A project I designed to study biodiesel production in algae

1. A
chemical
genetics
approach
to
study
regulation
of
lipid
accumulation
and
cell
growth
in
C.
reinhardtii
Summary
Algae
have
the
potential
to
be
a
green
and
renewable
source
for
biodiesel
production
but
in
order
to
make
it
economically
sustainable
and
competitive
many
scientific
challenges
need
to
be
undertaken.
One
of
them
most
relevant
issues
is
the
necessity
to
manipulate
algal
metabolism
in
order
to
maximize
the
yield
of
neutral
lipids,
in
particular
tryacilyglycerols
(TAG),
the
biodiesel
precursors.
The
unveiling
of
regulative
mechanisms
controlling
cell
growth
and
neutral
lipid
biosynthesis
at
molecular
level
is
an
essential
key
factor
in
algal
biodiesel
research.
To
accomplish
this
goal
I
propose
to
use
a
chemical
genetic
approach
screening
diverse
compound
libraries
in
order
to
identify
“in
vivo”
chemical
modulators
of
lipid
accumulation
and
cell
growth.
Deconvolution
strategies
will
be
applied
to
identify
proteins
targeted
by
the
most
efficient
molecules
and
hence
the
putative
regulative
genes
involved
in
these
biological
processes.
The
results
of
this
approach
have
the
potential
to
allow
researchers
to
manipulate
algal
metabolism
with
two
different
complementary
strategies:
direct
“pharmacological”
employment
of
these
chemical
modulators
in
algal
cultures
and
mutagenesis/silencing
of
regulatory
genes
in
engineered
algal
strains.
In
the
future
a
combination
of
these
two
strategies
could
be
the
best
solution
to
guarantee
simultaneous
growth
robustness
and
high
lipid
content,
maximizing
biodiesel
productivity.
Carrying
out
this
research
in
an
american
lab
will
give
me
the
possibility
to
work
in
a
stimulating
scientific
environment
where
the
confront
of
different
ideas
and
the
access
to
material
and
intellectual
resources
will
help
me
to
develop
not
only
the
project
itself,
but
also
my
ability
of
critical
thinking.
This
will
strongly
contribute
to
my
professional
and
personal
enrichment.
Furthermore
I
am
interested
in
exploring
the
dynamics
that
are
turning
the
San
Diego
area
in
a
“hub”
for
algal
biodiesel
research,
where
academia
and
industry
work
in
synergy
with
a
continuous
exchange
of
resources
and
knowledge
to
turn
biodiesel
from
algae
in
a
reality.
The
lack
of
this
common
effort
and
synergistic
exchange
between
universities
and
private
companies
is
one
of
major
factors
preventing
the
italian
research
to
become
more
competitive
in
the
global
contest.

2. Research
Plan
a. Specific
aims
The
long-­‐term
goal
of
this
research
is
to
maximize
the
yield
of
neutral
lipids
in
algae
to
make
these
organisms
a
competitive
and
sustainable
source
for
biodiesel
production.
This
goal
will
be
accomplished
identifying
and
characterizing
chemical
and/or
genetic
modulators
of
neutral
lipid
accumulation
and
cell
growth
promotion,
using
a
forward
chemical
genetic
approach
coupled
to
a
proteomic-­‐based
target
deconvolution
strategy.
A
chemical
high
throughput
screening
will
be
performed
to
identify
small
molecules
able
to
trigger
either
lipid
accumulation
or
stimulate
cell
growth.
In
order
to
maximize
sustainable
oil
production
will
be
tested
if
the
activity
of
single
molecules
inducing
different
phenotypes
(e.g.
lipid
accumulation
and
cell
growth)
can
be
maintained
and
combined
in
vivo
when
cells
are
exposed
to
such
molecules
simultaneously.
A
key
goal
of
the
research
is
to
identify
the
putative
targets
of
the
most
effective
molecules
and
hence
unveiling
one
or
more
genes
involved
in
the
regulation
of
lipid
accumulation
and
cell
growth.
RNAi
silencing
of
the
candidate
target
genes
will
provide
additional
information
regarding
the
regulative
mechanisms
of
the
biological
processes
investigated.
