Sialic Acid Production by Metabolically Engineered Escherichia coli Benjamin R. Lundgren and Christopher N. Boddy * Syracuse University, Syracuse, NY 13244

Sialic acid is a key molecule in cell adhesion and cell signaling N-acetylneuraminic acid, Neu5Ac, undergoes extensive modifications to generate the diversity of the sialic acid family

Sialic acid encompasses a large family of cell-surface carbohydrates Biological Function Cell Adhesion Cell Signaling Glycoprotein Stability Bacterial Virulence Tumor Metastasis Medical Importance Influenza Inhibitors Marker for Disorders Biologics Diagnostics Limited supply of sialic acid analogs has hindered advancement in basic research, diagnostic development and therapeutic production

Current resources for sialic acid are scarce, costly, and non-scalable Natural Resources Chemical Synthesis Enzymatic Synthesis Microbial Fermentation? low yield & purity costly & not readily scalable challenging

Fermentation as an alternative route in sialic acid production Low cost Scalable Can be crystallized from aqueous solutions at concentrations > 150 g/L Bacterial sialic acid metabolism is well characterized Harness the chemistry of biological pathways in bacteria to produce sialic acid

Gene Expression Plasmid Host DNA Lacks genes for sialic acid degradation E. coli Cell Encodes sialic acid biosynthesis Feedstock Sialic Acid

Sialic Acid Sialic Acid ManNAc + Pyruvate ManNAc-6-P GlcNAc-6-P GlcN-6-P Fructose-6-P NanA NanK NanE NagA NagB NanT OUTIN Removal of sialic acid catabolism in E. coli is crucial for sialic acid production ATP ADP Deletion of transporter and aldolase eliminates sialic acid degradation Transporter Aldolase

NeuC and NeuB catalyze the de novo biosynthesis of intracellular sialic acid in N. meningitidis group B UDP-GlcNAcManNAc Sialic Acid CMP-Sialic Acid [Sialic Acid] n NeuCNeuB NeuA NeuS PEPPi CTP PPi Export Polysialic Acid Capsule Insertion of NeuB and NeuC allows in vivo synthesis of sialic acid H2OH2OUDP

Glucosamine synthase increases the synthesis of key metabolite for sialic acid biosynthesis Fru-6-P GlcN-6-PGlcN-1-P GlcNAc-1-PUDP-GlcNAc GlmS GlnGlu GlmM Ac-CoA CoA GlmU UTPPPi NeuBC pathway Sialic Acid Increases production of sialic acid from low-cost feedstocks

Sialic acid production by genetically & metabolically engineered E. coli

Various carbon sources lead to sialic acid production Sialic acid production from glucose is the cheapest

Multiple feedings of glucose leads to a sialic acid titer of 1.7 grams per liter Production costs: <$1 per gram of sialic acid

Sialic Acid Analogs: Tools for discovery in sialic acid research Serve as biological probes, components of drugs and diagnostics N-azido sialic acidN-acyl sialic acid imaging of cells in vivomodulate cell-cell interactions

Analogs can be produced using chemically modified feedstocks N-acyl glucosamineN-acyl sialic acid GlmS NeuC NeuB Feeding of N-acyl glucosamines to engineered E. coli results in production of N-acyl sialic acids

Analogs can be produced in vivo by expressing tailoring enzymes GlmS NeuC NeuB Hydroxylase GlucoseSialic Acid Produce gram quantities of important analogs N-glycolyl sialic acid

Sialic acid production by microbial fermentation Is efficient, rapid, and cost effective Higher yields from dense-cell cultures Generate large amounts of analogs Shows the feasibility to produce complex, unavailable small molecules

Acknowledgements Christopher N. Boddy The Blattner lab at University of WI-Madison Timothy J. Durfee Kinya Hotta The Borer and Doyle labs at Syracuse University Syracuse University and the Structural Biology, Biochemistry and Biophysics (SB3) graduate program.