Functional expression of horseradish peroxidase in Saccharomyces cerevisiae and Pichia pastoris

The ability to engineer proteins by directed evolution requires functional expression of the target polypeptide in a recombinant host suitable for con struction and screening libraries of enzyme variants. Bacteria and yeast ar e preferred, but eukaryotic proteins often fail to express in active form i n these cells. We have attempted to resolve this problem by identifying mut ations in the target gene that facilitate its functional expression in a gi ven recombinant host. Here we examined expression of HRP in Saccharomyces c erevisiae, Through three rounds of directed evolution by random point mutag enesis and screening, we obtained a 40-fold increase in total HRP activity in the S. cerevisiae culture supernatant compared with wild-type, as measur ed on ABTS [2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)] (260 unit s/I/OD600). Genes from wild-type and two high-activity clones were expresse d in Pichia pastoris, where the total ABTS activity reached 600 units/I/OD6 00 in shake flasks. The mutants show up to 5.4-fold higher specific activit y towards ABTS and 2.3-fold higher specific activity towards guaiacol.