Page 1
Small Molecule Modulators of Lipid Production in
Microalgae and NMR Spectroscopy of Lipids
Lisa A. Anderson
PhD Candidate, University of California, Davis
8th Annual Algae Biomass Summit
San Diego, California
Progress in Algae Research, Metabolic Regulation
September 30, 2014
Page 2
Algae biofuel research in the Franz Group
Lipid AnalysisIncreasing Lipid Production Understanding Lipid
MetabolismBiofuel Conversion
Anderson, L. A.; Franz, A. K., Energy Fuels
2012, 26, 6404.Wong, D. M.; Franz, A. K. J Microbiol Meth
2013, 95, 122.
Danielewicz, M. A.; Anderson, L. A.; Franz, A.
K. J. Lipid Res. 2011, 52, 2101.Franz, A. K.; Danielewicz, M. A.; Wong, D. M.;
Anderson, L. A.; Boothe, J. R. ACS Chem. Biol.
2013, 8, 1053.
Flu
ore
scence
biodiesel
CF =100´IME
(IME + 9´ ITG )
Anderson et al., unpublished
Page 3
Microalgae are Photosynthetic Factories
Chisti, Y. Biotechnology Advances 2007, 25, 294 Wijffels, R. H.; Barbosa, M. J. Science 2010, 329, 796.
Sheehan, J., Dunahay, T., Benemann, J., Roessler, P.. National Renewable Energy Laboratory, 1998, 328.
CO2
Microalgae
100x
T. suecica100x
Nannochloris sp.100x
N. oculata 100x
N. salina
100x
P. tricornutum
Industrially relevant
bio-products
lipids
nutraceuticals
biodiesel
carbohydrates ethanol
materials
value-added feedstocksH2
proteins
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Methods and Barriers for Modulating and
Understanding Microalgae Lipid Metabolism
Nutrient limitation or “stress”
Heterotrophic growth conditions
Genetic manipulation
Difficult but advancing
Algae model species
Chlamydomonas reinhardtii (“the green yeast”)
Phaeodactylum tricornutum
Starchless mutants: Work, V. H., et al. Eukaryot. Cell 2010, 9, 1251.
Li, Y.; Han, D.; Hu, G.; Sommerfeld, M.; Hu, Q. Biotechnol. Bioeng. 2010, 107, 258.
RNAi: Moellering and Benning, Eukaryot. Cell 2010, 9, 97.
Yu, W.-L.; Ansari, W.; Schoepp, N.; Hannon, M.; Mayfield, S.; Burkart, M. Microb. Cell Fact. 2011, 10, 91.
Radakovits et al., Euk Cell, 2010
Subcellular localization and multigene stacking: Rasala and Mayfield et al., PLoSONE, 2014
Wong, D. M.; Franz, A. K. J Microbiol Meth 2013, 95, 122.
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Chemical Genetic Approach to Modulate Algae
Lipid Production
Mutant Gene
Protein Wild-Type “Mutant”
Phenotype
Classical GeneticsFeatures of Chemical Genetics
1) Direct changes in real-time2) Phenotype can be reversible3) Concentration effects 4) Temporal effects5) Synergistic compound effects6) Combine for use with engineered strains
Small Molecule
Chemical Genetics
Schreiber, 1998, Bioorg Med Chem
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Organic Molecules for Dynamic Information Flow
DNARNA
replication
transcription translation
proteins
small molecules (signaling, probes, medicines)
small molecules:signaling
communicationprobes
medicines
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Screening Approach
Add algae
and media
Grow in microplatesAdd chemical trigger
Select
chemical
triggers
Add Nile Red
(lipophilic
dye)
Measure lipid production
Phase I: microplate screening & Phase II: dose response screening
Extract lipids
Harvest
algae
Grow in batch cultures
Analyze lipids by
MS, NMR, and
microscopy
Phase III: 10+ selected lead compounds for 500 mL cultures
Add chemical trigger
Increase understanding of algae lipids and
metabolism
Franz, A. K.; Danielewicz, M. A.; Wong, D. M.; Anderson, L. A.; Boothe, J. R. ACS Chem. Biol. 2013, 8, 1053.