The
great
advantage
of
this
experimental
approach
is
that
it
will
potentially
enable
characterization
of
genes
involved
in
a
lipid
accumulation
and
cell
growth
and
at
the
same
time
will
provide
chemical
tools
to
modulate
their
functions.
b. Background
and
significance
Neutral
lipids,
and
in
particular
triacylglycerols
are
the
raw
material
for
biodiesel
production.
Currently
biodiesel
is
produced
mainly
out
of
field
crops
but
this
source
is
not
sustainable
to
satisfy
the
increasing
demand
of
the
market,
especially
if
the
goal
is
replacing
fossil
fuels.
Algae
have
the
potential
to
overcome
many
of
the
limits
affecting
crops
in
biodiesel
production,
but
in
order
to
become
a
competitive
and
commercial
reality
technologic
breakthroughs
are
needed
in
different
scientific
fields
[1].
Metabolic
engineering
of
algal
strains
in
order
to
re-­‐direct
as
much
as
possible
of
the
captured
solar
energy
into
the
highly
energetic
chemical
bounds
of
lipids
is
one
of
the
most
critical
issues.
To
accomplish
this
goal
many
biological
questions,
in
particular
related
to
biosynthesis
and
regulation
of
fatty
acids
and
triacylglycerols
(TAG),
need
to
be
addressed
[2].
The
pathways
and
the
enzymes
involved
in
fatty
acid
and
TAG
biosynthesis
are
poorly
studied
in
algae
but
are
very
well
characterized
in
higher
plants.
Computational
analysis
of
integrated
genomic,
proteomic
and
metabolomic
data
is
a
powerful
tool
that
can
help
mapping
the
metabolic
network
of
algae,
revealing
pathways
in
common
with
higher
plants
or
animals,
and
new
and
unique
ones
not
present
in
other
eukaryotic
organisms
[3,4].
If
charting
the
metabolic
maps
in
an
essential
knowledge
to
identify
structural

3. genes
involved
in
fatty
acids
and
TAG
biosynthesis,
this
knowledge
may
not
be
sufficient
to
efficiently
manipulate
a
given
metabolic
pathway
through
overexpression
of
one
or
more
structural
genes.
This
issue
was
highlighted
by
lack
of
significant
effect
on
lipid
accumulation
after
overexpression
in
Cyclotella
criptica
of
Acetyl-­‐CoA
Carboxylase
(ACCA)
a
key
enzyme
in
fatty
acid
biosynhesis
[5].
Therefore
the
“Holy
Grail”
in
biodiesel
research
is
to
understanding
regulation
of
TAG
biosynthesis
at
molecular
level,
and
in
particular
how
the
cells
direct
the
metabolic
flux
of
photosynthetically
fixed
carbon
towards
fatty
acids
biosynthesis
first,
and
towards
TAG
biosynthesis
afterwards.
Regulative
genes
involved
in
such
control
need
to
be
identified
in
order
to
create
rational
engineered
algae
with
superior
lipid
content
[2].
It
is
well
known
that
many
algal
species,
even
belonging
to
different
taxonomic
groups,
alter
their
lipid
metabolism
in
response
to
changes
in
environmental
factors,
such
as
nutrient,
pH,
temperature,
salinity
and
light
intensity.
In
particular
specific
stress
conditions,
such
as
nutrient
limitation,
lead
to
an
increase
in
de
novo
biosynthesis
of
neutral
lipids
and
a
conversion
of
membrane
polar
lipids
in
TAG.
In
many
cases
the
net
result
is
an
increase
in
total
lipid
content
of
two
or
three
folds.
The
most
critical
nutrient
whose
limitation
triggers
this
metabolic
shift
is
nitrogen.
The
drawback
is
that
under
stress
such
as
nitrogen
limitation
algae
slowly
decrease
their
growth
until
they
reach
a
complete
arrest
in
cell
cycle
and
a
quiescent
state
[5].
While
most
of
the
research
in
this
field
has
been
focusing
in
individuating
algal
strains
and
culture
conditions
leading
to
the
highest
yield
of
neutral
lipid
accumulation,
no
attempts
to
shed
light
into
the
molecular
biology
of
such
phenomena
have
been
made
yet.
Individuating
the
molecular
actors
involved
in
control
mechanisms
of
lipid
metabolism
is
an
essential
goal
to
design
rational
engineered
algal
strains
that
constitutively
synthesize
and
store
high
level
of
neutral
lipids.
