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
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
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 gt 150 g/L
• Bacterial sialic acid metabolism is well
  characterized

Harness the chemistry of biological pathways in
bacteria to produce sialic acid
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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
NanA
NanT
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
PEP
Pi
H2O
UDP
PPi
NeuA
NeuS
Export
Polysialic Acid Capsule
CMP-Sialic Acid
Sialic Acidn
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
GlcNAc-1-P
UDP-GlcNAc
UTP
PPi
NeuBC pathway
Sialic Acid
Increases production of sialic acid from low-cost
feedstocks
10
Sialic acid production by genetically
metabolically engineered E. coli
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Various carbon sources lead to sialic acid
production
Sialic acid production from glucose is the
cheapest
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Multiple feedings of glucose leads to a sialic
acid titer of 1.7 grams per liter
Production costs lt1 per gram of sialic acid
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Sialic Acid Analogs Tools for discovery in
sialic acid research

• Serve as biological probes, components of drugs
  and diagnostics

N-azido sialic acid
N-acyl sialic acid
imaging of cells in vivo
modulate cell-cell interactions
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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
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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
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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

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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.