Methanol independent induction in Pichia pastoris by simple derepressed overexpression of single transcription factors

Thomas Vogl, Lukas Sturmberger, Pia C Fauland, Patrick Hyden, Jasmin Elgin Fischer, Christian Schmid, Gerhard G Thallinger, Martina Geier, Anton Glieder

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Carbon source regulated promoters are well-studied standard tools for controlling gene expression. Acquiring control over the natural regulation of promoters is important for metabolic engineering and synthetic biology applications. In the commonly used protein production host Komagataella phaffii (Pichia pastoris), methanol-inducible promoters are used because of their tight regulation and exceptional strength. Yet, induction with toxic and flammable methanol can be a considerable safety risk and cannot be applied in many existing fermentation plants. Here we studied new regulatory circuits based on the most frequently used alcohol oxidase 1 promoter (PAOX1 ), which is tightly repressed in presence of repressing carbon sources and strongly induced by methanol. We compared different overexpression strategies for putative carbon source dependent regulators identified by a homology search in related yeasts and previously published literature in order to convert existing methanol dependent expression strains into methanol free systems. While constitutive overexpression showed only marginal or detrimental effects, derepressed expression (activated when the repressing carbon source is depleted) showed that three transcription factors (TFs) are single handedly suitable to strongly activate PAOX1 in P. pastoris without relying on any specifically engineered host strains. Transcriptome analyses demonstrated that Mxr1, Mit1 and Prm1 regulate partly overlapping and unique sets of genes. Derepressed overexpression of a single TF was sufficient to retrofit existing PAOX1 based expression strains into glucose/glycerol regulated, methanol-free systems. Given the wide applicability of carbon source regulated promoters, the simplicity and low cost of controlling carbon source feed rates in large scale bioreactors, similar approaches as in P. pastoris may also be useful in other organisms. This article is protected by copyright. All rights reserved.

LanguageEnglish
Pages1037-1050
JournalBiotechnology and Bioengineering
Volume115
Issue number4
Early online date27 Dec 2017
DOIs
StatusPublished - 1 Apr 2018

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Transcription factors
Pichia
Methanol
Transcription Factors
Carbon
alcohol oxidase
Synthetic Biology
Metabolic engineering
Metabolic Engineering
Poisons
Gene Expression Profiling
Bioreactors
Glycerol
Gene expression
Yeast
Fermentation
Glucose
Alcohols
Genes
Yeasts

Keywords

  • Journal Article

Fields of Expertise

  • Human- & Biotechnology
  • Information, Communication & Computing

Cite this

Methanol independent induction in Pichia pastoris by simple derepressed overexpression of single transcription factors. / Vogl, Thomas; Sturmberger, Lukas; Fauland, Pia C; Hyden, Patrick; Fischer, Jasmin Elgin; Schmid, Christian; Thallinger, Gerhard G; Geier, Martina; Glieder, Anton.

In: Biotechnology and Bioengineering, Vol. 115, No. 4, 01.04.2018, p. 1037-1050.

Research output: Contribution to journalArticleResearchpeer-review

Vogl T, Sturmberger L, Fauland PC, Hyden P, Fischer JE, Schmid C et al. Methanol independent induction in Pichia pastoris by simple derepressed overexpression of single transcription factors. Biotechnology and Bioengineering. 2018 Apr 1;115(4):1037-1050. Available from, DOI: 10.1002/bit.26529
Vogl, Thomas ; Sturmberger, Lukas ; Fauland, Pia C ; Hyden, Patrick ; Fischer, Jasmin Elgin ; Schmid, Christian ; Thallinger, Gerhard G ; Geier, Martina ; Glieder, Anton. / Methanol independent induction in Pichia pastoris by simple derepressed overexpression of single transcription factors. In: Biotechnology and Bioengineering. 2018 ; Vol. 115, No. 4. pp. 1037-1050
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