Repressible promoters – A novel tool to generate conditional mutants in Pichia pastoris

Marizela Delic,Diethard Mattanovich,Brigitte Gasser

doi:10.1186/1475-2859-12-6

Abstract

BackgroundRepressible promoters are a useful tool for down-regulating the expression of genes, especially those that affect cell viability, in order to study cell physiology. They are also popular in biotechnological processes, like heterologous protein production.ResultsHere we present five novel repressible Pichia pastoris promoters of different strength: PSER1, PMET3, PTHR1, PPIS1 and PTHI11. eGFP was expressed under the control of each of these promoters and its fluorescence could be successfully decreased in liquid culture by adding different supplements. We also expressed the essential genes with different native promoter strength, ERO1 and PDI1, under the control of two of the novel promoters. In our experiments, a clear down-regulation of both repressible promoters on transcriptional level could be achieved. Compared to the transcript levels of these two genes when expressed under the control of their native promoters, only ERO1 was significantly down-regulated.ConclusionOur results show that all of the novel promoters can be used for repression of genes in liquid culture. We also came to the conclusion that the choice of the repressible promoter is of particular importance. For a successful repression experiment it is crucial that the native promoter of a gene and the repressible promoter in its non-repressed state are of similar strength.

Highlights

Repressible promoters are a useful tool for down-regulating the expression of genes, especially those that affect cell viability, in order to study cell physiology
Based on literature search in S. cerevisiae [9,10,11,12] and own microarray experiments in P. pastoris we identified other potential repressible promoters (PPIS1, PTHR1, PSER1 and PMET3), which all could be of interest for studying physiology of the cell
P. pastoris strains expressing enhanced green fluorescent protein (eGFP) under the control of these promoters were cultivated in the synthetic M2 medium either without or with respective repressing supplements, in order to test the regulation of the chosen promoters by these compounds

Summary

Introduction

Repressible promoters are a useful tool for down-regulating the expression of genes, especially those that affect cell viability, in order to study cell physiology. They are popular in biotechnological processes, like heterologous protein production. The methylotrophic yeast Pichia pastoris is a favored microorganism for the production of heterologous proteins in biotechnology. It has gained its popularity, because this yeast is easy to manipulate, can grow to high cell densities on cheap cultivation media and is well suited for the production of secreted proteins [1,2].

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Conclusion