Download 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
OR5
OR 4
R3 O
R 2HN
O
R 1O
R1 = Ac
CO 2H
OH
R2 = Ac or -CO-CH 2OH
R3 = Ac
4
R = Ac, CH 3 or SO3
R5 = S-lactyl or PO3 2
1 R1 = R 3 = R 4 = R 5 = H, R 2 = 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
Chemical Synthesis
challenging
Natural
Resources
low yield
& purity
OH OH
HO
AcHN
O
CO 2H
OH
HO
Microbial
Fermentation?
Enzymatic
Synthesis
costly &
not readily
scalable
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
OH
HO
HO
OH
O
HO
AcHN
OH
NH 2
Encodes
sialic acid
biosynthesis
O
CO 2H
OH
HO
Sialic Acid
Feedstock
Plasmid
OH
Gene
Expression
E. coli Cell
Host DNA
Lacks genes for
sialic acid degradation
Removal of sialic acid catabolism in E. coli is
crucial for sialic acid production
OUT
IN
Sialic
Sialic NanA
NanT
ManNAc + Pyruvate
Acid
Acid
Aldolase ATP
Transporter
NanK
ADP
Fructose-6-P
NagB
GlcNAc-6-P
ManNAc-6-P
NanE
NagA GlcN-6-P
Deletion of transporter and aldolase eliminates
sialic acid degradation
NeuC and NeuB catalyze the de novo
biosynthesis of intracellular sialic acid in
N. meningitidis group B
NeuC
NeuB Sialic
UDP-GlcNAc
ManNAc
Acid CTP
H2O UDP
PEP Pi
PPi
NeuA
Polysialic Export [Sialic Acid] NeuS CMP-Sialic Acid
n
Acid Capsule
Insertion of NeuB and NeuC allows in vivo
synthesis of sialic acid
Glucosamine synthase increases the synthesis of
key metabolite for sialic acid biosynthesis
Fru-6-P
GlmS
GlcN-6-P
Gln Glu
UDP-GlcNAc
GlmM
GlcN-1-P
Ac-CoA
GlmU
CoA
GlmU
GlcNAc-1-P
PPi UTP
NeuBC pathway
Sialic Acid
Increases production of sialic acid from low-cost
feedstocks
Sialic acid production by genetically &
metabolically engineered E. coli
OH OH
HO
AcHN
O
OH OH
CO 2H
OH
HO
1, Neu5Ac
NanT
X
HO
AcHN
ux
E ffl
OH
NHAC
NeuB
O
HO
OH
HO
2, ManNAc
UDP
NeuC
H 2O
OH
CO2 H
O
OH
HO
1
X NanA
OH
NHAC
O
O
HO
OH
HO
2
CO2
4, Pyruvate
O
HO
HO
OUDP
NHAC
5, UDP-GlcNAc
2
OPO3
CO2
3, PEP
OH
GlmU HO
HO
PP
UTP
CoA
Ac-CoA
2
OPO 3
HO
HO
O
2
OH
Pgi
OH
7, Glc-6-P
phosphorylase
Maltose
O 3PO
O
HO
OH
NHAC
6, GlcNAc
GlmU
GlmM
2
OPO3
OH
GlmS
HO
HO
OH
HO
Gln Glu
8, Fru-6-P
gluconeogenesis
Glycerol
O
O
OH
NH 2
9, GlcN-6-P
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
OH OH
H3C
HO
NH
n
HO
O
OH OH
CO 2H
O
OH
N3
HO
NH
O
CO 2H
OH
HO
O
N-acyl sialic acid
N-azido sialic acid
modulate cell-cell
interactions
imaging of cells in vivo
Analogs can be produced using chemically
modified feedstocks
OH OH
OH
O
HO
HO
NH
OH
GlmS
NeuC NeuB
O
HO
R
NH
O
CO 2H
OH
HO
O
R
N-acyl glucosamine
N-acyl sialic acid
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
OH OH
OH
HO
HO
O
OH
OH
GlmS
NeuC NeuB
HO
NH
O
OH
HO
O
Glucose
CO 2H
Sialic Acid
Hydroxylase
Produce gram quantities
of important analogs
HO
OH
OH
HO
NH
O
CO 2H
OH
HO
O
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.