Efficient expression of heterologous protein by engineered Komagataella phaffii by harnessing a bioelectrical CO2 reduction system

Abstract

Methylotrophic yeast Komagataella phaffii (syn. Pichia pastoris) is an important host for heterologous protein expression although its traditional inducer methanol is toxic and flammable. Using green fluorescent protein (GFP) as a model of heterologous protein in this research, recombinant K. phaffii was constructed through wild-type formate dehydrogenase gene knockout, intracellular and extracellular expression of Candida boidinii formate dehydrogenase gene under sorbitol dehydrogenase promoter (PSDH) respectively to reduce carbon dioxide (CO2) to formate in a two-chamber H-shaped three-electrode bioelectrical reactor equipped with a solar panel stage by stage. Generated formate in the bioelectrical reactor was used as a safe inducer of engineered K. phaffii strains expressing GFP successfully. Further, the electrical potential was optimized and three cathodes, graphite disk electrode, graphite rod electrode, and glassy carbon electrode, were used and modified subsequently with poly(neutral red) to obtain the highest volumetric GFP intensity and specific GFP intensity as 4424.5 a.u. and 384.0 a.u./OD600, which was 1.7- and 3.2- fold of that of methanol induction respectively, 3.9-fold of that formate induction. Therefore, a safe recombinant K. phaffii system was developed successfully for heterologous protein expression, and demonstrated a new approach to utilize CO2.