Parallel
and
integrative
studies
aimed
to
increase
the
rate
of
cell
growth
are
required
to
further
boost
the
productivity
of
biodiesel
from
algae.
Complete
genomic
sequences
of
several
algal
species
are
available
[4]
allowing
classic
forward
and
reverse
genetic
studies,
where
mutagenesis
is
a
mean
to
elucidate
the
relationship
between
genes
and
phenotypes.
Chemical
genetics
is
an
emerging
powerful
technology
which
employs
diverse
small-­‐
molecule
compounds
(replacing
mutagenesis)
acting
as
“perturbers”
in
a
biological
system,
in
order
to
elucidate
a
biological
process
of
interest
and
identify
gene
products
involved
in
that
process.
This
approach
offers
several
advantages
over
classic
forward
and
reverse
genetics
potentially
also
in
relation
to
the
algal
biodiesel
research.
First
of
all
small
molecules
work
rapidly
and
often
reversibly,
commercial
compound
libraries
are
available
in
formats
that
allow
a
relatively
fast
analysis
and
possibility
of
automation,
effectively
reducing
the
time
of
the
screening
(especially
when
compared
with
the
time
required
to
create
and
screen
mutant
libraries).
Furthermore
while
mutagenesis
strategies
rely
on
complete
inactivation
of
a
gene,
chemicals
have
the
potential
to
block
only
a
specific
function
of
a
multifunctional
protein,
potentially
generating
phenotypes
not
reproducible
via
mutagenesis
or
silencing
[6].
Indeed
several
cases
where
small
molecules
and
mutations
targeting
the
same
proteins
produced

4. radically
different
phenotypes
have
been
reported
[7].
Another
important
advantage
is
that
active
compounds
may
be
tested
for
functionality
across
different
species
where
genomic
data
are
not
available
or
efficient
transgenic
technologies
are
not
fully
developed.
Last
but
not
least
the
discovery
of
compounds
with
biological
desired
activities
may
lead
to
industrial
applications
(so
far
applied
mainly
in
the
pharmaceutical
field)
and
this
might
be
a
crucial
benefit
in
biodiesel
research
as
well.
Based
on
their
efficacy
small
bioactive
molecules
could
potentially
find
a
practical
employment
in
biomass
and
biodiesel
precursor
production,
or
at
least
to
be
the
base
to
design
drugs
with
high
efficacy
and
specificity
in
modulating
the
biological
processes
of
interest
(lipid
accumulation
and
cell
growth
promotion).
Chemical
modulators
could
indeed
be
an
alternative
way
to
transgenesis,
in
order
to
increase
biodiesel
productivity
in
algae,
especially
given
the
reluctance
of
large
part
of
the
public
opinion
and
the
scientific
community
to
introduce
GMOs
in
the
environment.
On
the
other
side
identification
of
regulative
genes
could
allow
the
design
of
rational
metabolic
engineered
algal
strains,
able
to
accumulate
neutral
lipids
possibly
with
improved
cell
growth
performances,
in
standard
culture
conditions.
In
the
future
the
combination
of
transgenic
strains
and
chemical
modulators
could
be
the
best
strategy
to
maximize
the
yield
of
biodiesel
precursors
in
algae.
These
are
the
reasons
why
I
propose
to
use
this
approach
in
studying
lipid
accumulation
and
cell
growth
in
a
model
algal
species.
c. Preliminary
studies
No
preliminary
studies
are
available.
d. Research
design
and
methods
The
project
is
based
on
a
phenotype
driven
chemical
proteomic
approach,
which
consists
in
introducing
small
molecules
in
a
system
and
selecting
the
ones
able
to
induce
a
particular
phenotype.
The
only
algal
organism
for
which
extensive
biological
genomic
and
proteomic
data
exist
is
Chlamydomonas
reinhardtii
[4];
different
mutants
characterized
by
different
phenotypes
are
available,
among
these
the
mutant
CC-­‐503
cw92
mt+
is
characterized
by
the
absence
of
a
cell
wall
and
was
used
for
genomic
sequencing.
A
growth
inhibition-­‐based
drug
screening
performed
in
parallel
in
wild
type
and
wall
less
C.reinhardtii
cells
showed
that
the
latter
were
less
sensitive
to
drugs
compared
to
the
former.
The
authors
hypothesized
a
better
uptake
and
internalization
of
the
molecules
in
the
wall
less
cells
[8].
