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Metabolic Pathways Involved in

Storage Lipid Accumulation

Wayne Riekhof

National Jewish Medical and Research

Center, Denver, CO

(Formerly MSU-DOE Plant Research Laboratory and

Dept. Biochemistry, Michigan State Univ.)

Funding: NSF, MSU-CPPT, MSU-DOE PRL, NIH, ACS


Outline
• Overview of lipid gene annotation of
Chlamydomonas genome.
• Pathway from CO2 to triacylglycerol in
algae.
• Carbon partitioning (lipid, starch, and
protein) and lessons from other
organisms.
• Major unresolved questions and future
prospects.
Overview of Lipid Metabolism in

Chlamydomonas

CO 2
RuBisCO
To Glc-6-P for

3-PG
Starch synthesis
DGDG SQDG
PGM ASQD
2-PG Based on annotation of
Eno

PEP
UDP-SQ
SO3-
UDP-Glc genome version 3.
PK

Pyr MGDG DAG PtdGro

To TCA for
PDH
G-3-P
Major conclusions:

AA anabolism
Acetyl-CoA PtdOH CDP-DAG
ACCase
Malonyl-CoA
Acyl-ACP Lipid metabolism is
lyso-PtdOH
MCT
Acyl-ACP
Acyl-ACP
simpler than in seed
Malonyl- ACP FAS FAT G-3-P plants.
Free-FA
Plastid

Cytoplasm/ER
??
Isozyme numbers are
G-3-P
Free-FA Acyl-CoA GPAT much smaller than
DGTS
LACS
lyso-PtdOH Arabidopsis.
Acyl-CoA LPAAT
Met AdoMet
DAG
PAH
PtdOH
Glc-1-P
Many potential dual-
P-Etn CDP-Etn
targeted proteins.
Etn PtdEtn ACAT/
CDP-DAG
PDAT
Ins-3-P
Ser
PtdGro PtdIns
TAG

From Riekhof, Sears, and Benning, Euk. Cell, 2005

From CO2 to Triacylglycerol


CO 2 (via the most direct route)
RuBisCO
To Glc-6-P for

3-PG
Starch synthesis

PGM

2-PG
How to maximize the
PEP
Eno
synthesis of TAG while
PK

Pyr
minimizing the “side
PDH
To TCA for
reactions” of carbohydrate
Acetyl-CoA
ACCase
AA anabolism
and amino acid metabolism?
Malonyl-CoA
Acyl-ACP
MCT
FAS FAT
How to induce this pathway in
Malonyl- ACP
Free-FA
nutrient replete cells?
Plastid
??
Cytoplasm/ER
G-3-P
Free-FA Acyl-CoA GPAT
LACS
lyso-PtdOH
Acyl-CoA LPAAT
ACAT PAH
TAG PDAT DAG PtdOH
1. Production of Acetyl-CoA

Proposed pathway for direct


CO 2
formation of acetyl-CoA in the
RuBisCO
algal plastid.
3-PG
PGM

In contrast to seed plants,


there is no need to import
2-PG sucrose for conversion to
Eno
acetyl-CoA. This avoids the
PEP loss of up to 1/3 of the fixed
PK
carbon through PDH.
Pyr
Contributions to acetyl-CoA
PDH

pool by different pathways


Acetyl-CoA can be determined by
metabolic flux analysis.
1. Production of Acetyl-CoA
The carbon concentrating

mechanism is essential under

atmospheric pCO2:

From Moroney and Ynalvez, Euk Cell, 2007


HCO3 CO2
2. Acetyl-CoA carboxylase

-ACCase from Chlamydomonas, like that from


higher plants, is a heterotetrameric plastid enzyme.
-A cytosolic monomeric ACCase was not identified
in the genome.
2. Fatty Acid Synthase
Plastids use a type II multimeric (bacterial
type) FAS enzyme.

