH group of glucose, and a mutational backbone that helped to unlock its promiscuity toward other molecules, including secondary alcohols. In the current study, we have used the M-RQW mutant as a starting point to engineer a set of GOases that are very thermostable and that are easily produced at high titers in yeast, enzymes with latent activities applicable to sustainable chemistry. To boost the generation of sequence and functional diversity, the directed evolution workflow incorporated one-shot computational mutagenesis by the PROSS algorithm and ancestral sequence reconstruction. This synergetic approach helped produce a rapid rise in functional expression by <i>Pichia pastoris</i>, achieving g/L production in a fed-batch bioreactor while the different GOases designed were resistant to pH and high temperature, with <i>T</i>₅₀ enhancements up to 27 °C over the parental M-RQW. These designs displayed latent activity against glucose and an array of secondary aromatic alcohols with different degrees of bulkiness, becoming a suitable point of departure for the future engineering of industrial GOases.