New high-throughput screening tool may hold the key to increasing the biosynthetic potential of glycoengineered bacteria
Angineering. The article, “A flow cytometric approach to engineering Escherichia coli for eukaryotic protein glycosylation,” was the result of an NSF-funded collaboration with the Lucks lab at Northwestern to develop a flow cytometric assay capable of rapidly screening variants of the eukaryotic Man3GlcNAc2 protein glycosylation pathway. We used our assay to introduce new modifications to the biosynthetic pathway that help balance the “cost” of enzyme expression with the needs of the cell. In addition, we characterized the effects of overexpressing additional metabolic enzymes involved in GDP-mannose, a precursor molecule to the glycan biosynthesis pathway. Our flow cytometry screening was then corroborated with several analytical techniques, including fluorophore-assisted carbohydrate electrophoresis (FACE), mass spec, and protein glycosylation assays. We ultimately found that the modifications led to a large increase in glycosylation efficiency, from <1% in the previously reported pathway to nearly 100% efficiency in our optimized pathway.
We believe this work will be important as it lays the groundwork for future glycoengineering efforts that aim to develop novel vaccines and biotherapeutics in this simple organism. In addition, we think that that this work has taught us some very important lessons and principles on how we might apply RNA-based gene regulation to metabolic pathways in the future.
You can download our paper here: