{
  "abstract": "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.",
  "abstract_shown": true,
  "affiliations": [
    "College of Animal Science and Technology, China Agricultural University, Beijing 100083, China"
  ],
  "article_type": null,
  "arxiv": null,
  "author_total": 4,
  "authors": [
    {
      "affs": [
        0
      ],
      "name": "Bing Bing Fan"
    },
    {
      "affs": [
        0
      ],
      "name": "An Rong Zhang"
    },
    {
      "affs": [
        0
      ],
      "name": "Zhen Long Wu"
    },
    {
      "affs": [
        0
      ],
      "name": "Ying Yang"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/febdrfgq8861n.bib",
  "cited_by": 0,
  "doi": "10.3390/ijms27219403",
  "fulltext_url": null,
  "id": "febdrfgq8861n",
  "issns": [
    "1422-0067"
  ],
  "landing_url": "https://www.mdpi.com/1422-0067/27/21/9403",
  "language": null,
  "licence": "CC-BY-4.0",
  "licence_label": "CC BY 4.0",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "openalex",
      "2404|Antimicrobial Peptides and Activities"
    ]
  ],
  "openalex": "W7223403500",
  "pdf_url": null,
  "pmcid": null,
  "pmid": null,
  "published": "2026-10-22",
  "publisher": "MDPI AG",
  "ris": "https://research.draconitic.com/paper/febdrfgq8861n.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:18:57Z",
      "native_id": "10.3390/ijms27219403",
      "source": "crossref",
      "url": "https://www.mdpi.com/1422-0067/27/21/9403"
    }
  ],
  "subjects": [
    {
      "field": "biology",
      "origin": "openalex:2404|Antimicrobial Peptides and Activities",
      "subfield": "microbiology"
    }
  ],
  "title": "Engineering Modification Enhances the Antibacterial Activity of Yak Gastric Lysozyme and Its High-Level Expression in Pichia pastoris",
  "title_text": "Engineering Modification Enhances the Antibacterial Activity of Yak Gastric Lysozyme and Its High-Level Expression in Pichia pastoris",
  "type": "peer-reviewed",
  "updated_at": "2026-10-30T12:18:57Z",
  "url": "https://research.draconitic.com/paper/febdrfgq8861n",
  "venue": "International Journal of Molecular Sciences",
  "versions": []
}