{
  "abstract": "Escherichia coli features low cost, mature fermentation technology and high recombinant protein output, but the expression of glycosylation-dependent eukaryotic proteins therein is severely limited. Many glycosylated proteins are key tool enzymes for the biosynthesis of various pharmaceutical molecules, such as glycosaminoglycan-related synthases, but there is a lack of universal protein engineering methods to enable the adaptive evolution of these enzymes in E. coli. We established an evolution-guided optimization strategy that targets non-conserved N-glycosylation sites (EGO-NGly). The core of this strategy lies in the phylogeny-guided selective reversion of evolutionarily acquired N-glycosylation sequons to naturally occurring non-glycosylated residues from early-divergent orthologs, thereby improving heterologous production and residual activity stability for selected sulfotransferases in E. coli. Using heparin 3-O-sulfotransferase 1 (3OST-1) as a model enzyme apparatus in eukaryotic organisms, EGO-NGly extended the residual activity half-life by 4.5-fold. Structural analysis and molecular dynamics simulations indicate that greater surface rigidity and remodeling of intramolecular interaction networks contribute to the improved conformational stability of M7 in an aglycosylated host. EGO-NGly was further applied to a panel of 3OST and 6OST homologs, demonstrating its applicability to a subset of the tested enzymes. Furthermore, EGO-NGly can be orthogonally integrated with other complementary protein engineering strategies, as exemplified by its combination with the machine learning-aided Protein Repair One-Stop Service, generating variants with 8-fold and 17-fold extensions in residual activity half-life. Collectively, these results establish EGO-NGly as a practical and potentially cost-effective engineering strategy for improving the prokaryotic expression and functional performance of selected glycosylation-dependent eukaryotic sulfotransferases.",
  "abstract_shown": true,
  "affiliations": [
    "Shandong university"
  ],
  "article_type": "New results",
  "arxiv": null,
  "author_total": 10,
  "authors": [
    {
      "name": "C. Li"
    },
    {
      "name": "Y. Li"
    },
    {
      "name": "X. Li"
    },
    {
      "name": "Y. Zhao"
    },
    {
      "name": "J. Sun"
    },
    {
      "name": "Y. Cao"
    },
    {
      "name": "X. Guo"
    },
    {
      "name": "S. Zheng"
    },
    {
      "name": "J. Deng"
    },
    {
      "affs": [
        0
      ],
      "name": "J. Sheng"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/8e12acnb0rwd7.bib",
  "cited_by": 0,
  "doi": "10.64898/2026.10.18.759982",
  "fulltext_url": "https://www.biorxiv.org/content/10.64898/2026.10.18.759982v1.full",
  "id": "8e12acnb0rwd7",
  "issns": [],
  "landing_url": "https://www.biorxiv.org/content/10.64898/2026.10.18.759982v1",
  "language": null,
  "licence": "CC-BY",
  "licence_label": "CC BY",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "biorxiv",
      "biochemistry"
    ],
    [
      "openalex",
      "1312|Enzyme Catalysis and Immobilization"
    ]
  ],
  "openalex": "W7223057606",
  "pdf_url": "https://www.biorxiv.org/content/10.64898/2026.10.18.759982v1.full.pdf",
  "pmcid": null,
  "pmid": null,
  "published": "2026-10-18",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/8e12acnb0rwd7.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:28:46Z",
      "native_id": "10.64898/2026.10.18.759982",
      "source": "biorxiv",
      "url": "https://www.biorxiv.org/content/10.64898/2026.10.18.759982v1"
    }
  ],
  "subjects": [
    {
      "field": "biochemistry",
      "origin": "openalex:1312|Enzyme Catalysis and Immobilization",
      "subfield": "molecular-biology"
    }
  ],
  "title": "Evolution-Guided Engineering of Non-conserved N-Glycosylation Sites Improves Heterologous Production of Sulfotransferases",
  "title_text": "Evolution-Guided Engineering of Non-conserved N-Glycosylation Sites Improves Heterologous Production of Sulfotransferases",
  "type": "preprint",
  "updated_at": "2026-10-30T12:28:46Z",
  "url": "https://research.draconitic.com/paper/8e12acnb0rwd7",
  "venue": "bioRxiv",
  "versions": []
}