Efficient Synthesis of Sweet Protein Brazzein by Komagataella phaffii

In this study, the expression system of Komagataella phaffii GS115 was optimized for improved secretion of the sweet protein Brazzein. The recombinant strain K. phaffii Bra-1 was successfully obtained, which produced 90.2 mg/L of Brazzein in shake flask fermentation under 2% methanol, 0.08% biotin, 30 ℃ and initial pH 6.0. Selection and optimization of expression elements showed that the endogenous alcohol oxidase 1 (AOX1) promoter had the best expression performance. The αΔ57-70 signal peptide increased the expression of Brazzein to 183.2 mg/L, which was further increased to 209.3 mg/L by the co-expression regulatory element, disulfide bond isomerase (PDI). In a 5 L bioreactor, fed-batch fermentation at an initial OD₆₀₀ of 100 and a constant pH of 5.0 yielded 1.4 g/L of Brazzein. This study lays the foundation for the large-scale production and application of Brazzein.