A Toolbox of Diverse Promoters Related to Methanol Utilization: Functionally Verified Parts for Heterologous Pathway Expression in Pichia pastoris

Thomas Vogl, Lukas Sturmberger, Thomas Kickenweiz, Richard Wasmayer, Christian Schmid, Anna-Maria Hatzl, Michaela Agnes Gerstmann, Julia Pitzer, Marlies Wagner, Gerhard G Thallinger, Martina Geier, Anton Glieder

Research output: Contribution to journalArticle

Abstract

The heterologous expression of biosynthetic pathways for pharmaceutical or fine chemical production requires suitable expression hosts and vectors. In eukaryotes, the pathway flux is typically balanced by stoichiometric fine-tuning of reaction steps by varying the transcript levels of the genes involved. Regulated (inducible) promoters are desirable to allow a separation of pathway expression from cell growth. Ideally, the promoter sequences used should not be identical to avoid loss by recombination. The methylotrophic yeast Pichia pastoris is a commonly used protein production host, and single genes have been expressed at high levels using the methanol-inducible, strong, and tightly regulated promoter of the alcohol oxidase 1 gene (PAOX1). Here, we have studied the regulation of the P. pastoris methanol utilization (MUT) pathway to identify a useful set of promoters that (i) allow high coexpression and (ii) differ in DNA sequence to increase genetic stability. We noticed a pronounced involvement of the pentose phosphate pathway (PPP) and genes involved in the defense of reactive oxygen species (ROS), providing strong promoters that, in part, even outperform PAOX1 and offer novel regulatory profiles. We have applied these tightly regulated promoters together with novel terminators as useful tools for the expression of a heterologous biosynthetic pathway. With the synthetic biology toolbox presented here, P. pastoris is now equipped with one of the largest sets of strong and co-regulated promoters of any microbe, moving it from a protein production host to a general industrial biotechnology host.

LanguageEnglish
Pages172-86
Number of pages15
JournalACS Synthetic Biology
Volume5
Issue number2
DOIs
StatusPublished - 19 Feb 2016

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Pichia
Methanol
alcohol oxidase
Genes
Biosynthetic Pathways
Alcohols
Synthetic Biology
Proteins
Pentoses
Pentose Phosphate Pathway
DNA sequences
Cell growth
Biotechnology
Eukaryota
Yeast
Drug products
Genetic Recombination
Reactive Oxygen Species
Phosphates
Tuning

Keywords

  • Journal Article
  • Research Support, Non-U.S. Gov't

Cite this

A Toolbox of Diverse Promoters Related to Methanol Utilization : Functionally Verified Parts for Heterologous Pathway Expression in Pichia pastoris. / Vogl, Thomas; Sturmberger, Lukas; Kickenweiz, Thomas; Wasmayer, Richard; Schmid, Christian; Hatzl, Anna-Maria; Gerstmann, Michaela Agnes; Pitzer, Julia; Wagner, Marlies; Thallinger, Gerhard G; Geier, Martina; Glieder, Anton.

In: ACS Synthetic Biology , Vol. 5, No. 2, 19.02.2016, p. 172-86.

Research output: Contribution to journalArticle

Vogl, Thomas ; Sturmberger, Lukas ; Kickenweiz, Thomas ; Wasmayer, Richard ; Schmid, Christian ; Hatzl, Anna-Maria ; Gerstmann, Michaela Agnes ; Pitzer, Julia ; Wagner, Marlies ; Thallinger, Gerhard G ; Geier, Martina ; Glieder, Anton. / A Toolbox of Diverse Promoters Related to Methanol Utilization : Functionally Verified Parts for Heterologous Pathway Expression in Pichia pastoris. In: ACS Synthetic Biology . 2016 ; Vol. 5, No. 2. pp. 172-86
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