GAP Promoter Library for Fine-Tuning of Gene Expression in Pichia pastoris

Abstract

A library of engineered promoters of various strengths is a useful genetic tool that enables the fine-tuning and precise control of gene expression across a continuum of broad expression levels. The methylotrophic yeast Pichia pastoris is a well-established expression host with a large academic and industrial user base. To facilitate manipulation of gene expression spanning a wide dynamic range in P. pastoris, we created a functional promoter library through mutagenesis of the constitutive GAP promoter. Using yeast-enhanced green fluorescent protein (yEGFP) as the reporter, 33 mutants were chosen to form the functional promoter library. The 33 mutants spanned an activity range between ϳ0.6% and 19.6-fold of the wild-type promoter activity with an almost linear fluorescence intensity distribution. After an extensive characterization of the library, the broader applicability of the results obtained with the yEGFP reporter was confirmed using two additional reporters (␤-galactosidase and methionine adenosyltransferase [MAT]) at the transcription and enzyme activity levels. Furthermore, the utility of the promoter library was tested by investigating the influence of heterologous MAT gene expression levels on cell growth and S-adenosylmethionine (SAM) production. The extensive characterization of the promoter strength enabled identification of the optimal MAT activity (around 1.05 U/mg of protein) to obtain maximal volumetric SAM production. The promoter library permits precise control of gene expression and quantitative assessment that correlates gene expression level with physiologic parameters. Thus, it is a useful toolbox for both basic and applied research in P. pastoris.The creation of a library of engineered promoters of various strengths has enabled the precise control of gene expression in a broad range of activities required for detailed genotypephenotype characterization and the identification of an optimal gene expression level for the desired product yield (1,4,12,20,22,29,35). For example, a library of promoters has been used to assess the influence of phosphoenolpyruvate carboxylase levels on growth yield and deoxyxylulose-P synthase levels on lycopene production and to demonstrate that the optimal expression levels of deoxyxylulose-P synthase are different in a strain preengineered to produce lycopene and in the parental strain (1). In another example, random mutagenesis of the Pm promoter has been applied to improve production of the medically important granulocyte-macrophage colony-stimulating factor in Escherichia coli at an industrial level (4). Furthermore, promoter libraries coupled with in silico modeling have been used to guide predictable gene network construction in Saccharomyces cerevisiae (13). Therefore, the promoter library constitutes a valuable addition to the genetic toolbox for the study of metabolic engineering, functional genomics, and synthetic biology, as well as for various biotechnology applications. However, this powerful tool is implemented mainly in bacteria (2,4,12,20,35,36...

Molecular strategies to increase the levels of heterologous transcripts inKomagataella phaffiifor protein production

“…9,19 The P GAP sequence has also been manipulated through error-prone PCR for the construction of a GAP-promoter library with strengths varying from »0,6% to 19,6-fold of that of the wild-type P GAP . 20 …”

Analysis of the 5′ untranslated region (5′UTR) of the alcohol oxidase 1 (AOX1) gene in recombinant protein expression in Pichia pastoris

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Synthetic Biology and Metabolic Engineering Approaches and Its Impact on Non-Conventional Yeast and Biofuel Production

“…Random mutated library of pAOX1 has been constructed and resulted in less glucose repression and increased activity compared to wild pAOX1 (Berg et al, 2013). The constitutive pGAP (glyceraldehyde-3-phosphate dehydrogenase promoter) served as a scaffold, and this resulted in the development of a series of promoters with a range of activity from 0.01-to 19.6-fold of wild-type pGAP activity (Qin et al, 2011). Blazeck et al (2012) reported that the combination of core promoter elements with synthetic upstream activator sequence could increase the expression level.…”