Sialic Acid Production by Metabolically Engineered Escherichia coli

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

2. 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

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

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

5. 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

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

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

8. NeuC and NeuB catalyze the de novo biosynthesis of intracellular sialic acid in N. meningitidis group B NeuC NeuB Sialic Acid UDP-GlcNAc ManNAc CTP H2O UDP PEP Pi PPi NeuA Export NeuS Polysialic Acid Capsule CMP-Sialic Acid [Sialic Acid]n Insertion of NeuB and NeuC allows in vivo synthesis of sialic acid

9. Glucosamine synthase increases the synthesis of key metabolite for sialic acid biosynthesis GlmS GlmM GlcN-6-P GlcN-1-P Fru-6-P Ac-CoA Gln Glu GlmU CoA GlmU UDP-GlcNAc GlcNAc-1-P PPi UTP NeuBC pathway Sialic Acid Increases production of sialic acid from low-cost feedstocks

10. Sialic acid production by genetically & metabolically engineered E. coli

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

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

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

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

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

16. 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

17. 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.