On May 26, 2025, Hao, Liao, and Lyu from the State Key Laboratory of Pathogen and Biosecurity at the Beijing Institute of Biotechnology published a paper titled "Biosynthesis of Two Types of Exogenous Antigenic Polysaccharides in a Single Escherichia coli Chassis Cell" in Life. This work introduces a robust Synthetic Biology Platform that successfully engineers a single E. coli host to simultaneously produce two structurally distinct Antigenic Polysaccharides. Crucially, the engineered cell also performs the subsequent in vivo bioconjugation, resulting in the efficient, one-step production of two separate glycoprotein (conjugate vaccine) candidates within a single cellular environment, simplifying the historically complex process of multivalent vaccine manufacturing.

Overview

The urgency of this work is rooted in the accelerating global crisis of antimicrobial resistance (AMR), with pathogens like E. coli and Klebsiella pneumoniae posing serious public health threats. Conjugate vaccines, which rely on immunogenic bacterial surface polysaccharides (antigens) chemically linked to an immune-stimulating carrier protein, represent a highly effective preventive strategy. However, creating these vaccines, especially multivalent formulations targeting multiple strains or pathogens, is technically challenging, often requiring multiple separate fermentation and purification stages followed by inefficient chemical conjugation. The lack of scalable, cost-effective methods for producing multivalent glycoconjugates has been a major barrier, which this synthetic biology platform seeks to overcome.

Research Results

• Dual-Pathway Engineering for Polysaccharide Co-Expression

The core innovation was the parallel introduction of two distinct polysaccharide biosynthetic gene clusters, representing two major mechanisms: the Wzy/Wzx-dependent pathway and the ABC transporter-dependent pathway. The host, E. coli W3110, was successfully transformed and induced to express both pathways concurrently. Experimental evidence confirmed the functional production of both target polysaccharides, demonstrating that the single Bacterial Chassis can manage the complex, dual metabolic demands required for generating two structurally diverse carbohydrate antigens.

Fig.1 Construction and characterization of the ECO1 and KPO2α biosynthetic polysaccharide plasmid. (Hao, et al., 2025)

• One-Cell System for In Vivo Glycoprotein Bioconjugation

The ultimate goal of this research was to streamline the production of conjugate vaccines. To achieve this, the engineered E. coli Chassis was further modified to express carrier proteins and the necessary oligosaccharyl transferase enzymes. The synthesized polysaccharides were then successfully linked to their respective carrier proteins in vivo via a process known as bioconjugation. This established a self-contained, one-cell production system capable of producing two complete glycoprotein conjugates, eliminating the need for complex, costly, and yield-limiting in vitro chemical conjugation steps.

• Molecular Characterization and Pathway Optimization Insights

The researchers employed whole-cell proteomic profiling followed by MFUZZ clustering and gene ontology (GO) analysis to validate the engineered biosynthetic routes and understand potential metabolic bottlenecks. The analysis revealed that core biosynthetic genes segregated into two functional clusters, largely localized to the cytoplasm and enriched in translation and protein binding pathways. While a weak competitive interaction for shared membrane space or activated monosaccharide precursors was observed during initial induction, the overall polysaccharide yield remained stable over extended induction periods. This detailed molecular insight is invaluable for guiding future rational design and optimization efforts in synthetic glycobiology.

Conclusion

This study marks a significant leap forward for synthetic glycobiology, presenting a robust platform that successfully integrates two distinct polysaccharide production and conjugation systems into a single Microbial Cell Factory. By achieving the simultaneous biosynthesis and in vivo bioconjugation of two disparate glycoprotein vaccines, the research dramatically simplifies the production workflow, offering a scalable and cost-effective strategy for generating multivalent conjugate vaccines against multidrug-resistant pathogens. This innovation paves the way for the rational design of highly complex, next-generation glycan-based therapeutics and diagnostics, demonstrating the immense potential of microbial chassis cells as unified biological production systems.