肽-N-糖苷酶介导的胞内糖蛋白编辑技术在酿酒酵母中的构建及应用研究

杨雨晗; 逯浩睿; 田朝光

doi:10.12211/2096-8280.2026-026

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肽-N-糖苷酶介导的胞内糖蛋白编辑技术在酿酒酵母中的构建及应用研究

研究论文 | 更新时间：2026-06-11

- 肽-N-糖苷酶介导的胞内糖蛋白编辑技术在酿酒酵母中的构建及应用研究
- Construction and Preliminary Application of PNGase-Mediated Intracellular Glycoprotein Editing Technology in Saccharomyces cerevisiae
- 合成生物学 2026年页码：1-14
- 作者机构：
  
  1.中国科学院天津工业生物技术研究所，低碳合成工程生物学全国重点实验室，天津 300308
  2.合成生物国家技术创新中心，天津 300308
- 作者简介：
  
  杨雨晗（1999—），女，硕士研究生。研究方向为生物化学与分子生物学。
  田朝光（1973—），男，研究员，博士，博士生导师。研究方向为真菌合成生物学等。
- 基金信息：
  
  低碳合成工程生物学全国重点实验室开放课题(E5J4QZKF04)
- DOI：10.12211/2096-8280.2026-026
  中图分类号： Q939.9
- 收稿：2026-04-17，
  
  修回：2026-06-10，
  
  网络首发：2026-06-11，
- 稿件说明：
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杨雨晗，逯浩睿，田朝光. 肽-N-糖苷酶介导的胞内糖蛋白编辑技术在酿酒酵母中的构建及应用研究［J］. 合成生物学， 2026， 7. DOI： 10.12211/2096-8280.2026-026

YANG Yuhan， LU Haorui， TIAN Chaoguang. Construction and Preliminary Application of PNGase-Mediated Intracellular Glycoprotein Editing Technology in Saccharomyces cerevisiae［J］. Synthetic Biology Journal， 2026， 7. DOI： 10.12211/2096-8280.2026-026
杨雨晗，逯浩睿，田朝光. 肽-N-糖苷酶介导的胞内糖蛋白编辑技术在酿酒酵母中的构建及应用研究［J］. 合成生物学， 2026， 7. DOI： 10.12211/2096-8280.2026-026 DOI：

YANG Yuhan， LU Haorui， TIAN Chaoguang. Construction and Preliminary Application of PNGase-Mediated Intracellular Glycoprotein Editing Technology in Saccharomyces cerevisiae［J］. Synthetic Biology Journal， 2026， 7. DOI： 10.12211/2096-8280.2026-026 DOI：

摘要

N-糖基化是真核生物中广泛存在的一类翻译后修饰，在蛋白质折叠、分泌转运、环境响应等过程中发挥重要作用。肽-N-糖苷酶（PNGase，简称糖肽酶）催化糖蛋白完整N-糖链的去除，并伴随该位点天冬酰胺残基到天冬氨酸残基的序列编辑。本研究首先通过构建糖肽酶的表达菌株，结合定量糖蛋白组学分析，比较了酿酒酵母（

Saccharomyces cerevisiae

）Png1 H1结构域缺失突变体（Png1ΔH1）以及伊丽莎白金氏菌（

Elizabethkingia miricola

）PNGF在酿酒酵母中的去糖基化活性与去糖基化蛋白组，进一步比较研究了不同来源糖肽酶对选定靶标蛋白的去糖基化编辑效果，研究发现，不同糖肽酶存在显著差异。随后，本研究利用Coh-Doc蛋白间相互作用系统构建了针对糖蛋白Cdc50和Ecm38靶向去糖基化编辑器，结果显示，编辑器可显著增强糖肽酶对靶标蛋白的去糖基化编辑效率。总之，本研究探索利用糖肽酶的蛋白质去糖基化功能，以酿酒酵母为模式体系，开发了细胞内目标糖蛋白去糖基化编辑技术，实现了对目标糖蛋白去糖基化编辑，为未来进一步开发可用于生命科学研究和疾病治疗的糖蛋白编辑技术奠定了基础。

Abstract

N-linked glycosylation is a widespread post-translational modification in eukaryotes and plays essential roles in protein folding

secretory trafficking

and environmental responses. Peptide:N-glycanase (PNGase) catalyzes the removal of entire N-linked glycans from glycoproteins

notably converting the glycosylated asparagine residue to aspartic acid and thus achieving an amino acid substitution without altering the genome. This study utilized

the model eukaryote

Saccharomyces cerevisiae

taking advantage of its sophisticated genetic toolbox and numerous conserved eukaryotic characteristics

to investigate glycoproteomics and deglycosylation editors. In this study

we first knocked out the endogenous PNG1 and

on this basis

constructed PNGase-expressing

S. cerevisiae

strains. Using quantitative glycoproteomics

we compared the deglycosylation activities and deglycosylated proteomes of the endogenous Png1 H1-domain deletion mutant (Png1ΔH1) and

Elizabethkingia miricola

PNGase F (PNGF) expressed in

S. cerevisiae

. The results showed that PNGF and PNG1 shared some common substrates in

S. cerevisiae

whereas PNGF exhibited more distinct peptide- and site-level differences

suggesting that the exogenous PNGase possesses a substrate spectrum and application potential that are not fully consistent with those of the endogenous PNG1.Further comparative analyses revealed significant differences in the deglycosylation editing efficiencies of PNGases from different sources toward selected target proteins. To further improve deglycosylation efficiency and explore the potential of PNGases as protein-editing tools

using the cohesin-dockerin (Coh-Doc) protein interaction system

this study constructed targeted deglycosylation editors for the glycoproteins Cdc50 and Ecm38. The results showed that this system enhanced the interaction between PNGase and target proteins

and the editors markedly enhanced the deglycosylation efficiency of PNGase toward the target proteins. Moreover

this editor can

to some extent

compensate for the limited spatial accessibility of heterologous PNGases in host cells

demonstrating promising cross-species adaptability.Finally

by genomically inserting a Coh2 tag in situ at the Cdc50 locus

we achieved efficient deglycosylation editing under physiological conditions

reaching a deglycosylation efficiency of 44.8%. This study explores the protein deglycosyla

tion function of PNGase and

using

S. cerevisiae

as a model system

establishes an intracellular deglycosylation editing technology for target glycoproteins

laying a foundation for future development of glycoprotein editing tools for life science research and disease therapy.

关键词

Keywords

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