Expression Profiling of Recombinant Biofilm Surface Layer Protein A in Pichia pastoris Under Constant Dissolved Oxygen and Oxygen-Limited Fermentation

BslA (Biofilm surface layer protein A), a highly hydrophobic lipoprotein from Bacillus spp., self-assembles at fluid interfaces to form a crystalline film that reduces surface tension. In this study, we selected Pichia pastoris as a eukaryotic system for expressing recombinant BslA identified in Bacillus paralicheniformis BL-1. The secretory expression of recombinant BslA in the P. pastoris GS115 strain under the AOX1 promoter was confirmed in shake-flask cultivation. Next, two fed-batch fermentation strategies, constant dissolved oxygen strategy (DO-stat) and oxygen-limited fed-batch (OLFB) strategy, in a 5 L scale, were compared. The DO-stat process led to late-stage cell death and product degradation, limiting yields. Switching to the OLFB process by removing the glycerol feeding phase mitigated this issue, allowing extended fermentation and increasing the final recombinant BslA concentration to 657 mg/L. This study establishes P. pastoris with an OLFB strategy as an effective system for secreting recombinant BslA protein, providing a basis for future industrial-scale production.