Combinatorial Mutagenesis and Selection to Understand and Improve Yeast Promoters

Laila Berg,Trine Aakvik Strand,Svein Valla,Trygve Brautaset

doi:10.1155/2013/926985

Laila Berg, Trine Aakvik Strand + Show 2 more

Open Access

https://doi.org/10.1155/2013/926985

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Abstract

Microbial promoters are important targets both for understanding the global gene expression and developing genetic tools for heterologous expression of proteins and complex biosynthetic pathways. Previously, we have developed and used combinatorial mutagenesis methods to analyse and improve bacterial expression systems. Here, we present for the first time an analogous strategy for yeast. Our model promoter is the strong and inducible P AOX1 promoter in methylotrophic Pichia pastoris. The Zeocin resistance gene was applied as a valuable reporter for mutant P AOX1 promoter activity, and we used an episomal plasmid vector to ensure a constant reporter gene dosage in the yeast host cells. This novel design enabled direct selection for colonies of recombinant cells with altered Zeocin tolerance levels originating solely from randomly introduced point mutations in the P AOX1 promoter DNA sequence. We demonstrate that this approach can be used to select for P AOX1 promoter variants with abolished glucose repression in large mutant libraries. We also selected P AOX1 promoter variants with elevated expression level under induced conditions. The properties of the selected P AOX1 promoter variants were confirmed by expressing luciferase as an alternative reporter gene. The tools developed here should be useful for effective screening, characterization, and improvement of any yeast promoters.

Highlights

Microorganisms are widely used in biotechnology as cell factories for sustainable production of proteins, biopolymers, and chemicals for medical, pharmaceutical and industrial applications
Circular plasmids were transformed to P. pastoris GS115 and the transformation suspensions were plated on rich agar media with glucose or minimal agar media with methanol containing increasing concentrations of Zeocin to directly select for the transformants containing the PAOX1 promoter variants leading to stimulated expression compared to that of the wild-type
Plasmid pPPE22 was constructed by substituting the PAOX1 promoter in pPPE17 with the constitutive P. pastoris PGAP promoter, and it was included as a valuable control system

Summary

Introduction

Microorganisms are widely used in biotechnology as cell factories for sustainable production of proteins, biopolymers, and chemicals for medical, pharmaceutical and industrial applications. We have constructed expression vectors by combining xylS/Pm with the minimal replicon trfA/oriV of the natural broad host range plasmid RK2 and demonstrated industrial level production of several medical recombinant proteins in Escherichia coli under high-cell-density cultivations [3, 4]. Yeast promoters are in general longer and more complex than bacterial promoters, and the understanding of the about 950 base pairs long P. pastoris PAOX1 promoter DNA region is limited, both with regard to its cis-acting elements and the molecular mechanisms underlying its regulation. We demonstrate for the first time that this approach works in yeast and that it can be used both to alter the regulation properties as well as to improve the maximized expression level of the PAOX1 promoter. The methodology presented here should be valuable both to manipulate and improve yeast promoters, for promoter probe approaches, and for applied perspectives as well as to generate basic understanding of microbial promoters

Materials and Methods

Results and Discussion

Conclusions