Industrially relevant species
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Initial Screening Set
Other
(e.g. phytohormones)
Kinases/phosphorylases
Lipid metabolism
Lipid oxidation
Protein synthesis
Primary target
antioxidant
oxidative signaling
cell cycle regulation
plant growth
anti-carcinogenic
anti-proliferative
anti-inflammatory
anti-fungal
Bioactivity
Chemical Structure
phenolics
flavones
terpenoids
purine
derivatives
alkaloids
triazoles
oxindoles
fatty acids
Indole-derived
maleimides
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Diverse Compound Screening Reveals
Small Molecule ModulatorsScatter Plot
absorbance % change
nile r
ed
% c
hang
e a
t m
ax
-8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6
70
60
50
40
30
20
10
0
-10
-20
-30
-40
Color by
Molecule
Abscisic acid
acetomenophen
AG82
Alosine A
Apigenin
arctigenin
Atrazane
Baicalein
Benzylamine purine
BIPQ-II
Bohemine
BPDQ
Caffeine
cantharidin
CDC25
CDK2 Inhibitor II
Cerulenin
Citric Acid
Clotrimazole
cycloheximide
DMSO
epibrassinolide
E piga llocatechine g
Epinephrine
Erbstatin analog
FAAH Inhibitor II
Fluconazole
forskolin
gibberellic acid
...
Scatter Plot
Absorbance % change
Nile R
ed
% c
hang
e (
ma
x)-20 -15 -10 -5 0 5 10 15
50
40
30
20
10
0
-10
-20
-30
Color by
Molecule
Abscisic acid
acetomenophen
AG82
Alosine A
Apigenin
arctigenin
Atrazane
Baicalein
Benzylamine purine
BIPQ-II
Bohemine
BPDQ
Caffeine
cantharidin
CDC25
CDK2 Inhibitor II
Cerulenin
Citric Acid
cycloheximide
DMSO
epibrassinolide
E piga llocatechine g
Epinephrine
Erbstatin analog
Ethyl palmitate
FAAH Inhibitor II
Fluconazole
forskolin
gibberellic acid
...
N. oculata
Scatter Plot
absorbance % change
nile
red
% c
han
ge
at
max
-55 -50 -45 -40 -35 -30 -25 -20 -15 -10 -5 0 5
260
240
220
200
180
160
140
120
100
80
60
40
20
0
-20
Color by
Molecule
Abscisic acid
acetomenophen
AG82
Alosine A
Apigenin
arctigenin
Atrazane
Baicalein
Benzylamine purine
BIPQ-II
Bohemine
BPDQ
Caffeine
cantharidin
CDC25
CDK2 Inhibitor II
Cerulenin
Citric Acid
Clotrimazole
cycloheximide
DMSO
epibrassinolide
E piga llocatechine g
Epinephrine
Erbstatin analog
FAAH Inhibitor II
Fluconazole
forskolin
gibberellic acid
...
Nannochloris
sp.P. tricornutum
N. salina
Growth
Lipids
Growth
Lipids
Lipids
Growth
Growth
Lipids
Legend
Analysis performed with Spotfire
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Diverse Compound Screening Reveals
Small Molecule ModulatorsScatter Plot
absorbance % change
nile r
ed
% c
hang
e a
t m
ax
-8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6
70
60
50
40
30
20
10
0
-10
-20
-30
-40
Color by
Molecule
Abscisic acid
acetomenophen
AG82
Alosine A
Apigenin
arctigenin
Atrazane
Baicalein
Benzylamine purine
BIPQ-II
Bohemine
BPDQ
Caffeine
cantharidin
CDC25
CDK2 Inhibitor II
Cerulenin
Citric Acid
Clotrimazole
cycloheximide
DMSO
epibrassinolide
E piga llocatechine g
Epinephrine
Erbstatin analog
FAAH Inhibitor II
Fluconazole
forskolin
gibberellic acid
...
Scatter Plot
Absorbance % change
Nile R
ed
% c
hang
e (
ma
x)-20 -15 -10 -5 0 5 10 15
50
40
30
20
10
0
-10
-20
-30
Color by
Molecule
Abscisic acid
acetomenophen
AG82
Alosine A
Apigenin
arctigenin
Atrazane
Baicalein
Benzylamine purine
BIPQ-II
Bohemine
BPDQ
Caffeine
cantharidin
CDC25
CDK2 Inhibitor II
Cerulenin
Citric Acid
cycloheximide
DMSO
epibrassinolide
E piga llocatechine g
Epinephrine
Erbstatin analog
Ethyl palmitate
FAAH Inhibitor II
Fluconazole
forskolin
gibberellic acid
...
N. oculata
Scatter Plot
absorbance % change
nile
red
% c
han
ge
at
max
-55 -50 -45 -40 -35 -30 -25 -20 -15 -10 -5 0 5
260
240
220
200
180
160
140
120
100
80
60
40
20
0
-20
Color by
Molecule
Abscisic acid
acetomenophen
AG82
Alosine A
Apigenin
arctigenin
Atrazane
Baicalein
Benzylamine purine
BIPQ-II
Bohemine
BPDQ
Caffeine
cantharidin
CDC25
CDK2 Inhibitor II
Cerulenin
Citric Acid
Clotrimazole
cycloheximide
DMSO
epibrassinolide
E piga llocatechine g
Epinephrine
Erbstatin analog
FAAH Inhibitor II
Fluconazole
forskolin
gibberellic acid
...
Nannochloris
sp.P. tricornutum
N. salina
Growth
Lipids
Growth
Lipids
Lipids
Growth
Growth
Lipids
Legend
Compounds such as:epigallocatechin gallate
PTP Inhibitor IICDK2 Inhibitor 2MAP sb202190
quercetinkinetin
cycloheximide
Analysis performed with Spotfire
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Chemical Triggers in 500 mL Cultures
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
0 5 10 15 20 25
OD
68
0
Time (Days)
ControlNitrogen Deficient (33%)DMSO (0.4%)EGCG (40 μM)
Small MoleculeSpecific Growth
Rate (day-1)
Lipid Content
(% w/w)
Lipid Productivity
(mg L-1 day-1)
Control 0.23 ± 0.06 23.6 ± 9.0 4.7 ± 1.4
Quinacrine (40 nM) 0.25 ± 0.02 26.5 ± 3.9 4.5 ± 1.0
EGCG (40 μM) 0.22 ± 0.06 23.1 ± 10.6 6.3 ± 2.2
DMSO (0.4%) 0.26 ± 0.07 24.3 ± 7.5 6.5 ± 1.7
cAMP (4 μM) 0.22 ± 0.03 28.6 ± 10.7 7.9 ± 3.4
EGCG (in water) (4 μM) 0.24 ± 0.01 32.4 ± 0.1 7.1 ± 1.2
BHA (4 nM) 0.23 ± 0.00 28.8 ± 11.4 8.1 ± 0.6
Propyl gallate (40 nM) 0.23 ± 0.00 25.9 ± 11.4 8.3 ± 1.7
N. salina
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Cost Analysis for Lead Compounds
Compound Price/gram
Concentration
(identified in
screening)
Amount
neededPrice per dose
EGCG $1,200.00 4 mM 91.68 g $110,016.00
cAMP $107.00 4 mM 65.84 g $7,044.88
Forskolin $4,950.00 4 nM 0.0821 g $406.40
BHA $0.06 4 nM 0.036 g $0.002
Propyl gallate $0.09 40 nM 0.4244 g $0.04
DMSO $64.16/L 0.04% 20 L $1,283.20
*crude sources of chemical triggers*
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What pathway are our small molecules acting
through?
Pathways and targets:
• Antioxidant and oxidative stress
• Signaling kinases
• Lipid metabolism
• Fatty acid oxidation
• Nutrient transport
• Growth stimulation and cell division
Desired
Phenotype
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What metabolomics can tell us
Genome
Transcriptome
Proteome
Metabolome
DNA
RNA
Proteins
Sugars NucleotidesAmino
acidsLipids
Metabolites - metabolic intermediates
- hormones and signaling molecules
- secondary metabolites (e.g. pigments)
What has happened and
is happening
What can happen
What appears to be
happening
What makes it happen
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Metabolomics Work Flow
Sample collection and preparation Data acquisition
Bioinformatics
Fiehn, O. Plant molecular biology 2002, 48, 155.
