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Flip/flop mating-type switching in the methylotrophic yeast Ogataea polymorpha is regulated by an Efg1-Rme1-Ste12 pathway.

ABSTRACT: In haploid cells of Ogataea (Hansenula) polymorpha an environmental signal, nitrogen starvation, induces a reversible change in the structure of a chromosome. This process, mating-type switching, inverts a 19-kb DNA region to place either MATa or MAT? genes under centromeric repression of transcription, depending on the orientation of the region. Here, we investigated the genetic pathway that controls switching. We characterized the transcriptomes of haploid and diploid O. polymorpha by RNAseq in rich and nitrogen-deficient media, and found that there are no constitutively a-specific or ?-specific genes other than the MAT genes themselves. We mapped a switching defect in a sibling species (O. parapolymorpha strain DL-1) by interspecies bulk segregant analysis to a frameshift in the transcription factor EFG1, which in Candida albicans regulates filamentous growth and white-opaque switching. Gene knockout, overexpression and ChIPseq experiments show that EFG1 regulates RME1, which in turn regulates STE12, to achieve mating-type switching. All three genes are necessary both for switching and for mating. Overexpression of RME1 or STE12 is sufficient to induce switching without a nitrogen depletion signal. The homologous recombination genes RAD51 and RAD17 are also necessary for switching. The pathway controlling switching in O. polymorpha shares no components with the regulation of HO in S. cerevisiae, which does not involve any environmental signal, but it shares some components with mating-type switching in Kluyveromyces lactis and with white-opaque phenotypic switching in C. albicans.

SUBMITTER: Hanson SJ

PROVIDER: S-EPMC5720833 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Flip/flop mating-type switching in the methylotrophic yeast Ogataea polymorpha is regulated by an Efg1-Rme1-Ste12 pathway.

Hanson Sara J SJ Byrne Kevin P KP Wolfe Kenneth H KH

PLoS genetics 20171127 11

In haploid cells of Ogataea (Hansenula) polymorpha an environmental signal, nitrogen starvation, induces a reversible change in the structure of a chromosome. This process, mating-type switching, inverts a 19-kb DNA region to place either MATa or MATα genes under centromeric repression of transcription, depending on the orientation of the region. Here, we investigated the genetic pathway that controls switching. We characterized the transcriptomes of haploid and diploid O. polymorpha by RNAseq i ...[more]

PMID: 29176810

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Project description:BackgroundOgataea (Hansenula) polymorpha is one of the most thermotolerant xylose-fermenting yeast species reported to date. Several metabolic engineering approaches have been successfully demonstrated to improve high-temperature alcoholic fermentation by O. polymorpha. Further improvement of ethanol production from xylose in O. polymorpha depends on the identification of bottlenecks in the xylose conversion pathway to ethanol.ResultsInvolvement of peroxisomal enzymes in xylose metabolism has not been described to date. Here, we found that peroxisomal transketolase (known also as dihydroxyacetone synthase) and peroxisomal transaldolase (enzyme with unknown function) in the thermotolerant methylotrophic yeast, Ogataea (Hansenula) polymorpha, are required for xylose alcoholic fermentation, but not for growth on this pentose sugar. Mutants with knockout of DAS1 and TAL2 coding for peroxisomal transketolase and peroxisomal transaldolase, respectively, normally grow on xylose. However, these mutants were found to be unable to support ethanol production. The O. polymorpha mutant with the TAL1 knockout (coding for cytosolic transaldolase) normally grew on glucose and did not grow on xylose; this defect was rescued by overexpression of TAL2. The conditional mutant, pYNR1-TKL1, that expresses the cytosolic transketolase gene under control of the ammonium repressible nitrate reductase promoter did not grow on xylose and grew poorly on glucose media supplemented with ammonium. Overexpression of DAS1 only partially restored the defects displayed by the pYNR1-TKL1 mutant. The mutants defective in peroxisome biogenesis, pex3Δ and pex6Δ, showed normal growth on xylose, but were unable to ferment this sugar. Moreover, the pex3Δ mutant of the non-methylotrophic yeast, Scheffersomyces (Pichia) stipitis, normally grows on and ferments xylose. Separate overexpression or co-overexpression of DAS1 and TAL2 in the wild-type strain increased ethanol synthesis from xylose 2 to 4 times with no effect on the alcoholic fermentation of glucose. Overexpression of TKL1 and TAL1 also elevated ethanol production from xylose. Finally, co-overexpression of DAS1 and TAL2 in the best previously isolated O. polymorpha xylose to ethanol producer led to increase in ethanol accumulation up to 16.5 g/L at 45 °C; or 30-40 times more ethanol than is produced by the wild-type strain.ConclusionsOur results indicate the importance of the peroxisomal enzymes, transketolase (dihydroxyacetone synthase, Das1), and transaldolase (Tal2), in the xylose alcoholic fermentation of O. polymorpha.

Dataset Information

Flip/flop mating-type switching in the methylotrophic yeast Ogataea polymorpha is regulated by an Efg1-Rme1-Ste12 pathway.

Publications

Flip/flop mating-type switching in the methylotrophic yeast Ogataea polymorpha is regulated by an Efg1-Rme1-Ste12 pathway.

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