Regulation of alcohol oxidase gene expression in methylotrophic yeast Ogataea minuta

Abstract

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.

Keywords: Alcohol oxidase; Catabolite repression; Methylotrophic yeast; Ogataea minuta; Transcription factor.