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Methylotrophic yeasts such as  Komagataella phaffii  (syn.  Pichia pastoris ,  Pp ),  Hansenula polymorpha  ( Hp ),  Candida boidinii  ( Cb ) and  Pichia methanolica  ( Pm ) are widely used protein production platforms. Typically, strong, tightly regulated promoters of genes coding for their methanol utilization (MUT) pathways are used to drive heterologous gene expression. Despite highly similar open reading frames in the MUT pathways of the four yeasts, the regulation of the respective promoters varies strongly between species. While most endogenous  Pp  MUT promoters remain tightly repressed after depletion of a repressing carbon,  Hp ,  Cb  and  Pm  MUT promoters are derepressed to up to 70% of methanol induced levels, enabling methanol free production processes in their respective host background. Here, we have tested a series of orthologous promoters from  Hp ,  Cb  and  Pm  in  Pp . Unexpectedly, when induced with methanol, the promoter of the  HpMOX  gene reached very similar expression levels as the strong methanol, inducible, and most frequently used promoter of the  Pp alcohol oxidase 1  gene ( P   PpAOX1  ). The  HpFMD  promoter even surpassed  P   PpAOX1   up to three-fold, when induced with methanol, and reached under methanol-free/derepressed conditions similar expression as the methanol induced  P   PpAOX1  . These results demonstrate that orthologous promoters from related yeast species can give access to otherwise unobtainable regulatory profiles and may even considerably surpass endogenous promoters in  P. pastoris .

Orthologous promoters from related methylotrophic yeasts surpass expression of endogenous promoters of Pichia pastoris