Methylotrophic yeast engineering for secreted protein production via precision fermentation: food ingredient proteins and synthetic biology strategies.

Methylotrophic yeasts are outstanding platforms for the production of recombinant proteins through precision fermentation. Growing demand for protein-based food ingredients requires economically viable secretion efficiency and space-time yields, as well as correct post-translational modifications to ensure proper food functionality. Here, we review recent advances and future potential in engineering methylotrophic yeasts for food protein production, with particular focus on Komagataella phaffii. We first summarise the production of three categories of protein food ingredients: recombinant dairy proteins, proteins for plant-based and cultivated meat applications, and sweet-tasting proteins. Addressing the dual goals of improving both yield and quality, we then review synthetic biology and metabolic engineering strategies in methylotrophic yeasts, with emphasis on genome engineering and gene expression tools, protein secretory pathway engineering, and glycoengineering. Further advances in synthetic biology, integrated with industrial process development, will be critical to unlocking future precision fermentation systems based on methylotrophic yeasts.