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

Thomas Vogl,Anton Glieder,Jasmin Elgin Fischer,Richard Wasmayer,Lukas Sturmberger,Patrick Hyden

doi:10.1186/s13568-020-00972-1

Abstract

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 (PPpAOX1). The HpFMD promoter even surpassed PPpAOX1 up to three-fold, when induced with methanol, and reached under methanol-free/derepressed conditions similar expression as the methanol induced PPpAOX1. 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.

Highlights

Recombinant proteins such as biopharmaceuticals or industrially relevant biocatalysts are commonly produced by heterologous gene expression in microorganisms
These promoters have been reported to be amongst the strongest methanol inducible promoters and at the same time the most derepressed promoters in the respective organisms [reviewed by (Hartner and Glieder 2006)]. These promoters were compared to state of the art endogenous promoters which were so far most frequently used in P. pastoris, i.e. the methanol inducible PAOX1, constitutive PGAP, and derepressed/methanol inducible PCAT1 (Vogl et al 2016) (Table 1)
DNA sequencing showed that the promoter sequences contained minor differences compared to previous reports (Additional file 1: S2). These differences are possibly arising from the use of genomic DNA from Hansenula polymorpha (Hp), Candida boidinii (Cb) and Pichia methanolica (Pm) strains from different strain collections than previously reported as PCR templates

Summary

Introduction

Recombinant proteins such as biopharmaceuticals or industrially relevant biocatalysts are commonly produced by heterologous gene expression in microorganisms. The promoter of the alcohol oxidase 1 gene in P. pastoris (PPpAOX1) is only activated at 2–4% compared to methanol induced levels (Vogl and Glieder 2013). The promoter of the orthologous gene (named differently: methanol oxidase, MOX) in H. polymorpha (PHpMOX) shows derepressed expression up to 70% of methanol induced levels, even in presence of glycerol whereas PPpAOX1 is fully repressed by glycerol. The promoters of the orthologous genes in C. boidinii (alcohol oxidase 1, abbreviated AOD1) and P. methanolica (methanol oxidase 1/2, abbreviated MOD1/2) were reported to be activated by derepression, reaching up to 70% of methanol induced levels (Hartner and Glieder 2006). We are keeping these identifiers in addition to the prefixes Pp, Hp, Cb and Pm to differentiate between the organisms

Methods

Results

Conclusion