Metabolic Engineering of Escherichia coli for Efficient Eukaryotic Glycan Biosynthesis

One of the most important modifications for many proteins is N-linked glycosylation, which modulates an array of therapeutic properties from pharmacokinetic activity to immunogenicity. Large-scale production of therapeutic glycoproteins is currently accomplished using eukaryotic cell culture platforms; however, recent advances in Escherichia coli-based glycoprotein synthesis could complement existing platforms with high yields and better control over glycoform. These E. coli-based platforms involve heterologous, multi-enzyme pathways for glycan synthesis that require optimization for production-scale yields. The aim of this study is to optimize a previously reported pathway for eukaryotic core mannose₃-N-acetylglucosamine₂ (Man₃GlcNAc₂) N-linked glycosylation in E. coli with synthetic small regulatory RNAs (sRNAs), gene overexpression, and rational enzyme selection. We first constructed a panel of synthetic sRNAs to repress the endogenous expression of E. coli genes that were either rationally-selected or predicted by a genome-scale flux balance model to reduce sugar precursor levels. We then screened our sRNA panel for improved glycan production with a fluorescence-based assay of cell-surface displayed glycans and identified E. coli strains with over 2-fold increases in fluorescence intensity. Further, we overexpressed two E. coli genes involved in GDP-mannose biosynthesis to achieve an ~2.5-fold improvement glycan production, which increased to ~3.5-fold when combined with our best sRNAs. Finally, we expected that translocation of lipid-linked oligosaccharides (LLOs) into the periplasm was a bottleneck in our pathway. To overcome this, we selected an LLO flippase from Campyobacter jejuni that is known to exhibit relaxed substrate specificity and expressed this enzyme alongside our optimized system. This strategy resulted in an ~10-fold improvement in translocated glycan production. Future efforts will attempt to show that optimized glycan production significantly increases Man₃GlcNAc₂ protein glycosylation in E. coli.