Given
these
premises
I
propose
to
use
C.
reinhardtii
cw92
mt+
as
experimental
model.
The
research
will
be
essentially
divided
in
two
stages:
1) High
throughput
screening:
I
intend
to
use
the
fluorescent
dye
Nile
Red
to
screen
one
or
more
bioactive
compound
libraries
in
order
to
identify
molecules
that
are
able
to
trigger
neutral
lipid
accumulation
in
the
model
species.
Nile
Red
is
a
lipid

5. extrinsic
fluorescent
dye
whose
maximum
emission
is
blue-­‐shifted
as
the
polarity
of
the
surrounding
environment
decreases.
Recently
Chen
et
al.
proposed
an
optimized
protocol
with
increased
accuracy
and
sensitivity,
suitable
for
high
throughput
quantitative
screening
of
neutral
lipid
content
in
algae.
Reliability
of
the
method
was
demonstrated
by
a
direct
comparison
with
the
conventional
gravimetric
technique
[9].
This
protocol
can
be
easily
formatted
for
96
well
plates
and
used
with
a
microplate
reader
to
screen
compound
libraries.
In
addition
to
the
selected
fluorescence
emission,
cell
proliferation
will
be
monitored
either
checking
the
O.D.
at
750
nm
or
using
one
of
the
several
commercially
available
cell
viability/proliferation
assay
(colorimetric
or
fluorescent)
formatted
for
96
well
plates.
The
screening
will
be
performed
in
parallel
using
cells
growing
in
standard
condition
and
cells
growing
under
nitrogen
limitation.
For
each
condition
and
each
molecule
data
regarding
lipid
accumulation
and
cell
growth
will
be
acquired
at
least
at
three
different
time
points
(to
be
determined
empirically
and
very
likely
different
for
the
two
conditions)
in
a
time
course
manner.
Untreated
cells
growing
under
standard
condition
or
nitrogen
limitation
will
be
used
as
controls.
Dose-­‐response
studies
will
be
carried
out
to
analyze
the
potency
of
the
positive
compounds
using
fluorescence
intensity
and
standard
growth
curves,
respectively
for
neutral
lipid
accumulation
and
cell
growth.
During
the
screening
I
expect
to
find
molecules
affecting
lipid
metabolism
that
can
be
divided
in
3
main
categories
according
to
their
activities:
a. Able
to
promote
lipid
accumulation
in
standard
culture
conditions
but
not
to
further
increase
lipid
accumulation
in
nitrogen
starving
cells
b. Able
to
promote
lipid
accumulation
in
standard
culture
conditions
and
to
further
increase
lipid
accumulation
in
nitrogen
starving
cells
c. Able
to
inhibit
lipid
accumulation
in
nitrogen
starving
cells
In
the
same
way
I
am
interested
in
individuate
molecules
affecting
cell
growth
with
two
distinct
activities:
d. Able
to
promote
cell
growth
under
standard
conditions
but
not
in
nitrogen
starved
cells
e. Able
to
rescue
the
growth
defective
phenotype
of
nitrogen
starving
cells
but
not
to
promote
growth
of
cells
cultured
in
standard
conditions.
f. Able
to
rescue
the
growth
defective
phenotype
of
nitrogen
starving
cells
and
to
promote
growth
of
cells
cultured
in
standard
conditions
While
finding
single
molecules
exerting
both
desired
activities
(cell
growth
promotion
and
lipid
accumulation)
seems
unlikely,
the
combination
of
two
or
more
moelcules
characterized
by
different
biological
activities
will
be
tested
in
vivo
to
study
if
the
two
different
activities
can
be
combined
and
maintained
in
a
additive
or
synergistic
way.
Several
factors
may
affect
the
result
of
all
high-­‐throughput
screenings.
First
of
all
failure
in
identifying
compound
with
the
desired
activity
might
be
due
to
an
incorrect
design
of
the
screening
itself.
In
order
to
avoid
this
possibility
control
measurement
will
be
acquired
for
each
time
point
and
for
each
condition
and
replicates
will
be
used.
The
two
critical
factors
that
may
have
the
greater
impact
in
the
identification
of
molecules
inducing
the
desired
phenotype
are
drug

6. concentration
and
drug
exposure
time.