- Mechanistic insight
- Toxicology
- Classification
- Prediction
- Functional genomics
Modeling metabolic
interactions
Following biochemical
pathways and networks
Database curation and
statistical analysis
Collaboration with Fiehn Lab
“quenching”
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Pattern of regulated features was detected
according to nitrogen deficiency
more
less
P. tricornutum
Q1 Q3
= control = N-def
Q2
Metaboanalyst 2.0 www.metaboanalyst.ca
Collaboration with Fiehn LabAnderson et al., unpublished
Starch metabolism
Amino acid
metabolismCitrate
metabolism
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Lipidomics of P. tricornutum under nitrogen
deficiency displays distinct differences
Collaboration with Fiehn LabAnderson et al., unpublished
On going experiments: treatment with antioxidants such as EGCG will
increase metabolites and lipids relating to antioxidant defense system,
membrane integrity, and osmoprotection, along with changes in TCA cycle
intermediates that correlate to fatty acid biosynthesis.
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Control
Glycerol
CH3 of
w3
PUFAs
CH3 of
MUFAs
and SFAs
T. suecica
Lipid Composition by 1H NMR Spectroscopy
SFAs
(%)
UFAs
(%)
w3 PUFAs
(%)
Olive Oil 15 85 6
Peanut Oil 14 86 2
N. salina 31 60 8
N. oculata 24 76 28
T. suecica 21 79 16
T. suecica (glycerol) 44 56 9
P. tricornutum 21 68 11
Representative TAG
SFA (C16:0)
MUFA (C18:1)
PUFA (C18:3)
Danielewicz, M. A.; Anderson, L. A.; Franz, A. K. J. Lipid Res. 2011, 52, 2101.
Pollesello, P. et al. J. Appl. Phycol. 1992, 4, 315.
Anderson et al., unpublished
Page 19
Conclusions
Lipid AnalysisIncreasing Lipid Production Understanding Lipid
MetabolismBiofuel Conversion
Anderson, L. A.; Franz, A. K., Energy Fuels
2012, 26, 6404.Wong, D. M.; Franz, A. K. J Microbiol Meth
2013, 95, 122.
Danielewicz, M. A.; Anderson, L. A.; Franz, A.
K. J. Lipid Res. 2011, 52, 2101.Franz, A. K.; Danielewicz, M. A.; Wong, D. M.;
Anderson, L. A.; Boothe, J. R. ACS Chem. Biol.
2013, 8, 1053.
Flu
ore
scence
biodiesel
CF =100´IME
(IME + 9´ ITG )
Anderson et al., unpublished
Page 20
Acknowledgments
PI: Dr. Annaliese Franz
Brittany Armstrong
Nick Ball-Jones
Andrew Burch
Kayla Diemoz
Elisa Gutierrez
Julia Jennings
Jacob MacDonald
Ngon Tran
Alex Schramm
John Schreiber
Ben Shupe
Sarah Tang
Funding:
Algae Foundation Student Travel Award
Chevron Technology Ventures
NSF Graduate Research Fellowship
Bradford Borge Fellowship
Former:
Dr. Diana Wong
Dr. Megan Danielewicz
Dr. Joseph Badillo
Dr. Taewoo Min
Dr. Nadine Hanhan
Dr. Sean Wilson
Elaine Villena
Kate Gibson
Jordan Boothe
Catherine Pham
Andres Rosales
Barbara Murta
Collaborators:
Fiehn Lab, UCD
Page 22
Nitrogen limitation increases TCA cycle intermediates
Citric acid
Collaboration with Fiehn LabDong, H. P., et al., Plant physiol 2013, 162, 1110.
Page 23
Dose-Dependent Activity
Microplate screening in water
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Small molecule modulation of lipid metabolism in other organisms
Kim et al., ACS Comb. Sci. 2012, 14, 395.
Triazine library for improved biofuel generation in yeast
Screening for small-molecule modulators of lipid storage in
C. elegans for disease applications
Lemieux et al., Nat. Chem. Biol. 2011, 7, 206.
Triggering lipid droplet fusion with small molecules
for disease applications
Murphy et al., PLoS ONE 2010, 5, e15030.
DMSO
E4
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Beyond Biofuels
Avasthi et al., ACS Chem. Biol. 2012, 7, 911.
Chemical Screen to Identify GPCRs as Regulators of Cilia
in C. reinhardtii
Torres et al., Ecotox. Environmen. Safe. 2008, 71, 1.
Microalgal Bioremediation and Pollution Monitoring
Maucourt et al., Biochem. Pharmacol. 2002, 64, 1125.
C. reinhardtii mutants for anticancer drug screening Phytohormone signaling, photosynthesis and protection
indomethacin
Piotrowska, et al., Plant Growth Regul 2008, 55, 125.