From Shaker To Fermenter: Methanol-Free Pichia Pastoris Protein Production Workflow

By Ying Yang, Ma Sha, Bioprocess Applications Lab, Eppendorf, Inc.

The yeast Pichia pastoris has become an important and convenient workhorse for genetically engineered protein production in the biotechnology industry. Currently, most P. pastoris promoters used for efficient expression of heterologous proteins are derived from genes in the methanol metabolism pathway. For example, P_AOX1, the most widely used promotor, requires the switch to methanol feeding to activate protein production, referred to as methanol induction. Due to the flammable nature of methanol, this causes safety concerns in both academia and industry, especially when dealing with large volumes.

We explored the feasibility of a safer protein production alternative using a new P. pastoris strain carrying a strong methanol-independent promoter called pUPP. The pUPP promoter requires glycerol feeding for protein production, thus eliminating methanol induction and its affiliated safety risks. We carried out fed-batch P. pastoris fermentation runs for lipase production in the absence of methanol. This workflow was initiated in an Innova® S44i Biological Shaker for inoculum preparation, and followed with BioFlo ® 120 and BioFlo 320 bioprocess control systems for bench scale fermentation.