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 journalArticleResearchpeer-review

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

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Pichia
Libraries
Gene Expression
Synthetic Biology
Metabolic Engineering
Proteins
Green Fluorescent Proteins
Recombinant Proteins
Transcription Factors
Binding Sites
Enzymes

Treatment code (Nähere Zuordnung)

  • Basic - Fundamental (Grundlagenforschung)

Cite this

Hartner, F. S., Ruth, C., Langenegger, D., Johnson, SN., Hyka, P., Lin-Cereghino, GP., ... Lin-Cereghino, J. (2008). Promoter library designed for fine-tuned gene expression in Pichia pastoris. Nucleic acids research, 36(12), e76. https://doi.org/10.1093/nar/gkn369

Promoter library designed for fine-tuned gene expression in Pichia pastoris. / Hartner, Franz Stefan; Ruth, Claudia; Langenegger, D.; Johnson, SN; Hyka, P.; Lin-Cereghino, GP; Kovar, K.; Cregg, JM; Glieder, Anton; Lin-Cereghino, Joan.

In: Nucleic acids research, Vol. 36, No. 12, 07.2008, p. e76.

Research output: Contribution to journalArticleResearchpeer-review

Hartner, FS, Ruth, C, Langenegger, D, Johnson, SN, Hyka, P, Lin-Cereghino, GP, Kovar, K, Cregg, JM, Glieder, A & Lin-Cereghino, J 2008, 'Promoter library designed for fine-tuned gene expression in Pichia pastoris' Nucleic acids research, vol. 36, no. 12, pp. e76. https://doi.org/10.1093/nar/gkn369
Hartner FS, Ruth C, Langenegger D, Johnson SN, Hyka P, Lin-Cereghino GP et al. Promoter library designed for fine-tuned gene expression in Pichia pastoris. Nucleic acids research. 2008 Jul;36(12):e76. https://doi.org/10.1093/nar/gkn369
Hartner, Franz Stefan ; Ruth, Claudia ; Langenegger, D. ; Johnson, SN ; Hyka, P. ; Lin-Cereghino, GP ; Kovar, K. ; Cregg, JM ; Glieder, Anton ; Lin-Cereghino, Joan. / Promoter library designed for fine-tuned gene expression in Pichia pastoris. In: Nucleic acids research. 2008 ; Vol. 36, No. 12. pp. e76.
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