Development of a protein production system using <i>Ogataea minuta</i> alcohol oxidase-deficient strain under reduced-methanol-consumption conditions

Methylotrophic yeast is a useful host for producing heterologous proteins using the unique and strong alcohol oxidase 1 (AOX1) promoter, which is induced by methanol and repressed by various carbon sources. However, methanol is preferably avoided in industrial-scale fermentation given its toxicity, flammability, and explosiveness. To develop a protein production system under reduced methanol supply conditions, we attempted to characterize the AOX1 promoter induction activity by comparing derepression conditions with methanol induction conditions. This comparison is important because decreasing methanol consumption would enhance the industrial value of Ogataea minuta for heterologous protein production. For such a comparison, an alcohol oxidase-deficient (Δaox) strain was generated, with methanol only being used for AOX1 promoter induction. We also developed a culture process in a jar fermentor using the O. minuta Δaox strain under mixed feed conditions to achieve heterologous protein production comparable to that of the wild-type strain under low-methanol conditions.