Pichia pastoris regulates its gene-specific response to different carbon sources at the transcriptional, rather than the translational, level

Prielhofer, Roland, Cartwright, Stephanie, Graf, Alexandra B., Valli, Minoska, Bill, Roslyn, Mattanovich, Diethard and Gasser, Brigitte (2015). Pichia pastoris regulates its gene-specific response to different carbon sources at the transcriptional, rather than the translational, level. BMC Genomics, 16 (1),

Abstract

Background: The methylotrophic, Crabtree-negative yeast Pichia pastoris is widely used as a heterologous protein production host. Strong inducible promoters derived from methanol utilization genes or constitutive glycolytic promoters are typically used to drive gene expression. Notably, genes involved in methanol utilization are not only repressed by the presence of glucose, but also by glycerol. This unusual regulatory behavior prompted us to study the regulation of carbon substrate utilization in different bioprocess conditions on a genome wide scale. Results: We performed microarray analysis on the total mRNA population as well as mRNA that had been fractionated according to ribosome occupancy. Translationally quiescent mRNAs were defined as being associated with single ribosomes (monosomes) and highly-translated mRNAs with multiple ribosomes (polysomes). We found that despite their lower growth rates, global translation was most active in methanol-grown P. pastoris cells, followed by excess glycerol- or glucose-grown cells. Transcript-specific translational responses were found to be minimal, while extensive transcriptional regulation was observed for cells grown on different carbon sources. Due to their respiratory metabolism, cells grown in excess glucose or glycerol had very similar expression profiles. Genes subject to glucose repression were mainly involved in the metabolism of alternative carbon sources including the control of glycerol uptake and metabolism. Peroxisomal and methanol utilization genes were confirmed to be subject to carbon substrate repression in excess glucose or glycerol, but were found to be strongly de-repressed in limiting glucose-conditions (as are often applied in fed batch cultivations) in addition to induction by methanol. Conclusions: P. pastoris cells grown in excess glycerol or glucose have similar transcript profiles in contrast to S. cerevisiae cells, in which the transcriptional response to these carbon sources is very different. The main response to different growth conditions in P. pastoris is transcriptional; translational regulation was not transcript-specific. The high proportion of mRNAs associated with polysomes in methanol-grown cells is a major finding of this study; it reveals that high productivity during methanol induction is directly linked to the growth condition and not only to promoter strength.

Publication DOI: https://doi.org/10.1186/s12864-015-1393-8
Divisions: Life & Health Sciences > Biosciences
Life & Health Sciences
Additional Information: © 2015 Prielhofer et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Funding: Federal Ministry of Science, Research and Economy (bmwfw), the Federal Ministry of Traffic, Innovation and Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol and ZIT - Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG; Erasmus (internship grant); BBSRC (targeted priority studentship).
Uncontrolled Keywords: carbon substrate repression,crabtree-negative yeast,glucose repression,methanol induction,methylotrophic yeast,microarray analysis,pichia pastoris,polysome profiling,transcriptome,Biotechnology,Genetics
Full Text Link: http://www.biom ... 471-2164/16/167
Related URLs: http://www.scop ... tnerID=8YFLogxK (Scopus URL)
Published Date: 2015-03-11
Authors: Prielhofer, Roland
Cartwright, Stephanie
Graf, Alexandra B.
Valli, Minoska
Bill, Roslyn ( 0000-0003-1331-0852)
Mattanovich, Diethard
Gasser, Brigitte

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