Evolution-Guided Engineering of Non-conserved N-Glycosylation Sites Improves Heterologous Production of Sulfotransferases
- 1Shandong university
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.
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BibTeX
@misc{li2026evolutionguided,
title = {{Evolution-Guided Engineering of Non-conserved N-Glycosylation Sites Improves Heterologous Production of Sulfotransferases}},
author = {C. Li and Y. Li and X. Li and Y. Zhao and J. Sun and Y. Cao and X. Guo and S. Zheng and J. Deng and J. Sheng},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.18.759982},
url = {https://www.biorxiv.org/content/10.64898/2026.10.18.759982v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Evolution-Guided Engineering of Non-conserved N-Glycosylation Sites Improves Heterologous Production of Sulfotransferases
AU - C. Li
AU - Y. Li
AU - X. Li
AU - Y. Zhao
AU - J. Sun
AU - Y. Cao
AU - X. Guo
AU - S. Zheng
AU - J. Deng
AU - J. Sheng
PY - 2026
DA - 2026/10/18
PB - bioRxiv
DO - 10.64898/2026.10.18.759982
UR - https://www.biorxiv.org/content/10.64898/2026.10.18.759982v1
AB - 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.
ER -