Optimizing Glycoengineered E. coli with Synthetic Small Regulatory RNAs

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 enable higher 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 E. coli for increased glycan production with synthetic small regulatory RNAs (sRNAs). We constructed a panel of 41 synthetic sRNAs to repress the endogenous expression of E. coli genes that were predicted by a genome-scale flux balance model to reduce sugar precursor levels in the N-linked heptasaccharide biosynthetic pathway from Campylobacter jejuni. 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 50% increases in fluorescence intensity. Further work is anticipated to identify even greater improvements by screening multiplexed sRNA libraries with fluorescence-activated cell sorting (FACS)-based monitoring of glycan production. Parallel work is ongoing to optimize an engineered biosynthetic pathway that produces eukaryotic core mannose₃-N-acetylglucosamine₂ (Man₃GlcNAc₂) glycans in E. coli. Our approach of using sRNAs for metabolic flux optimization provides an effective strategy for increasing glycan yields and opens the door for more complex regulation of E. coli-based glycosylation by integration within genetic circuits.