On Apr 21, 2022, Kay et al., primarily from the London School of Hygiene & Tropical Medicine (LSHTM), published a paper in the journal Microbial Cell Factories titled "Engineering a suite of E. coli strains for enhanced expression of bacterial polysaccharides and glycoconjugate vaccines". The paper details the successful refactoring of the Campylobacter jejuni N-linked protein glycosylation (pgl) pathway within Escherichia coli. By systematically decoupling key biosynthetic genes from their native controls and optimizing their expression combinatorially, the researchers developed superior production strains. This innovation resulted in an up to two-fold enhancement in glycan output, offering a modular, high-yield platform essential for the future of cost-effective glycoconjugate vaccine manufacturing.

Overview

Glycoconjugate vaccines, which rely on chemically linking bacterial capsular polysaccharides or O-antigens to a carrier protein, have proven incredibly effective against numerous pathogens. However, the manufacturing process is hampered by two significant limitations: supply and consistency. Traditional methods either rely on complex and expensive chemical synthesis or require purifying the desired glycans from large, dangerous cultures of pathogenic bacteria.

Synthetic Glycobiology offers a transformative solution through glycoengineering—harnessing the genetic tools of safe, fast-growing hosts, notably E. coli, to biosynthesize these complex sugar structures. The challenge lies in porting the foreign, multi-gene pathways responsible for donor sugar creation, such as the C. jejuni N-linked heptasaccharide. These long biosynthetic pathways are often subject to complex native regulation in their original hosts, leading to unpredictable or sub-optimal yields when simply expressed heterologously in E. coli. To truly industrialize this technology, researchers must optimize every step of the metabolic pathway to ensure maximum flux toward the final, high-value glycan product. This paper addresses this crucial expression efficiency bottleneck head-on using cutting-edge DNA assembly techniques.

Research Results

• Rational Decoupling of the Biosynthesis Cluster

In the native context, the expression levels of the 10 glycosyltransferases and accessory enzymes are tightly co-regulated, which may not be optimal for maximum heterologous expression in E. coli. The researchers first successfully decoupled these genes, removing them from their native regulatory elements. This set the stage for one of the most significant innovations of the work: utilizing combinatorial start-stop assembly (CSSA).

This scarless, modular DNA assembly system allowed the team to rebuild the entire pgl cluster from the bottom up. Critically, each of the ten coding sequences was paired with a library of synthetic promoters and ribosome binding sites (RBSs) of varied, characterized strengths. This created a vast combinatorial library of expression constructs, testing millions of possible gene expression balance points that the native system could never achieve. This synthetic biology approach transforms the single-solution native pathway into a tunable, highly optimized expression platform.

• High-Throughput Screening Identifies High-Performance Strains

Generating a large library of combinatorial constructs is only half the battle; the other half is rapid, high-throughput screening to identify the best performers. The team developed and deployed a dual-platform screening strategy that allowed them to efficiently search the constructed library. Initial screening involved measuring gene expression levels and then correlating these expression profiles with the actual production of the glycan.

The researchers used whole-cell enzyme-linked immunosorbent assay (ELISA) assays to quantify the amount of the desired heptasaccharide displayed on the E. coli cell surface. This step was vital, as it ensured that the combinatorial tuning was effectively driving metabolic flux and not just increasing unstable protein levels. Clones like pgl1.C7 and pgl1.C8 were identified as having superior Glycan Production, exhibiting a two-fold increase in cell-surface exposed glycan relative to the traditional strain carrying the unmodified pgl cluster. This quantitative improvement validated the necessity of synthetic regulatory optimization over simply expressing the native pathway.

Fig.1 Engineering a family of glycoengineering strains. (Kay, et al., 2022)

• Translating Glycan Yield into Enhanced Glycoconjugate Titer

The ultimate goal of this research is not simply to produce free glycans, but to create functional glycoconjugate vaccines. The optimized strains were subsequently transformed into E. coli hosts that also expressed the C. jejuni oligosaccharyltransferase (PglB) and a model acceptor protein. PglB is the essential enzyme that "couples" the synthesized glycan onto the carrier protein.

The strains with the superior, combinatorially optimized pgl clusters successfully demonstrated a marked improvement in the yield of the final glycoconjugate protein product. This crucial result confirmed that the enhanced synthesis of the donor sugar effectively relieved a major bottleneck in the entire glycoengineering system. By maximizing the availability of the precursor glycan, the entire enzymatic coupling reaction was significantly boosted, leading directly to a higher titer of the desired vaccine candidate. This successful integration of synthetic regulation, high-throughput screening, and final product evaluation highlights the creation of a robust, production-ready Microbial Cell Factory.

Fig.2 Analysis of purified AcrA-SP4 glycoconjugates produced from different E. coli strains. (Kay, et al., 2022)

Conclusion

This study represents a significant milestone in synthetic glycobiology, providing a modular, plug-and-play blueprint for optimizing complex sugar biosynthesis pathways. By leveraging the scarless combinatorial DNA assembly approach, the researchers have demonstrated a powerful method for overcoming inherent metabolic limitations in heterologous hosts like E. coli. The successful, quantitative enhancement in both glycan production and final glycoconjugate yield directly addresses the key industrial challenges of scalability and cost. This engineered suite of strains paves the way for a more reliable, sustainable, and economically viable supply chain for advanced glycoconjugate vaccines, accelerating the development of critical immunizations against emergent and established infectious diseases worldwide.