Trm2p-dependent derepression is essential for methanol-specific gene activation in the methylotrophic yeast Candida boidinii

Sasano, Yu; Yurimoto, Hiroya; Kuriyama, Masami; Sakai, Yasuyoshi

doi:10.1111/j.1567-1364.2010.00640.x

Cited by 19 publications

(32 citation statements)

References 37 publications

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“…AOD1 gene expression was derepressed in glycerol-containing medium, to ϳ10% of full induction in methanol-containing medium. Disruption of the CbTRM1 gene reduced P AOD1 transcriptional activity to the derepressed level, whereas disruption of the CbTRM2 gene reduced P AOD1 transcriptional activity to the basal level (11). These results suggest that CbHap2p, CbHap3p, and CbHap5p are responsible for methanol-specific induction rather than glucose derepression.…”

Section: Discussionmentioning

confidence: 47%

“…In our previous study, we performed a genetic screen for C. boidinii gene-tagging mutants with reduced levels of methanol-inducible gene expression, using strain FM as the host (9,11). Strain FM expresses the S. cerevisiae PHO5 gene under the control of the modified FDH1 promoter fused with six copies of UAS-FM, which is a promoter element that responds to both methanol and formate.…”

Section: Resultsmentioning

confidence: 99%

“…The key enzymes of methanol metabolism in methylotrophic yeasts are highly induced by methanol and are virtually absent when cells are growing on glucose. We have studied the regulation of the methanol-inducible genes necessary for methanol metabolism and characterized several transcription factors involved in their regulation in C. boidinii (8)(9)(10)(11). Based on our previous results, we have proposed three modes of regulation during methanol-inducible gene expression, that is, CbMig1p- dependent glucose repression, CbTrm2p-dependent derepression, and CbTrm1p-dependent methanol-specific induction.…”

Section: Discussionmentioning

confidence: 99%

“…In previous studies, we identified several transcription factors responsible for methanol-inducible gene expression in C. boidinii and revealed the specific function of each transcription factor, i.e., CbMig1p in glucose repression, CbTrm2p in glucose derepression, and CbTrm1p in methanol-specific induction (8)(9)(10)(11). Among these three modes of regulation, the molecular mechanism of glucose repression/derepression has been extensively studied in the budding yeast Saccharomyces cerevisiae after a shift of the carbon source from glucose to the nonfermentable carbon source ethanol or glycerol (12,13).…”

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confidence: 99%

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Molecular Characterization of Hap Complex Components Responsible for Methanol-Inducible Gene Expression in the Methylotrophic Yeast Candida boidinii

et al. 2015

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cWe identified genes encoding components of the Hap complex, CbHAP2, CbHAP3, and CbHAP5, as transcription factors regulating methanol-inducible gene expression in the methylotrophic yeast Candida boidinii. We found that the Cbhap2⌬, Cbhap3⌬, and Cbhap5⌬ gene-disrupted strains showed severe growth defects on methanol but not on glucose and nonfermentable carbon sources such as ethanol and glycerol. In these disruptants, the transcriptional activities of methanol-inducible promoters were significantly decreased compared to those of the wild-type strain, indicating that CbHap2p, CbHap3p, and CbHap5p play indispensable roles in methanol-inducible gene expression. Further molecular and biochemical analyses demonstrated that CbHap2p, CbHap3p, and CbHap5p localized to the nucleus and bound to the promoter regions of methanol-inducible genes regardless of the carbon source, and heterotrimer formation was suggested to be necessary for binding to DNA. Unexpectedly, distinct from Saccharomyces cerevisiae, the Hap complex functioned in methanol-specific induction rather than glucose derepression in C. boidinii. Our results shed light on a novel function of the Hap complex in methanol-inducible gene expression in methylotrophic yeasts.

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Section: Discussionmentioning

confidence: 47%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Molecular Characterization of Hap Complex Components Responsible for Methanol-Inducible Gene Expression in the Methylotrophic Yeast Candida boidinii

et al. 2015

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“…Exhaustion of glucose releases glucose repression, resulting in activation of methanol-regulated genes by CbTrm2p, which does not require methanol for gene activation (derepression). In addition, the presence of methanol induces maximum activation of methanolregulated genes via CbTrm1p (methanol induction; methanol-specific induction) (Hartner & Glieder, 2006;Sasano et al, 2008;Yurimoto, 2009;Sasano et al, 2010;Zhai et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Unique C-terminal region of Hap3 is required for methanol-regulated gene expression in the methylotrophic yeast Candida boidinii

et al. 2016

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The Hap complex of the methylotrophic yeast Candida boidinii was found to be required for methanol-regulated gene expression. In this study, we performed functional characterization of CbHap3p, one of the Hap complex components in C. boidinii. Sequence alignment of Hap3 proteins revealed the presence of a unique extended C-terminal region, which is not present in Hap3p from Saccharomyces cerevisiae (ScHap3p), but is found in Hap3p proteins of methylotrophic yeasts. Deletion of the C-terminal region of CbHap3p (D256-292 or D107-237) diminished activation of methanol-regulated genes and abolished the ability to grow on methanol, but did not affect nuclear localization or DNA-binding ability. However, deletion of the N-terminal region of CbHap3p (D1-20) led to not only a growth defect on methanol and a decreased level of methanol-regulated gene expression, but also impaired nuclear localization and binding to methanol-regulated gene promoters. We also revealed that CbHap3p could complement the growth defect of the Schap3D strain on glycerol, although ScHap3p could not complement the growth defect of a Cbhap3D strain on methanol. We conclude that the unique C-terminal region of CbHap3p contributes to maximum activation of methanol-regulated genes, whilst the N-terminal region is required for nuclear localization and binding to DNA.

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

Vogl

Sturmberger

Fauland

et al. 2018

Biotech & Bioengineering

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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 (P ), 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 P 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 P 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.

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Trm2p-dependent derepression is essential for methanol-specific gene activation in the methylotrophic yeast Candida boidinii

Cited by 19 publications

References 37 publications

Molecular Characterization of Hap Complex Components Responsible for Methanol-Inducible Gene Expression in the Methylotrophic Yeast Candida boidinii

Molecular Characterization of Hap Complex Components Responsible for Methanol-Inducible Gene Expression in the Methylotrophic Yeast Candida boidinii

Unique C-terminal region of Hap3 is required for methanol-regulated gene expression in the methylotrophic yeast Candida boidinii

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

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