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Sialic Acid Production by Metabolically Engineered Escherichia coli
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Transcript of Sialic Acid Production by Metabolically Engineered Escherichia coli
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
OR3O
OR5OR4
R2HNOH
R1O
CO2HR1 = AcR2 = Ac or -CO-CH2OHR3 = AcR4 = Ac, CH3 or SO3
R5 = S-lactyl or PO32
1 R1 =R3 =R4 =R5 = H, R2 = Ac
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
OHO
OH OH
AcHNOH
HO
CO2H
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
OHO
OHOH
AcHNOH
HO
CO2HOHO
HONH2
OH
OH
GeneExpression
Plasmid
Host DNALacks genes for
sialic acid degradation
E. coli CellEncodes sialic acid biosynthesis
Feedstock Sialic Acid
SialicAcid
SialicAcid ManNAc + Pyruvate
ManNAc-6-P
GlcNAc-6-P GlcN-6-P
Fructose-6-P
NanA
NanK
NanENagA
NagB
NanT
OUT IN
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
TransporterAldolase
NeuC and NeuB catalyze the de novo biosynthesis of intracellular sialic acid in
N. meningitidis group B
UDP-GlcNAc ManNAc SialicAcid
CMP-Sialic Acid[Sialic Acid]n
NeuC NeuB
NeuANeuS
PEP Pi CTPPPi
ExportPolysialic Acid Capsule
Insertion of NeuB and NeuC allows in vivo synthesis of sialic acid
H2O UDP
Glucosamine synthase increases the synthesis of key metabolite for sialic acid biosynthesis
Fru-6-P GlcN-6-P GlcN-1-P
GlcNAc-1-PUDP-GlcNAc
GlmS
Gln Glu
GlmM
Ac-CoA
CoAGlmU
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
OHO
OH OH
NHOH
HO
CO2H
O
N3
N-azido sialic acid
OHO
OH OH
NHOH
HO
CO2H
O
H3C n
N-acyl sialic acid
imaging of cells in vivomodulate cell-cell interactions
Analogs can be produced using chemically modified feedstocks
OHOHO
NHOH
OH
RO
OHO
OH OH
NHOH
HO
CO2H
R
O
N-acyl glucosamine N-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
OHOHO
OHOH
OH
OHO
OH OH
NHOH
HO
CO2H
O
OHO
OH OH
NHOH
HO
CO2H
O
HO
GlmS NeuC NeuB
Hydroxylase
Glucose Sialic 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.