Cellular factors mediating the
production of astaxanthin by
Haematococcus pluvialis
Claude Aflalo & Sammy Boussiba
Thanks to: Bing Wang, Yuval Meshulem, Aliza Zarka, Ben Friehoff
Microalgal Biotechnology Laboratory
Blaustein Institutes for Desert Research
Ben Gurion University,
Sde Boker, Israel
Green algae under stress
Red snow
Haematococcus
Chlamydomonas nivalis
5 mm
5 mm
Chloromonas nivalis
Haematococcus pluvialis
CA
Chlamy06
2
Synchronized cultures and Life Cycle
40
40
10
10
1
0
10
20
30
40
50
Time (h)
Normal Light
60
70
80
1
0
Lag
enhanced productivity
20
40
60
80
100
Time (h)
High Light
CA
Chlamy06
3
The ratio light/cell as a growth limiting factor
Light intensity (mE m-2 s-1)
100
100
1000
10
100
1
Cell density (Mc/L)
Chl (mg/L)
10000
10
0.1
Cells
Chl
Chl
1
0
7
• The growth rate depends on
both incident light intensity
and culture density as interdependent variables.
500
14
Biomass
0.01
21
Continuous illumination (day)
Biomass dry weight (g/L)
25
• The combined limiting factor
is in fact the light available
locally to the average cell
under nutrient-replete
conditions.
• The light/cell availability
represents also a signal for
appropriate cell response:
dormancy or division (low
ratios, green cells), else
encystment (high ratio, red
cells).
CA
Chlamy06
4
Effect of different stresses on growth and astaxanthin
accumulation
Cells – 105/mL
30
Under stress, division stops and
secondary carotenoids accumulate as
astaxanthin. While high irradiance is
the most effective elicitor, its outcome
is transient and reversible due to
acclimatation and the decrease of
light/cell upon further growth.
A
NL
HL
-N
-P
-S
10
3
10
B
41
TCar:Chl – w/w
Chlorophyll – mg/mL
Final yield – mg/ml
C
100
30
10
8
39
6
4
41
2
3
0
1
2
3
Time - day
4
5
0
53
13
0
1
2
3
4
Time - day
CA
5
Chlamy06
5
Basic carotenogenesis:
primary and secondary products
Chloroplast
8 Pyr + 8 GA3P
Phytoene
Activation
(ATP)
Condensation
Condensation
Oxidation
6 IPP + 2 DMAPP
Reduction
(NADPH)
Lycopene
Cyclization
a carotene
b carotene
Export
Lipid globule
Zeaxanthin
Cyt-P450
Cyt-P450
Net
Net oxidation
oxidation
Canthaxanthin
?
O
Astaxanthin
Lutein
Xanthophylls
HO
O
OH
CA
Chlamy06
6
Lipid globules traffic in the cytosol
During exposure to
high light intensity,
the globules are
deployed at the cell
periphery…
Low light
High light
… with relatively
fast kinetics, as a
‘sun screen’ to the
exposed chloroplast.
0 time
5 min
10 min
The mechanism of
chloroplast-crossing
remains unresolved.
CA
Chlamy06
7
Electron flow in the thylakoid membrane
NADP+
2H+
DCMU
Fdx
NADPH
+ H+
PQ
PS II
Cyt
b6f
PS I
PQH2
DBMIB
H2 O
2H+ +1/2 O2
PC
2H+
CA
Chlamy06
8
Effect of electron flow inhibitors
10
While both inhibitors effectively stop
growth, the action of DBMIB that
promotes accumulation of reduced
plastoquinone (PQH2), results in
substantial astaxanthin accumulation,
at high – but not at normal – light
intensity.
Cells – 105/mL
NL HL
Control
DCMU
DBMIB
3
DBMIB
PQ
PS II
PQH2
Fdx
Cyt
b6f
PS I
PC
DCMU
4
TCar:Chl – w/w
Chlorophyll – mg/mL
1
20
10
6
4
0
12
24
Time - hour
36
3
2
1
0
0
12
24
36
Time - hour
CA
Chlamy06
9
Antioxidative enzymes activities in soluble extracts
Activity - U/mg prot
NL
HL
20
10
Activity - U/mg prot
SOD
30
0
0
1
2
3
4
APX
1.5
1.0
0.5
0
5
0
1
Activity - mU/mg prot
Activity - mU/mg prot
60
40
0
0
DHAR
1
2
3
3
4
5
4
5
Time - day
Time - day
20
2
4
Time - day
5
100
80
60
40
A large variation
of antioxidative
enzymes activity
is observed upon
growth, indicating
a response to light
stress for dilute
cultures. Except
for glutathione
reductase, no
obvious change is
prominent upon
high irradiance.
GR
20
0
0
1
2
3
Time - day
CA
Chlamy06
10
Chloroplast antioxidative enzymes activities
Stroma
PSI
O2
O2-
O2-
SOD
SOD
H2O2
H2O2
APX
APX
H2O
H2O
DHA
Asc
DHA
Asc
DHAR
Thylakoid
PSI
Fdox
Fdr
FdR
GSSG GSH
NADP+
GR
NADPH
The results are not conclusive
since they do not reflect
changes in the full complement
of the enzymes (the bound
activities are missing).
Nevertheless, on a kinetic point
of view, the observed changes
are competent as mediators in a
signal transduction cascade
leading to the induction of
astaxanthin accumulation.
CA
Chlamy06
11
Effect of singlet oxygen generator
N
N
20
Chlorophyll – mg/mL
A
–
Methylene blue generates reactive
singlet oxygen upon illumination.
The dye is taken up by algae.
+MB
HL
15
NL
H12NL
The inhibitory effect of MB on
growth, while mimicking high
irradiance can be prevented by a
pulse of high light intensity.
10
5
B
TCar:Chl – w/w
N
ClS+
Nevertheless, the action of the dye
promotes sustained astaxanthin
build-up in both stationary and
growing cultures.
4
2
0
0
12
24
Time - hour
36
48
Finally, it is important to note that
astaxanthin accumulation under
stress is not prevented by ROS
scavengers added to the medium.
CA
Chlamy06
12
Cell response to stress in the green alga Haematococcus
Mode of action
Environmental Stresses:
 Nutrient deprivation
 Salt stress
 High light
 Low temperature
 Drought
 Aging
Slowdown of cell division
+
LIGHT
Excess of light
Generation of ROS
Cellular sensing,
mediators
Activation of cell response
Encystment
1. xanthophyll cycle
2. ROS quenching enzymes
3. antioxidants
Motile cell
Palmelloid
Red cyst
Change in cell
anabolism
(lipids)
Astaxanthin accumulation
CA
Chlamy06
13
Thank you
☺
CA
Chlamy06
14