Regulation of two distinct alcohol oxidase promoters in the methylotrophic yeast Pichia methanolica.
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.
Nakagawa
Tomoyuki
T
Department of Food Science and Technology, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri, Hokkaido 099-2493, Japan.
Inagaki
Atsushi
A
Ito
Takashi
T
Fujimura
Shuki
S
Miyaji
Tatsuro
T
Yurimoto
Hiroya
H
Kato
Nobuo
N
Sakai
Yasuyoshi
Y
Tomizuka
Noboru
N
D000135:Acid Phosphatase / Q000235:genetics / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D006651:Histocytochemistry; D000432:Methanol / Q000378:metabolism; D016254:Mutagenesis, Insertional; D010100:Oxygen / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*
D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D000135:Acid Phosphatase; D010100:Oxygen; D000432:Methanol