A. Claisen condensation:
KAS1, KAS2, KAS3

B. Reduction of -keto:
KAR1

C. Dehydration of -hydroxy:
HAD1

D. Reduction of - double bond:


ENR1
3. Export of Fatty Acid From Plastid

Acyl-ACP Acyl-ACP

FAT
FAT
Free-FA
Plastid

?? Chnl
Cytoplasm/ER

Free-FA
LACS
LACS

Acyl-CoA Acyl-CoA

Kinetic studies with isolated spinach Cp give


evidence of substrate channeling, but the
nature of the channel is not well defined.
Chlamydomonas might be an ideal candidate

with which to dissect this process genetically.

4. Glycerolipid Assembly

-
HO O
In mammalian liver and adipose
O
OH
HO P tissue, GPAT is a major control
G-3-P O point for flux into TAG.
No GPAT homolog is present in
Acyl-CoA GPAT the Chlamydomonas genome.
-
HO O

1
O
OH A recently identified
O P
R
lysophospholipid acyltransferase
O
O
lyso-PtdOH enzyme with LPAAT activity was
recently described in Yeast
Acyl-CoA LPAAT (Riekhof et al., JBC, 2007, and
others.)
2

PtdOH
R
O
This gene has a homolog in
O O
- Chlamydomonas, and may
R

1
O
O
P
OH represent the unidentified LPAAT
O
activity.
O

5. Formation of Triacylglycerol

PtdOH hydrolase is a major control point for


TAG synthesis in adipose tissue. Disruption
causes lipodystrophy (lack of body fat) in mice.
2 2
R R 2
O O R
O
O
O O -
O O
O OH
1
O 3 1
O O OH
R R R 1
O P
R
O
O O
O
DGAT PAH
TAG PDAT DAG PtdOH

Major question: Does betaine lipid (a non-


hydrolyzable ether lipid) get turned over as a
source of DAG for TAG synthesis?
Betaine Lipid Vs. PtdCho

Plant-type pathway: PtdCho is an intermediate in TAG synthesis, subject to FAD.

R
2
PtdCho DAG
O 2
R
- O
O O
CH3
PLC/CT
O O +
1 P N O
R O CH3 OH
O CH3 1
O
R
O
O

Chlamy. pathway: Role of betaine lipid as TAG intermediate is unknown


2
2 BTA1 O
R
DAG
DGTS O
R
O -
O
O
O OH
O +
CH3 ??? 1
O
N R
1
R O CH3
CH3 O
O

Packaging of TAG Into Lipid Droplets

To avoid coalescence
upon seed desiccation,
the surface of the lipid
droplet in oilseeds is
coated in oleosin
proteins.
No known lipid droplet
surface proteins have
been identified in the
Chlamydomonas genome.
Protein composition of
algal lipid droplets is
unknown.
Starch Versus TAG Accumulation

From Hicks et al., Plant Physiol, 2001

Starchless mutants of Chlamydomonas are


available. Do they make more oil under
inducing conditions?
Summary

• TAG and starch synthesis are induced by nutrient


limitation.
• Acetyl-CoA can be produced directly from 3-PGA in
the algal plastid.
• ACCase and FAS are multmeric bacterial-type
enzymes of the plastid.
• Fatty acids are exported from the plastid for
synthesis of glycerolipids in the ER.
• TAG is synthesized by ER enzymes and packaged
into lipid droplets.
• Protein synthesis and carbohydrate storage
compete with TAG for fixed carbon.
Unresolved Questions and

Future Research

• How is fatty acid exported from the plastid?


• Is betaine lipid an intermediate in the TAG synthesis
pathway of Chlamydomonas and other Volvocales?
• How is carbon partitioning between starch and TAG
controlled, and can it be manipulated?
• What are the changes in gene expression and
enzyme activities during oil accumulation, and can
we turn these pathways on under nutrient replete
conditions?
• What is the protein composition of the algal lipid
droplet?
• Do starchless mutants push more photosynthate
into lipid than the wild-type?