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Sialic acid overproduction by metabolic engineering of Escherichia coli

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Title: Sialic acid overproduction by metabolic engineering of Escherichia coli


1
Sialic acid overproduction by metabolic
engineering of Escherichia coli
  • Benjamin R. Lundgren and Christopher N. Boddy

2
Talk Outline
  • importance and significance of sialic acid
  • sialic acid biosynthesis
  • engineering E. coli to overproduce sialic acid
  • conclusions and remarks

3
Sialic acids a family of natural and synthetic
acidic sugars
Schauer, R. Zoology 2004, 107, 4964
N-acetylneuraminic acid or NANA is parent
compound for all sialic acids
4
Cell-surface carbohydrates often terminate with
sialic acids
  • because of their structure and terminal position,
    sialic acids are very biologically active sugars

5
Sialic acids in biology and medicine
  • cell signaling, cell adhesion, cellular
    immunity
  • stabilizes proteins
  • serve as self-recognition markers
  • diagnostic markers in disease
  • components in antiviral drugs/vaccines
  • stabilizer of peptide drugs

6
Sialic acid is available in limited quantities
J. BioSci Bioeng. 2002, 93, 258265
high demand for sialic acid but sialic acid is in
short supply metabolically engineered E. coli
are a relatively inexpensive source for sialic
acid production?
7
Bacterial sialic acid metabolism is well
characterized
Re-construct known enzymes into a pathway that
oveproduces sialic acid and does not degrade
product
8
Pathway engineering for sialic acid production in
E. coli
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Efflux
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Removed catabolic proteins (dashed
lines) Introduced anabolic proteins (bold lines)
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9
Recombinant technology used to create sialic acid
overproducing E. coli strain
  • Isolated neuB and neuC genes from Nesseria
    meningitidis type B strain MC58
  • Isolated glmS gene from E. coli K12
  • Cloned genes into single plasmid
  • Purchased nanT- E. coli mutant
  • Inactivated nanA in mutant using gene replacement
  • Inserted T7 RNA polymerase gene into chromosome
    of nanA- nanT- E. coli mutant

10
Induce gene expression with IPTG Uncertain of
efflux processes
11
SDS-PAGE of NeuB-NeuC overexpression in
engineered E. coli strain
marker
time after induction (hr)
NeuB
0 1 2 4 6 8 10 24
  • NeuB 40 kDa
  • NeuC 42 kDa

50 35
Overexpressed NeuB NeuC
pBRL22
12
Small-scale liquid fermentations in vivo studies
of engineered E. coli
carbon source NH4OH
30 C, 150 rpm
Can screen several sugars easily and readily
13
Effect of feed-stock sugar on sialic acid
production in engineered E. coli strain
BRL02/pBRL22 single feeding of 0.5 sugar
14
Cell growth and sialic acid production in shake
flask cultures
BRL02/pBRL47 grown on glucose
Induced with 0.1 mM IPTG
15
Sialic acid analogs novel agents in research
  • serve as probes in biological processes
  • improve stability of drugs
  • novel agents in medical diagnostics
  • will use engineered E. coli strain to produce

N-propanoylneuraminic acid N-pentanoylneuraminic
acid N-levulinolylneuraminic acid N-azidoacetylne
uraminic acid
16
Conclusions and remarks
  • engineered E. coli to overproduce sialic acid to
    gt 1 gram per liter in shake flask
  • expect a 10-fold increase in titer under
    high-cell density fermentation (bioreactor)
  • expect to be able to generate sialic acid analogs
    in sufficient yields for downstream processes
  • currently addressing sialic acid efflux

Metabolically engineered cells that overproduce
sialic acid will reduce overall costs in sialic
acid research!
17
Acknowledgements
  • Christopher N. Boddy
  • Timothy Durfee (Univ. WI-Madison)
  • Kenji Watanabe Kenji Hotta (Univ. Southern
    California)
  • The Phil Borer lab (Syracuse Univ.)
  • Jack Milton (Visiting Scientist, Syracuse Univ.)
  • Members of the Boddy lab (Syracuse Univ.)
  • SB3 program (structural biology, biochemistry,
    biophysics) at Syracuse Univ.
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