Genetic engineering of E. coli K-12 for heterologous carbohydrate antigen production

作者：Caixia Li, Hongxu Zha, Ziyan Jiao, Keyan Wei, Huaiyu Gao, F.A. Lai, Zuoyong Zhou, Hongyan Luo, Pei Li · 发表于：Microbial Cell Factories · 年份：2025 · DOI：10.1186/s12934-025-02749-2 · 被引用次数：3 · 研究领域：Escherichia coli research studies、Cancer Research and Treatments、Transgenic Plants and Applications

BACKGROUND: Carbohydrate-based vaccines have made a remarkable impact on public health over the past three decades. Efficient production of carbohydrate antigens is a crucial prerequisite for the development of such vaccines. The enzymes involved in the synthesis of bacterial surface carbohydrate antigens are usually encoded by large, uninterrupted gene clusters. Non-pathogenic E. coli glycoengineering starts with the genetic manipulation of these clusters. Heterologous gene cluster recombination through an expression plasmid has several drawbacks, including continuous antibiotic selection pressure, genetic instability, and metabolic burdens. In contrast, chromosome-level gene cluster expression can minimize the metabolic effects on the host and reduce industrial costs. RESULTS: In this study, we employed the suicide vector-mediated allelic exchange method to directly replace the native polysaccharide gene clusters in E. coli with heterologous ones. Unlike previously strategies, this method does not rely on I-SceI endonuclease or CRISPR/Cas system to release the linearized DNA insert and λ-red recombinase to promote its homologous recombination. Meanwhile, the vectors could be conveniently constructed by assembling multiple large DNA fragments in order in vitro. The scarless chromosomal insertions were confirmed by whole-genome sequencing and the polysaccharide phenotypes of all glycoengineered E. coli mutants were evaluated through growth curves, silver staining, western blo...