Engineering Modification Enhances the Antibacterial Activity of Yak Gastric Lysozyme and Its High-Level Expression in Pichia pastoris

To broaden the antibacterial spectrum of yak gastric lysozyme (YGL) against Gram-negative bacteria and improve its secretory expression in Pichia pastoris, this study employed a combined strategy of functional short-peptide fusion and host engineering. Four hydrophobic peptides, FFVAP, GVSLANWVCLAK, VVYP, and AWVAWK, were individually fused to the C-terminus of YGL, and the enzymatic activity and antibacterial performance of the modified proteins were compared. AW-YGL was subsequently selected for sequential optimization of the host strain, secretion signal peptide, endoplasmic-reticulum-associated molecular chaperones, and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated host-gene knockout, followed by physicochemical stability evaluation of the optimized product. The results showed that AWVAWK modification produced the most pronounced enhancement of YGL activity against Gram-negative bacteria. AW-YGL exhibited inhibition rates of 65%, 68%, and 67% against Escherichia coli, Pseudomonas aeruginosa, and Salmonella enteritidis, respectively. During expression-system optimization, the GS115 host significantly improved AW-YGL secretion, with total enzyme activity and specific activity in the culture supernatant reaching 3.1 × 105 U/L and 983 U/mg, respectively. Replacement with the HSS signal peptide further increased total enzyme activity to 4.3 × 105 U/L and specific activity to 1264 U/mg. Co-expression of the endoplasmic reticulum molecular chaperone binding immunoglobulin protein (BIP) further promoted functional AW-YGL expression, increasing the specific activity to 1560.3 U/mg. On this basis, knockout of the cell-wall-related gene FKS1 further increased the total enzyme activity in the culture supernatant to 6.9 × 105 U/L and the specific activity to 1638.3 U/mg, indicating that stepwise optimization of the signal peptide, folding assistance, and host-cell engineering effectively enhanced AW-YGL secretory expression and enzymatic activity. The optimized AW-YGL retained relatively high activity over defined pH and temperature ranges and showed tolerance to several metal ions and digestion-related proteases. Collectively, C-terminal fusion with hydrophobic short peptides enhanced the inhibitory activity of YGL against Gram-negative bacteria, while the combined strategies of signal-peptide optimization, folding assistance, and cell-wall engineering further improved its secretory expression in P. pastoris. These findings provide an experimental basis for the engineering and scalable production of novel broad-spectrum lysozymes.