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Sialic Acid Overproduction by Metabolic Engineering of Escherichia coli Benjamin Robert Lundgren and Christopher N. Boddy* Department of Chemistry, Syracuse University Syracuse, NY 13244 Sialic acid is a complex sugar found in several biological contexts HO COOH Isolated genes using polymerase-chain reaction (PCR) and inserted into cloning vectors Checking for the presence of the bicistronic in a recombinant vector OH HO •E.g., nanA-nanE: Genes can be either forwards or backwards O H N OH C H3C O • Forwards = start codons are distal Backwards = start codons are juxtaposed key component of N- & O-glycans HO 1) PCR Functions •EcoR I sites flank stop codons and allow for directionality to be assessed PCR thermocycler 2) analyzed on Gel cell recognition cellular immunity EcoR I 3) purified PCR product 4) ligated into cloning vector genomic DNA of either E. coli or N. meningitidis protein stability bacterial capsules in vivo expression studies on engineered E. coli Induce expression with IPTG EcoRI nanA nanE 1.7 kb bacterial virulence glycoproteins available in limited quantities and at high cost EcoR I EcoR I Cloning Vector • high copy number nanE 0.7 kb Harness natural biosynthetic pathways to overproduce sialic acid •exploit the enzymatic machinery of bacteria to synthesize sialic acid Genes need to be cloned into expression vectors to produce protein cut insert (gene) and expression vector with same enzymes Biosynthetic genes expressed and proteins can be purifed in vitro expression studies on engineered E. coli • Recombinant proteins fused to a His-tag: 6 repeating histidine residues • His-tag adopts conformation that supports its binding to Ni2+ • Use recombinant proteins to check for in vitro activity •two different, potential pathways will be studied: 1) Catabolic pathway from E. coli + NH ligate cut vector with cut insert N CH2 O N H 2) anabolic pathway from N. meningitids CH C HN CH H N C O CH O CH2 C H2C NH N N Ni 2+ N N 1) transfrom into E. coli *in vitro biochemistry show the enzyme works in reverse 2) select for antibiotic resistance NANA = N-acetyneuraminic acid = sialic acid GlcNAc = N-acetyl-glucosamine ManNAc= N-acetyl-mannosamine PEP = phosphenolpyruvate UDP = uridine diphosphate 6P = phosphate at 6’ position FT = flowthrough (total protein) Engineered E. coli strain provides a scalable, cheap route to sialic acid •efflux pump crucial to remove cytotoxic sialic acid from cell Multiple genes need to be placed in a single plasmid • Genes for sialic acid biosynthetic pathway were cloned into the same expression vector •simultaneously overexpress pump and biosynthetic enzymes in nanTE. coli mRNA with ribosomes Quantification of in vivo sialic acid production using an enzyme-coupled colorimetric assay •Measure the loss of absorbance at 340 nm due to NADH consumption Sialic acid biosynthetic enzymes NanA-NanE NanA-NanE-NanK NeuB-NeuC •test different combinations of pumps and biosynthetic enzymes to maximize sialic acid production COOH OH HO H3C (yellow) NADH ManNAc (colorless) NAD COO O H N C O HO OH sialic acid sialic acid lyase C COO O CH3 lactate dehydrogenase pyruvate Plasmid with tricistronic insert: nanA-nanE-nanK Tricistronic is transcribed into one mRNA Each gene has ribosome-binding site, thus all are translated simultaneously Prof. Clay C. C. Wang (University of Southern California) Dr. Kinya Hotta (University of Southern California) •This loss is proportional to the amount of sialic acid present HO Efflux Pumps NorM SetA Acknowledgements d[NADH] d[pyruvate] [sialicacid] dt dt HC OH Mike Henry (Syracuse University) The Boddy Lab (Syracuse University) The Borer Lab (Syracuse University) CH3 lactate Mike Cabbage (University of California Riverside) Schimadzu Scientific Instruments SB3 Program (Syracuse University) Syracuse University