High-efficiency patatin expression strategies in Komagataella phaffii (Pichia pastoris): Expression cassette toolbox and regulation of protein secretion

Patatin, a prominent food protein derived from potatoes, is renowned for its exceptional nutritional value. Patatin has been characterized for its diverse physiological attributes, including esterase activity, antioxidative properties, cholesterol-lowering effects, and high lysine content, alongside notable physicochemical traits such as foaming, emulsification, and gelation capabilities. Conventional methods for patatin extraction are fraught with inefficiencies, elevated costs, and detrimental impacts on protein structural and functional integrity. Herein, we leveraged an optimized strategy integrating an expression cassette toolbox and regulation of protein secretion to harness Komagataella phaffii as the expression host and achieved an expression level of 3.2 g per litre (g/L) in a 5-Litre bioreactor, which is the highest yield of patatin production using engineered bacteria and funguses that has been reported thus far. In this study, we innovatively refined the endogenous promoter P_CAT1, and its efficacy in driving heterologous protein expression under methanol induction surpassed that of the conventional AOX1 promoter. Furthermore, crucial nodes for patatin heterologous expression in yeast were identified, substantially curtailing the production costs associated with patatin synthesis.