Methanol-Inducible Promoter of Thermotolerant Methylotrophic Yeast Ogataea thermomethanolica BCC16875 Potential for Production of Heterologous Protein at High Temperatures

Abstract

Methanol-utilizing metabolism is generally found in methylotrophic yeasts. Several potential promoters regulating enzymes in this pathway have been extensively studied, especially alcohol oxidase. Here, we characterized the alcohol oxidase gene promoter from thermotolerant Ogataea thermomethanolica (OthAOX). This promoter can be induced by methanol, and was shown to regulate expression of phytase up to 45 °C. The pattern of heterologous phytase N-glycosylation depends on the induction temperature. Unlike the AOX promoter from Pichia pastoris, this OthAOX initially turns on the expression of the heterologous protein at the de-repression stage in the presence of glycerol. Full induction of protein is observed when methanol is present. With this methanol-inducible promoter, target protein can be initially produced prior to the induction phase, which would help shorten the time for protein production. Being able to drive protein expression at various temperatures prompts this newly identified AOX promoter to be potential tool for heterologous protein production in high temperature conditions.