Abstract
High-cell-density fermentation is a critical aspect of industrial protein production, requiring the selection of an optimal growth medium and carbon source. Pichia pastoris, a methylotrophic yeast, has been established as a widespread recombinant protein expression system in the food and pharmaceutical industries. The primary objective of this work was to create a superior platform for producing alternative proteins thus contributing to future innovation in these sectors. This study compared three wild-type strains, with two of them also analyzed in their diploid versions, using shake flasks and bioreactors. It investigated glucose and glycerol as carbon sources using mCherry as a protein model. Glycerol emerged as the preferred carbon source, resulting in over 40% increase in biomass concentrations compared to glucose across all strains. Notably, wild-type strain Y-7556 reached an exceptional biomass concentration of 244 g DCW/L in just 48 h, the highest reported to date, highlighting the potential of high-cell-density fermentation in P. pastoris. Regarding protein expression, the diploid version of Y-11430 produced >43% of purified mCherry protein after 123 h of fermentation, compared to the haploid counterpart. Our findings underscore the advantages of diploid strains, optimized fermentation media, and carbon source selection, effectively addressing crucial gaps in the literature.