Retrofitting of AOX1 Promoter-Based Pichia pastoris Expression Strains for Methanol-Independent Protein Production

The classical Pichia pastoris expression system is built on derivative strains of Komagataella phaffii NRLL Y-11430 and relies on strong methanol-inducible promoters that can be tightly regulated like the alcohol oxidase 1 promoter (P_AOX1). The P_AOX1 is tightly repressed by carbon sources such as glucose or glycerol and is strongly induced upon methanol induction in absence of repressing carbon sources. However, induction with toxic and flammable methanol is a considerable safety risk during storage and handling, and was also discussed to cause metabolic stress and increased cell lysis. Furthermore, methanol is not compatible with many existing production plants; therefore, prompting the search for innovative processes that allow alternative inducible, derepressed, or constitutive high-level protein production without the use of methanol. Alternatively, methanol-free protein production has been achieved by the overexpression of single transcription factors, which among others regulate diverse methanol utilization genes like the AOX1 gene. This approach also allows to convert (retrofit) existing methanol-dependent production strains to efficient methanol-independent systems or even to increase the productivity of such strains by simple overexpression of intrinsic Pichia pastoris genes. In this chapter, we present an approach to retrofit P_AOX1-based expression strains for methanol-independent expression based on a simple example using the intracellular reporter protein eGFP.