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

• Published in: Yeast (Chichester, England) Vol. 38; no. 6; pp. 382 - 387
• Main Authors: Williams, Kim E., Olsen, David R.
• Format: Journal Article
• Language: English
• Published: England Wiley Subscription Services, Inc 01.06.2021
• Subjects: Binding sites; Cell density; Copper; CUP1 gene; Fermentation; Gelatin; Heat shock factors; Pichia; Pichia pastoris; Proteins; response elements; Saccharomyces cerevisiae; Salts; Yeast; binding sites; copper; response elements; Saccharomyces cerevisiae; gelatin; Pichia
• ISSN: 0749-503X; 1097-0061
• DOI: 10.1002/yea.3554

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. Take Away Multimerization of the Ace1p binding site in the of S. cerevisiae CUP1 promoter in a human gelatin expression vector significantly increased copper‐dependent expression in P. pastoris. This engineered promoter has desirable attributes for fermentation as copper dose‐dependently induced gelatin expression at a range of cell densities in standard fermentation media. The P. pastoris homolog of S. cerevisiae heat shock factor (Hsf1p) may regulate this promoter as removal of the Hsf1p binding site reduced the induction of gelatin in a copper‐dependent fashion in P. pastoris.