Gelatin expression from an engineered Saccharomyces cerevisiae CUP1 promoter in Pichia pastoris

Abstract

The methylotrophic yeast Pichia pastoris (reclassified as Komagataella phaffii) is a versatile protein expression system, yet many commonly used promoters have attributes undesirable for fermentation or its optimization. Hence, the copper‐inducible CUP1 gene promoter from the related yeast Saccharomyces cerevisiae was used to express human gelatin. Multimerization of a potential copper response element in the CUP1 promoter, a S. cerevisiae Ace1p binding site, significantly increased gelatin expression. Expression was induced by copper in a dose‐dependent fashion and was not dependent on cell density. Gelatin was additionally induced in standard copper‐containing fermentation basal salts media. Removal of a S. cerevisiae heat shock factor (Hsf1p) binding site reduced copper‐dependent gelatin induction suggesting that a similar protein may regulate this promoter in P. pastoris. This engineered copper inducible promoter expands the yeast recombinant protein production tool kit.