On March 19, 2024, Bao et al. from Dalian University in China published a paper titled "Construction of an Escherichia coli chassis for efficient biosynthesis of human-like N-linked glycoproteins" in Frontiers in Bioengineering and Biotechnology. This paper details the successful construction of a stable, high-performance Escherichia coli Chassis cell factory designed for the efficient biosynthesis of human-like N-linked glycoproteins. Their core achievement is establishing an integrated genomic platform that overcomes the stability and yield limitations of previous multi-plasmid systems, culminating in the production of high-titer, homogeneously glycosylated and terminally sialylated therapeutic protein mimics.

Overview

Glycoproteins, which are proteins post-translationally modified by carbohydrates (glycans), are the most complex and important class of biopharmaceuticals, including therapeutic antibodies and hormones. Historically, these drugs have been produced in expensive and complex mammalian cell cultures (like CHO cells), which often result in heterogeneous mixtures of glycan structures. This heterogeneity compromises drug efficacy, immunogenicity, and stability. The field of microbial glyco-engineering seeks to address this by using robust and cost-effective bacteria, such as E. coli, as a stable chassis to produce specific, single, "homogeneous" Human-like Glycan Structures. This study builds upon decades of foundational work, aiming to surpass limitations like low glycosylation efficiency and the instability of multi-plasmid systems, which were previously bottlenecks for industrial scalability.

Research Results

• Genomic Integration for System Stability

A critical challenge in microbial glycoprotein production is maintaining the stability and copy number of multiple plasmids encoding the requisite glycosylation pathway enzymes. The authors addressed this by adopting a genomic integration strategy. They successfully performed the tandem integration of the oligosaccharyltransferase pglB (essential for linking the glycan to the protein) and the full glycosyltransferase cluster lsgCDEF into the E. coli XL1-Blue genome. This integration replaced the native enterobacterial common antigen (ECA) and the nanKETA gene clusters, eliminating competing metabolic pathways and providing a stable, "plug-and-play" expression scaffold that is inherently superior to transient plasmid-based systems.

• Optimizing Promoter Strength for Maximum Output

Achieving maximum glycosylation efficiency and yield requires fine-tuning the expression levels of the integrated glycosylation genes. The team systematically optimized the regulatory elements by testing promoters of different strengths (such as Ptac and PlacUV5) to control the transcription of the integrated genes. This meticulous genetic optimization resulted in an Engineered Chassis that, when paired with a target protein expressed on a separate plasmid, achieved a 100% tetrasaccharide modification efficiency. Furthermore, this engineered strain produced the highly modified protein at a remarkable yield of approximately 320 mg/L—an outstanding output for a complex recombinant glycoprotein in a bacterial system.

Fig.1 Enhancement of biosynthesizing human-like glycoproteins by promoter optimization in chassis strains. (Bao, et al., 2024)

• Constructing and Sialylating the Human-Like Glycan

The ultimate goal for biotherapeutic agents is to mimic full human glycoforms, which are often capped by sialic acid residues to enhance pharmacokinetics. The paper demonstrates this functional extension by constructing the metabolic pathway necessary for sialylation (CMP-sialic acid synthesis) and expressing alpha-2,3-sialyltransferase within the high-yield chassis. This modification effort resulted in the successful terminal alpha-2,3-sialylation of the glycoprotein with an efficiency of 40%. Achieving this specific alpha-2,3 linkage is highly significant for generating biologically active compounds, and the process yielded a substantial 65 mg/L of the homogeneously sialylated product in flask cultures.

Fig.2 Production of human-like sialylated N-glycoproteins in glyco-engineered E. coli. (Bao, et al., 2024)

Conclusion

By successfully integrating the complete synthesis pathway for a core human-like tetrasaccharide (Gal-beta-1,4-GlcNAc-beta-1,3-Gal-beta-1,3-GlcNAc-) and optimizing its expression for maximum yield and 100% efficiency, Bao et al. have created a truly "plug-and-play" system. The subsequent success in establishing terminal alpha-2,3-sialylation is particularly exciting, as this specific linkage is vital for the in vivo function of many therapeutic drugs. The stability and high productivity of this glyco-engineered E. coli chassis provide a strong, cost-effective foundation for advancing the development of a diverse range of next-generation sialylated glycoprotein pharmaceuticals.