Cellular factors mediating the

production of astaxanthin by

Haematococcus pluvialis

Claude Aflalo & Sammy Boussiba

Microalgal Biotechnology Laboratory

Blaustein Institutes for Desert ResearchBen Gurion University, Sde Boker, Israel

Thanks to: Bing Wang, Yuval Meshulem, Aliza Zarka, Ben Friehoff

CA Chlamy06 2Haematococcus pluvialis

Haematococcus

Green algae under stress

5 m

Red snow

Chlamydomonas nivalis

Chloromonas nivalis5 m

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Synchronized cultures and Life Cycle

0 10 20 30 40 50 60 70 80

Time (h)

1

10

40

Cel

l (x

105/m

l)C

hlo

rop

hyl

l (µ

g/ m

l )

1

10

40

Cel

l (x

105/m

l)C

hlo

rop

hyl

l (µ

g/ m

l )

0 20 40 60 80 100Time (h)

High LightNormal Light

Lag enhanced productivity

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50025

Light intensity (E m-2 s-1)

1

10

100

1000

10000

0 7 14 21

Continuous illumination (day)

Cel

l den

sity

(M

c/L

) C

hl (

mg

/L)

0.01

0.1

1

10

100

Bio

mas

s d

ry w

eig

ht

(g/L

)Cells Chl Biomass

100

Chl

The ratio light/cell as a growth limiting factor

• The growth rate depends on both incident light intensity and culture density as inter-dependent variables.

• 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).

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Cells – 105/mL

10

3

30

NL

HL

- N

- P

- S

A

10

100

30

3

Chlorophyll – g/mL

B

Time - day 0 1 2 3 4 5

Effect of different stresses on growth and astaxanthin accumulation

Time - day

0

2

4

6

8

10

0 1 2 3 4 5

TCar:Chl – w/w

C

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.

1353

41

39

41

Final yield – g/ml

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Basic carotenogenesis: primary and secondary products

8 Pyr + 8 GA3P

6 IPP + 2 DMAPP

Activation(ATP)

Condensation

Reduction (NADPH)

Phytoene

Lycopene

Condensation

Oxidation

carotene carotene

Cyclization

Lutein

Zeaxanthin

Chloroplast

Xanthophylls

Cyt-P450 Net oxidation

Canthaxanthin

Astaxanthin

Lipid globule

O

OH

HO

O

Export

?

Cyt-P450 Net oxidation

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Lipid globules traffic in the cytosol

During exposure to high light intensity, the globules are deployed at the cell periphery…

… with relatively fast kinetics, as a ‘sun screen’ to the exposed chloroplast.

0 time 5 min 10 min

Low light High light

The mechanism of chloroplast-crossing remains unresolved.

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H2O

PQ

PS I PS II

NADP+

NADPH+ H+

PC

2H+

2H+ +1/2 O2

2H+

Cyt b6f

PQH2

Fdx

Electron flow in the thylakoid membrane

DBMIB

DCMU

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Time - hour

0

1

2

3

4

0 12 24 36

TCar:Chl – w/w

4

20

Chlorophyll – g/mL

6

10

Time - hour0 12 24 36

HL

3

Cells – 105/mL

1

10

NL

DCMU

DBMIB

Control

PQFdx

PS I PS II

PCDCMU

DBMIB

Cyt b6fPQH2

Effect of electron flow inhibitorsWhile 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.

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Antioxidative enzymes activities in soluble extracts

0

20

40

60

0 1 2 3 4 5

Time - day

Activity - mU/mg prot

DHAR

0 1 2 3 4 5

Time - day

Activity - mU/mg prot

0

20

40

60

80

100

GR

0

0.5

1.0

1.5

0 1 2 3 4 5

Activity - U/mg prot

APX

Time - day Time - day

0

10

20

30

0 1 2 3 4 5

Activity - U/mg prot

NL

SOD

HL

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.

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Chloroplast antioxidative enzymes activities

Fdr

Fdox

O2-O2

-

DHAR

GSHGSSG

GRNADP+

NADPH

PSI FdR

H2O2

SOD

H2OAPX

Asc

DHA

PSIO2

H2O2

SOD

H2OAPX

DHA

Asc

Thylakoid

Stroma

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.

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Cl-

N

N

NS+

TCar:Chl – w/w

0

2

4

0 12 24 36 48

Time - hour

B

5

10

15

20

Chlorophyll – g/mL

A

NL

HL

+MB

H12NL

–

Effect of singlet oxygen generator

Methylene blue generates reactive singlet oxygen upon illumination. The dye is taken up by algae.

Nevertheless, the action of the dye promotes sustained astaxanthin build-up in both stationary and growing cultures.

The inhibitory effect of MB on growth, while mimicking high irradiance can be prevented by a pulse of high light intensity.

Finally, it is important to note that astaxanthin accumulation under stress is not prevented by ROS scavengers added to the medium.

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Excess of light

Generation of ROS

Cellular sensing,Cellular sensing, mediators mediators

Activation of cell responseActivation of cell response

+ LIGHT

Environmental Stresses: Nutrient deprivation Salt stress High light Low temperature Drought Aging

Cell response to stress in the green alga Cell response to stress in the green alga HaematococcusHaematococcus Mode of action Mode of action

Slowdown of cell division

11. . xanthophyll cycle xanthophyll cycle 2. ROS quenching enzymes2. ROS quenching enzymes3. antioxidants3. antioxidants

Change in cellChange in cellanabolismanabolism

(lipids)(lipids)

PalmelloidPalmelloidMotile cellMotile cell Astaxanthin accumulationAstaxanthin accumulation

EncystmentEncystment

Red cystRed cyst

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Thank you

☺