EnhancedCatecholDegradationvia MetabolicChannelingin E.coli - - PowerPoint PPT Presentation

Enhanced
Catechol
Degradation
via
 Metabolic
Channeling
in
E.
coli


Elena
Pasko,
Anish
Kapadia,
Kris
Hon,
Farhan
Raja,
 Daniel
Wong
and
Graham
Cromar



Oil
Sands
are
an
important,
naturally


• ccurring
petroleum
resource


Bitumen,
a
heavy
 and
very
viscous
 form
of
crude
oil
 Mixed
with
sand
and
clay
=
oil
sands


Major
contaminants:



• Hydrocarbons


• Naphthenic
Acid

• Heavy
Metals

























































Are
toxic
to:













 





















































aquatic
organisms
 





















































and
animals


• e.g.
Pseudomonas


Putida



• Generates
a


common
toxic
 intermediate‐ Catechol


• Reaction
rates
are


typically
slow


Polycyclic
aromatic
hydrocarbons
can
be
 degraded
by
some
bacteria


Catechol


 Protein
Scaffolds
  Immobilizing
 Enzymes
  Fusion
proteins


Metabolic
channeling
can
overcome
some
 limitations
by
shuttling
intermediates
between
 consecutive
enzymes


Dueber et al. Nat Biotech 27: 753-759 Conrado et ak. Curr. Opin. In Biotech. 19: 492-499

Experiments
done
in
silico
predict
it
is
possible
 to
overcome
pathway
limitations
by
co‐localizing
 some
pairs
of
enzymes.



No
channelling

 Channelling



Work
by
Chris
Sanford.


Aims



Augment
the
existing
capabilities
of
an
aromatic
hydrocarbon
 degrading
microbe
(e.g.
Pseudomonas
putida)
using
metabolic
 channeling.

We
will
first
demonstrate
this
process
in
E.coli
 using
pathway
reconstruction.




1.
Develop
a
generalizable
methodology
for
the
analysis,
modeling
and
 prediction
of
enzyme
candidates
for
metabolic
channeling.
 2.
Demonstrate
metabolic
channeling
of
a
relevant
bioremediation
pathway
in
a
 model
organism
(Escherichia
coli).
 3.
Further
characterize
of
our
encapsulin
micro‐compartment.


Ortho‐cleavage
of
catechol
is
amenable
to
 metabolic
channeling


• 1. Pathway
is
absent
in
E.coli
therefore
any
breakdown
of
catechol
is


the
result
of
the
reconstructed
pathway.


• 2. 
E.coli
is
the
chassis
of
choice.

Other
parts
in
the
library
including,


importantly,
our
encapsulator
are
designed
and
available.


• 3. There
is
a
genome‐scale
model
of
E.coli
metabolism.


Pairs
of
enzymes
will
be
fused
in
a
series
of
 pathway
reconstructions


CFU
/
ml
 Time
hrs


E.Coli
DH5a
is
sensitive
to
catechol


Treated
50mM
Catechol
 Untreated
(Control)


Reconstituted
pathway
leads
to
Catechol
 degradation
in
E.
coli


Flux
Balance
Analysis
(FBA)
was
used
to
predict
the
 effect
of
our
manipulations
on
E.coli
metabolism


• Flux
Balance
Analysis:


– Models
stoichiometric
 reaction
system
of
cell
 – Determines
metabolic
flux
 through
reaction
network



• FBA
used
to
determine


growth
rate
for
specified
 uptake
levels
of
glucose
and
 catechol


• Catechol
transport
and


degredation
reactions
added
 to
modeled
E.
Coli
 metabolism
flux
model
 (iJR904).


Catechol + O2 + H2O + CoA --> Acetyl-CoA + Succinate

Catechol
degradation
increases
growth
 rate
given
certain
conditions


1. Determine
optimal
glucose
 &
catechol
uptake
rates


Objective
#1


Flux
predominantly
influences
the
TCA
cycle


• Able
to
identify
reactions
where
there
is
a
change
in
flux


variability
or
magnitude
when
catechol
degradation
pathway
 is
introduced


Determine
what
reaction
 pathways
will
be
affected
 by
incorporation
of
 catechol
degradation
 pathway
into
E.
Coli.


