Optimization of heterologous protein production process using genetically engineered Ogataea minuta toward industrial-scale manufacturing.

We have developed the methylotrophic yeast Ogataea minuta as a useful host for producing heterologous proteins. In this study, a double mutant that lacks the Prb1 protease and alcohol oxidase was generated and applied for heterologous protein production. Upon our optimization of the fermentation conditions, such as feeding of carbon and nitrogen sources and pH control, this mutant showed increased production of human serum albumin, resulting in a yield of approximately 7.5 g/L at 21 days in the production phase. The established optimal fermentation condition with the double mutant was successfully applied to manufacture a candidate biologic protein: lipocalin derivative which is designed to attach to the calcitonin gene-related peptide, on an industrial scale. The candidate biologic protein was successfully manufactured at an industrial scale in a 4500 L bioreactor under well-controlled conditions. This first successful case highlights the potential of our O. minuta-based production system, and this study is helpful for a large-scale cultivation of a methylotrophic yeast for protein production.