Abstract
BackgroundThe 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.ResultsWe 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.ConclusionsP. 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1393-8) contains supplementary material, which is available to authorized users.
Highlights
The methylotrophic, Crabtree-negative yeast Pichia pastoris is widely used as a heterologous protein production host
P. pastoris strain X-33 was cultivated in shake flasks under four different bioprocess conditions (Table 1): excess glycerol or glucose; limiting glucose; and periodic methanol addition
Our current knowledge of translational regulation comes from studies on S. cerevisiae cells [74,75,76,77], where stress conditions have been found to induce a global translational down-regulation that is mediated by translation initiation factors
Summary
The methylotrophic, Crabtree-negative yeast Pichia pastoris is widely used as a heterologous protein production host. Genes involved in methanol utilization are repressed by the presence of glucose, and 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. Transcriptional profiling of a recombinant strain harboring Rhizomucor miehei lipase (RML) under the control of the methanol-driven PAOX1 promoter revealed that cells grown on methanol induce genes involved in protein production and energy metabolism more than cells grown on glycerol.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
