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Small Molecule Modulators of Lipid Production in Microalgae and NMR Spectroscopy of Lipids Lisa A. Anderson PhD Candidate, University of California, Davis 8 th Annual Algae Biomass Summit San Diego, California Progress in Algae Research, Metabolic Regulation September 30, 2014
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Page 1: Small Molecule Modulators of Lipid Production in ...algaebiomass.org/wp-content/gallery/2012-algae-biomass-summit/201… · Small Molecule Modulators of Lipid Production in Microalgae

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

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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Anderson et al., unpublished

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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

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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

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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

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Nitrogen limitation increases TCA cycle intermediates

Citric acid

Collaboration with Fiehn LabDong, H. P., et al., Plant physiol 2013, 162, 1110.

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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.