Abstract: 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.

Key words: sweet protein; Brazzein; Komagataella phaffii; promoter; signal peptide