Regulation of two distinct alcohol oxidase promoters in the methylotrophic yeast Pichia methanolica

In this study, two Pichia methanolica alcohol oxidase (AOD) promoters, P MOD 1 and P MOD 2 , 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 MOD 1 and P MOD 2 promoters was found to be strong and tightly regulated by carbon source at the transcriptional level. P MOD 1 was induced not only by methanol but also by glycerol. P MOD 2 was induced only by methanol, although it was not repressed on the addition of glycerol to a methanol medium, suggesting that P MOD 2 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 MOD 1 and P MOD 2 . P MOD 1 expression was optimal at low methanol concentrations, whereas P MOD 2 was predominantly expressed at high methanol and high oxygen concentrations. Based on these results, both P MOD 2 and P MOD 1 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 MOD 1 and P MOD 2 in the same host cell and in the same flask. Copyright © 2006 John Wiley & Sons, Ltd.