Promoter library designed for fine-tuned gene expression in Pichia pastoris

Franz Stefan Hartner, Claudia Ruth, D. Langenegger, SN Johnson, P. Hyka, GP Lin-Cereghino, K. Kovar, JM Cregg, Anton Glieder, Joan Lin-Cereghino

Research output: Contribution to journalArticle

Abstract

Although frequently used as protein production host, there is only a limited set of promoters available to drive the expression of recombinant proteins in Pichia pastoris. Fine-tuning of gene expression is often needed to maximize product yield and quality. However, for efficient knowledge-based engineering, a better understanding of promoter function is indispensable. Consequently, we created a promoter library by deletion and duplication of putative transcription factor-binding sites within the AOX1 promoter (P(AOX1)) sequence. This first library initially spanned an activity range between approximately 6% and >160% of the wild-type promoter activity. After characterization of the promoter library employing a green fluorescent protein (GFP) variant, the new regulatory toolbox was successfully utilized in a 'real case', i.e. the expression of industrial enzymes. Characterization of the library under repressing, derepressing and inducing conditions displayed at least 12 cis-acting elements involved in P(AOX1)-driven high-level expression. Based on this deletion analysis, novel short artificial promoter variants were constructed by combining cis-acting elements with basal promoter. In addition to improving yields and quality of heterologous protein production, the new P(AOX1) synthetic promoter library constitutes a basic toolbox to fine-tune gene expression in metabolic engineering and sequential induction of protein expression in synthetic biology.

Original languageEnglish
Pages (from-to)e76
JournalNucleic acids research
Volume36
Issue number12
DOIs
Publication statusPublished - Jul 2008

Treatment code (Nähere Zuordnung)

  • Basic - Fundamental (Grundlagenforschung)

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