Engineering of Promoters for Gene Expression in Pichia pastoris

The availability of exceptionally strong and tightly regulated promoters is a key feature of Komagataella phaffii (syn. Pichia pastoris), a widely applied yeast expression system for heterologous protein production. Most commonly, the methanol-inducible promoter of the alcohol oxidase 1 gene (P_AOX1) and the constitutive promoter of the glyceraldehyde 3 phosphate dehydrogenase gene (P_GAP) have been used. Recently, also promising novel constitutive (P_GCW14), regulated (P_GTH1, P_CAT1), and bidirectional promoters (histone promoters and synthetic hybrid variants) have been reported.As natural promoters showed so far limited tunability of expression levels and regulatory profiles, various promoter engineering efforts have been undertaken for P. pastoris . P_AOX1, P_DAS2, P_GAP, and P_GCW14 have been engineered by systematic deletion studies or random mutagenesis of upstream regulatory sequences. New engineering strategies have focused on P_AOX1 core promoter modifications by random or rational approaches and transcriptional regulatory circuits to render P_AOX1 independent of methanol induction. These promoter engineering efforts in P. pastoris have resulted in improved, sequence-diversified synthetic promoter variants allowing coordinated fine-tuning of gene expression for a multitude of biotechnological applications.