Objective
#2


FBA
helps
us
predict
conditions
that
may
 further
optimize
catechol
degradation


Assumes
we
can
solve
the
problems
associated
with
 enzyme
assembly...


Oxygen
splurging
 Genetic
knockouts


Molecular
modeling
is
being
used
to
 approximate
expression
levels
based
on
 rates
of
subunit
assembly


• Need
to
determine
ratio
of
monomer‐to‐multimer
for
each


enzyme


• Need
to
model
all
possible
multimer
dissassociation


arrangements


• We
also
need
to
determine
the
expected
level
of
linked
enzyme


formation
in
relation
to
unlinked
and
tangle
formation


Catechol
1,2‐Dioxygenase
(Dimer)
 ΔH
=
‐31,918
kJ/mol
 K
=
390,780
mol‐1


We
coupled
our
project
with
a
relevant
 human
practices
component


• Teach
synthetic
biology.


• Experience
life
in
China.

• Have
fun!



Our
goals:
 Toronto
iGEM
team
members
in
Guangzhou
China
2010


Teaching
in
China!


We
developed
and
delivered
a
six
part
 course
focusing
on
key
concepts


Units:


Evolutionary
biology
 Cell
biology
 Molecular
biology
 Bioinformatics
 Systems
biology
 Synthetic
biology
 Note:

Students
were
 exposed
to
basic
 engineering
 principles
in
 workshops
by
other
 UTACCEL
seminar
 leaders.
 UTACCEL
Leaders
and
participants


Daily
Crude
Oil
demand
of
Canada,
China,
India,
Japan,
and
USA

Thousand
Barrels
per
day
(kb/d) Source:
Joint
Oil
Data
Initiative
 http://www.jodidata.org/

China
is
a
source
of
increasing
demand
for
oil.


Transforming
the
world
takes
more
than
a
 vision
statement


Transforming
the
 world
through
clean
 energy
and
technology
 is
fine.
 Doing
it
while
meeting
 basic
needs
is
the
 challenge!
 Making
some
new
friends
along
the
way
helps!


Summary
of
Results


• Identified
a
relevant
bioremediation
pathway
that
is
amenable
to


metabolic
channeling.




• Created
six
biobrick
parts
representing
the
pathway
from
P.
putida.


• Modeled
the
likely
effects
of
knocking
in
this
pathway
in
a
genome


scale
model
of
E.
Coli
metabolism
(increase
in
growth
rate).
We
 have
also
explored
the
required
amounts
of
expressed
monomers
 based
on
molecular
dynamics
of
their
assembly.


• Conducted
baseline
experiments
to
allow
us
to
measure
the
effects

• f
catechol
on
our
mutants.

• Continued
to
characterize
our
encapsulin
part,
demonstrating
it’s


expression
in
an
IPTG
inducible
construct.


• Contributed
significantly
to
human
practices
through
our
outreach


initiative.


Acknowledgements


• Parkinson
Lab


– Farhan
Raja
 – Stacy
Hung
 – Yen
Leung
 – Kenny
Zhan


• Advisors


– Dr.
John
Parkinson
 – Dr.
Alan
Davidson
 – Dr.
Amin
Zia


 Shuttles
metabolic
intermediates
between
 the
active
sites
of
consecutive
enzymes

 accelerating
unfavourable
reactions
  Some
naturally
occurring
examples
are:


Metabolic
channeling
can
be
used
to
overcome
 some
of
these
limitations


Tryptophan
synthase

 Bacterial
microcompartments


Encapsulation
of
enzymes
may
provide
an
 enhancement
to
channeling


Multimeric
Enzymes


• Challenges


– Most
crystallography
files
only
have
partial
 multimers
 – Some
enzymes
lack
crystallography
files


• Homology
modeling


ZDOCK


We
also
need
to
determine
the
expected
level
of
 linked
enzyme
formation


• In
relation
to
unlinked


enzyme
formation


• In
relation
to
tangle


formation