[Expression of recombinant fibronectin FNIII8-10 in Pichia pastoris and fermentation process optimization]

The 8-10 repeat of fibronectin (FN) type III domain (FNIII8-10) contains multiple integrin-binding sites and serves as the core module mediating cell adhesion. Currently, recombinant FNIII8-10 is expressed only in prokaryotic microorganisms. This study aims to establish a Pichia pastoris expression system for the high-level production of recombinant FNIII8-10. Although the recombinant FNIII8-10 was successfully expressed in Pichia pastoris, the secretion efficiency was low. Through rational signal peptides design and fermentation optimization, secretion efficiency was significantly improved. Screening of multiple signal peptides identified the α-factor signal peptide as the most efficient. Site-directed mutagenesis of its hydrophobic core generated the high-efficiency mutant α-V50A, which increased secreted protein yield by 44.8%. Using the response surface methodology, fermentation conditions were optimized to 117.46 h, pH 6.91, and 1.91% (V/V) methanol supplementation. Meanwhile, with sorbitol as an auxiliary carbon source to alleviate methanol stress, the yield of the recombinant FNIII8-10 reached 65.49 mg/L. Finally, high-cell-density fermentation achieved a yield of 532.82 mg/L, representing a 7-fold increase compared with the highest yield in shake flasks. This study lays a technical foundation for the industrial-scale production of recombinant FNIII8-10.