acid-phosphatase and alcohol-oxidase

Ogataea minuta is a methylotrophic yeast that is closely related to Ogataea (Hansenula) polymorpha. Like other methylotrophic yeasts, O. minuta possesses strongly methanol-inducible genes, such as AOX1. We have focused on O. minuta as a host for the production of heterologous glycoproteins. However, it remained unknown how the AOX1 promoter is regulated in O. minuta. To elucidate regulation mechanisms of the AOX1 promoter, we adopted an assay system to quantitate AOX1 promoter activity using the PHO5 gene, which encodes an acid phosphatase, of Saccharomyces cerevisiae. The promoter activity assay revealed that glycerol, as well as glucose, cause strong catabolite repression of AOX1 expression in O. minuta. To investigate what factors are involved in transcription of the AOX1 promoter in O. minuta, we cloned three putative transcription factor genes, TRM1, TRM2, and MPP1, as homologues of other methylotrophic yeast species. Deletion mutants of these genes all showed decreased induction of the AOX1 promoter when methanol was added as the sole carbon source, indicating that these genes are indeed involved in AOX1 promoter regulation in O. minuta. Double deletion and constitutive expression of these transcription factor genes indicated that TRM1 and MPP1 regulate the transcription of AOX1 in the same pathway, while TRM2 regulates it in another pathway. By reverse transcription-qPCR, we also found that these two pathways compensate for each other and have crosstalk mechanisms with each other. A possible model for regulation of the AOX1 promoter in O. minuta was shown.

Topics: Acid Phosphatase; Alcohol Oxidoreductases; Deoxyribonucleases; Gene Expression; Gene Expression Regulation, Fungal; Methanol; Pichia; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Saccharomycetales; tRNA Methyltransferases

Pichia pastoris is a methylotrophic yeast that is widely used for the expression of heterologous proteins. Here, we have investigated the dependence of AOX1 and CAT1 expression on the source of nitrogen. It has shown that the expression of AOX1 and CAT1 depends on the source of nitrogen. In the presence of a rich nitrogen source, such as glutamine or ammonium sulfate, the AOX1 and CAT1 expression was dramatically induced. With proline as the nitrogen source AOX1 and CATI expression was reduced by more than threefold. The expression of these genes is controlled at the level of transcription. We have shown the impact of the nitrogen source on the growth rate in the presence of methanol as a sole carbon source. The specific growth rate was highest on media that contained proline as a nitrogen source compared to media with a rich nitrogen source.

Topics: Acid Phosphatase; Alcohol Oxidoreductases; Ammonium Sulfate; Catalase; Fungal Proteins; Gene Expression Regulation, Fungal; Glutamine; Methanol; Nitrogen; Pichia; Promoter Regions, Genetic; Saccharomyces cerevisiae Proteins; Urea

In this study, two Pichia methanolica alcohol oxidase (AOD) promoters, P(MOD1) and P(MOD2), were evaluated in a promoter assay system utilizing the acid phosphatase (AP) gene from Saccharomyces cerevisiae (ScPHO5) as a reporter. Heterologous gene expression driven by the P(MOD1) and P(MOD2) promoters was found to be strong and tightly regulated by carbon source at the transcriptional level. P(MOD1) was induced not only by methanol but also by glycerol. P(MOD2) was induced only by methanol, although it was not repressed on the addition of glycerol to a methanol medium, suggesting that P(MOD2) is regulated in a manner distinct from that of other AOD-gene promoters. On the other hand, methanol and oxygen level-influenced gene expression mediated by P(MOD1) and P(MOD2). P(MOD1) expression was optimal at low methanol concentrations, whereas P(MOD2) was predominantly expressed at high methanol and high oxygen concentrations. Based on these results, both P(MOD2) and P(MOD1) should be useful tools for controlling heterologous gene expression in P. methanolica. In particular, it should be possible to differentially control the production phases of two heterologous proteins, using P(MOD1) and P(MOD2) in the same host cell and in the same flask.

Topics: Acid Phosphatase; Alcohol Oxidoreductases; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Fungal; Histocytochemistry; Methanol; Mutagenesis, Insertional; Oxygen; Pichia; Promoter Regions, Genetic

We isolated the promoter regions of five methanol-inducible genes (P(AOD1), alcohol oxidase; P(DAS1), dihydroxyacetone synthase; P(FDH1), formate dehydrogenase; P(PMP20), Pmp20; and P(PMP47), Pmp47) from the Candida boidinii genome, and evaluated their strength and studied their regulation using the acid phosphatase gene of Saccharomyces cerevisiae (ScPHO5) as the reporter. Of the five promoters, P(DAS1) was the strongest methanol-inducible promoter whose strength was approximately 1.5 times higher than that of the commonly used P(AOD1) in methanol-induced cells. Although the expression of P(AOD1) and P(DAS1) was completely repressed by the presence of glucose, formate-induced expression of P(FDH1) was not repressed by glucose. Expression under P(PMP47), another methanol-inducible promoter, was highly induced by oleate. The induction kinetics of P(PMP47) and P(DAS1) revealed that methanol induces the expression of peroxisome membrane protein Pmp47, earlier than the expression of matrix enzyme dihydroxyacetone synthase (Das1p), and that this information is contained in the promoter region of the respective gene. This is the first report which evaluates several methanol-inducible promoters in parallel in the methylotrophic yeast.

Topics: Acid Phosphatase; Alcohol Oxidoreductases; Aldehyde-Ketone Transferases; Candida; Consensus Sequence; Enzyme Induction; Formate Dehydrogenases; Fungal Proteins; Gene Expression Regulation, Enzymologic; Genes, Reporter; Glucose; Membrane Proteins; Methanol; Peroxidases; Peroxiredoxins; Polymerase Chain Reaction; Promoter Regions, Genetic; Saccharomyces cerevisiae; Sequence Alignment