Response Surface Methodological Approach for Optimizing the Expression of Recombinant Ganoderma leucocontextum Immunomodulatory Protein in Pichia pastoris

Abstract

Ganoderma leucocontextum immunomodulatory protein 2 (FIP-gle2) is a recently identified small molecule protein. Based on the successful expression of FIP-gle2 in Pichia pastoris GS115, this study optimized the expression conditions of the engineered P. pastoris using response surface methodology (RSM). Initially, single-factor experiments determined that the optimal parameters for subsequent optimization were a cultivation period of 4 days, a temperature range of 26 to 30 °C, an initial pH of 6.0 to 7.0, and a methanol (MeOH) concentration of 0.75% to 1.25%. Subsequently, a box-behnken design (BBD)-based RSM was employed, with the total protein concentration in the fermentation broth serving as the detection indicator. The optimal fermentation conditions were determined to be a cultivation temperature of 27 °C, an initial pH of 6.7, and a MeOH concentration of 1.11%. Under these conditions, the actual operational parameters were set at a fermentation temperature of 27 °C, an initial pH of 6.5, and a MeOH concentration of 1%, resulting in an average total protein concentration of 225.12 mg·L-1. This study provides preliminary optimization of the cultivation conditions for P. pastoris, offering valuable data support for the industrial production of this protein.