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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
Ni2+
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]

 [sialic acid]
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