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. 2015 Mar 11;16(1):167.
doi: 10.1186/s12864-015-1393-8.

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

Roland Prielhofer  1   2 , Stephanie P Cartwright  3 , Alexandra B Graf  4   5 , Minoska Valli  6   7 , Roslyn M Bill  8 , Diethard Mattanovich  9   10 , Brigitte Gasser  11   12
Affiliations

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

Roland Prielhofer et al. BMC Genomics. .

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.

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Figures

Figure 1
Figure 1
Differentially expressed genes. The bar chart (A) shows the number of differentially expressed genes in excess glycerol (G), methanol (M) and limiting glucose (X) compared to the excess glucose condition. Venn diagrams illustrate the number of up-regulated (B) and down-regulated genes (C) in the conditions and intersections. Significantly-regulated genes were identified from total RNA fold changes compared to the excess glucose condition (cutoff ±50% fold change and adjusted p-values < 0.05; [23]).
Figure 2
Figure 2
Principal component analysis (PCA) bi-plots of microarray intensities from the green channel. Red vectors indicate variable (condition) correlation of all analyzed replicates and the grey data points indicate observations (genes). Replicate correlation fits very well already before data normalization. The components one and two (A) and two and three (B) are compared, which explain 78, 14 and 5% of the total variation, respectively. Similar PCA biplots are obtained from microarray intensities of the red channel.
Figure 3
Figure 3
Polysome profiles and P:M ratios for P. pastoris grown in different conditions. (A) Representative polysome profiles and (B) a bar chart presenting P:M ratios (with sd) of the four different cultivation conditions (excess glucose, D; excess glycerol, G; limiting glucose, X; methanol, M). Corresponding peaks (40S, 60S, 80S/monosomes and polysomes) are indicated in the first (D) polysome profile. P:M ratios were calculated from areas beneath the profile curve using ImageJ.
Figure 4
Figure 4
Translationally-enriched and depleted genes. Bar chart representing the number of translationally enriched and depleted genes in excess glycerol, limiting glucose and methanol conditions related to the excess glucose condition (cutoff ±50% change of the translational state and adjusted p-values < 0.05).
Figure 5
Figure 5
Schematic illustration of relations between transcript level, translation, UTR frequency and codon usage bias in P. pastoris genes. In contrast to genes with long coding sequences, shorter genes are more highly expressed, more efficiently translated, possess UTR’s less frequently and are more codon biased than longer genes.
Figure 6
Figure 6
Central carbon metabolism pathways in Pichia pastoris. Transcriptional log2 fold changes of genes significantly regulated in excess glycerol, methanol and limiting glucose compared to excess glucose are presented in bar charts (cutoff ±50% fold change and adjusted p-values < 0.05; [23]). According to cellular localization, peroxisomal, cytosolic and mitochondrial enzymes are colored in red, black and green, respectively. Metabolites: G-6-P: glucose 6-phosphate; F-1,6-P: fructose 1,6-phosphate; DHA(P): dihydroxy acetone (phosphate); G-3-P: glycerol 3-phosphate; GA-3-P: glyceraldehyde 3-phopshate; 1,3-bPG: 1,3-bisphosphoglycerate; 3-PG: 3-phosphoglycerate; 2-PG: 2-phosphoglycerate; PEP: phosphoenolpyruvate; PYR: pyruvate; OAA: oxaloacetate; CIT: citrate; ICIT: isocitrate; AKG: alpha-keto glutarate; SUC: succinate; SUC-CoA: succinyl-Coenzyme A; FUM: fumerate; MAL: malate; GLYO: glyoxylate; Enzymes: AOX1/2: alcohol oxidase; CTA1: catalase A; FLD: bifunctional alcohol dehydrogenase and formaldehyde dehydrogenase; FGH1: S-formylglutathione hydrolase; FDH1: formate dehydrogenase; DAK2: dihydroxyacetone kinase; DAS1/2: dihydroxyacetone synthase; GUT1: glycerol kinase; GUT2: glycerol-3-phosphate dehydrogenase; GPD1: glycerol-3-phosphate dehydrogenase; PCK1: phosphoenolpyruvate carboxykinase; GTH1: high-affinity glucose transporter; HXT1: low-affinity glucose transporter; HXK1: hexokinase; PGI1: phosphoglucose isomerase; PFK1/2: phosphofructokinase; FBP1: fructose-1,6-bisphosphatase; FBA1-1/1-2: fructose 1,6-bisphosphate aldolase; TPI1: triose phosphate isomerase; TDH3: glyceraldehyde-3-phosphate dehydrogenase; PGK1: 3-phosphoglycerate kinase; GPM1/3: phosphoglycerate mutase; ENO1: enolase I, phosphopyruvate hydratase; CDC19: pyruvate kinase; PDC1 pyruvate decarboxylase; PDA1: E1 alpha subunit of the pyruvate dehydrogenase (PDH) complex; ALD2: cytoplasmic aldehyde dehydrogenase; ALD4-1/4-2/5: mitochondrial aldehyde dehydrogenase; ACS1/2: acetyl-coA synthetase; PYC2: pyruvate carboxylase; CIT1: citrate synthase; ACO1/2: aconitase; ICL1: isocitrate lyase; DAL7: malate synthase; IDH1/2: isocitrate dehydrogenase; KGD1: alpha-ketoglutarate dehydrogenase complex; KGD2: dihydrolipoyl transsuccinylase; LSC1: succinyl-CoA ligase; SDH1/2/4: succinate dehydrogenase; FUM1: fumarase; MDH1: mitochondrial malate dehydrogenase; MDH3: malate dehydrogenase; MAE1: mitochondrial malic enzyme.
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