While
the
former
should
be
kept
as
low
as
possible
(low
µmolar
range)
to
increase
the
stringency
of
the
screening
and
limit
off
target
effects,
the
latter
should
be
long
enough
(at
least
72h)
to
allow
accumulation
of
lipids.
A
prescreening
setup
to
optimize
time
points
using
control
cells
grown
in
the
two
different
conditions
will
be
essential.
Another
critical
factor
affecting
the
success
of
a
chemical
screening
is
the
number
of
compounds
and
their
structural
variability.
A
starting
candidate
could
be
the
Diverset
library
(ChemBridge,
San
Diego)
since
many
successful
screenings
have
been
reported,
and
in
particular
two
independent
groups
were
recently
able
to
identify
new
auxin-­‐like
compounds
affecting
plant
growth
within
this
library
[10,11].
In
the
hypothesis
that
C.
reinhardtii
shares
with
higher
plants
the
auxin
response
pathway,
some
of
the
molecules
identified
in
these
screenings
could
stimulate
algal
growth
too
and
could
be
used
as
putative
positive
controls.
The
most
interesting
compounds
or
a
combination
of
them
can
be
tested
in
other
algal
species
of
particularly
intereste
for
biofuel
production,
to
assess
if
they
can
exert
the
same
effect.
2) The
following
step
consists
in
individuating
the
target/targets
of
the
compound
and
understanding
their
role
in
the
biological
process
investigated.
This
process
called
“target
deconvolution”
remains
the
most
challenging
part
of
every
chemical
genetics
experiment.
In
particular
in
this
case
the
low
stringency
of
the
screening
conditions
could
lead
to
identifications
of
a
broad
spectrum
of
compound
acting
on
different
pathways
and
acting
with
different
mechanisms.
The
problem
of
the
low
stringency
is
mainly
due
to
the
lack
of
information
at
molecular
level
regarding
lipid
accumulation
and
cell
growth
in
algae,
gap
that
this
project
could
fill
at
least
partially.
A
first
step
in
target
deconvolution
is
database
mining,
to
verify
if
the
molecule
of
interest
has
already
been
characterized
and
which
are
the
putative
target
proteins
[12].
Several
deconvolution
strategies
have
been
proposed
but
each
of
them
can
be
either
time
consuming
or
lead
to
false
positives
[13].
In
alternative
I
propose
a
novel
chemicalproteomic
quantitative
method
to
identify
drug
targets,
based
on
the
principle
that
the
binding
of
a
small
molecule
to
a
protein
affects
its
sensitivity
to
protease
digestion
[14].
Briefly
cell
lysates
are
prepared
in
non-­‐
denaturing
conditions,
aliquots
remain
untreated
while
others
are
incubated
with
different
concentrations
of
the
drug
and
finally
all
lysates
are
digested
with
one
or
more
proteases.
Treated
and
untreated
peptide
mixtures
will
be
labeled
with
iTRAQ
(isobaric
tags
for
relative
and
absolute
quantification),
separated
by
OFFGEL
[15]
and
analyzed
by
mass
spectrometry
to
identify
quantitative
differences
among
the
samples
[16].
Peptides
showing
a
stechiometric
enrichment
or
depletion
after
the
drug
exposure
should
be
indicative
of
proteins
interacting
with
the
drug
itself.
This
method
has
only
been
validate
as
proof
of
concept
to
verify
predicted
targets
of
know
drugs,
and
its
application
in
identifying
novel
targets
of
uncharacterized
drugs
has
not
been
tested
yet
especially
coupled
to
quantitative
proteomics.
Nevertheless
the
fact
that
doesn’t
require
expensive,
labor
intensive
and
time
consuming
studies,
and
it
can
potentially
be
used
in
a
high
throughput
is
critical
then
the
potential
to
become
the
method
of
choice
in
target
deconvolution
analysis.
The
setup
and

7. optimization
of
the
quantitative
proteomic
analysis,
possibly
using
characterized
drug
will
be
essential
and
can
be
carried
out
in
parallel
with
the
screening
process.
The
success
of
this
method
could
allow
individuation
of
targets
of
several
small
molecules
in
a
relatively
short
time.
Finally
additional
information
about
mechanisms
of
action
of
candidate
target
proteins
can
be
collected
studying
the
effect
of
gene
silencing
on
the
phenotype.
Recentely
a
method
for
efficient
knockdown
emplying
miRNA
has
been
developed
in
C.
reinhardtii
[17]
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