(117f) Engineered Cynr Regulon for Enzymatic Biocatalysis Enables Milk Oligosaccharides Biosynthesis Using Azido Sugars

Autonomous detection of azide-tagged biomolecules like azido sugars can enable the fields of chemical biology and glycoengineering. Here, we report an engineered synthetic promoter system called cyn regulon, and complementary Escherichia coli engineered strains, to selectively detect azide ions and autonomously induce downstream expression of reporter genes for various glycoengineering applications (1). The engineered cyn operon allowed highly tunable reporter green fluorescent protein (GFP) expression over three orders of inducer azide ion concentrations (0.01–5 mM) and rapidly induced GFP expression by over 600-fold compared to the uninduced control. We showcase the superior performance of this engineered cyn-operon over the classical lac-operon for recombinant protein production. Finally, we highlight how the cyn-operon synthetic biology toolkit can enable glycoengineering-based applications by facilitating in vivo activity screening of mutant carbohydrate-active enzymes (CAZymes), called glycosynthases, using azido sugars as donor sugar substrates and various acceptor sugars (e.g., lactose) to synthesize targeted prebiotics like human milk oligosaccharides (HMOs).

1. Bandi, C. K., Skalenko, K. S., Agrawal, A., Sivaneri, N., Thiry, M. & Chundawat, S. P. S., "Engineered Regulon to Enable Autonomous Azide Ion Biosensing, Recombinant Protein Production, and in Vivo Glycoengineering," ACS Synthetic. Biology. 10, 682–689 (2021). DOI: 10.1021/acssynbio.0c00449. Weblink: https://pubs.acs.org/doi/10.1021/acssynbio.0c00449