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

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