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Claude Aflalo & Sammy Boussiba

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1 Claude Aflalo & Sammy Boussiba
4/13/2017 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

2 Green algae under stress
blush... Red snow Haematococcus pluvialis Haematococcus Chlamydomonas nivalis Chloromonas nivalis 5 mm 5 mm CA Chlamy06

3 Synchronized cultures and Life Cycle
4/13/2017 Synchronized cultures and Life Cycle 10 20 30 40 50 60 70 80 Time (h) 1 Cell (x105/ml) Chlorophyll (µg/ml) 1 10 40 Cell (x105/ml) Chlorophyll (µg/ml) 20 60 80 100 Time (h) Lag enhanced productivity Normal Light High Light Filtered (<20um) green flagellated cells as inoculum Thecae are counted as single cells. CA Chlamy06

4 The ratio light/cell as a growth limiting factor
4/13/2017 The ratio light/cell as a growth limiting factor 25 Light intensity (mE m-2 s-1) 1 10 100 1000 10000 7 14 21 Continuous illumination (day) Cell density (Mc/L) Chl (mg/L) 0.01 0.1 Biomass dry weight (g/L) Cells Chl Biomass 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). 100 Chl 500 Cell division and biomass growth: separate processes CA Chlamy06

5 Effect of different stresses on growth and astaxanthin accumulation
Cells – 105/mL 10 3 30 NL HL - N - P - S A 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. Time - day 2 4 6 8 10 1 3 5 TCar:Chl – w/w C 13 53 41 39 Final yield – mg/ml 10 100 30 3 Chlorophyll – mg/mL B Time - day 1 2 4 5 CA Chlamy06

6 Basic carotenogenesis: primary and secondary products
4/13/2017 Basic carotenogenesis: primary and secondary products 8 Pyr + 8 GA3P 6 IPP + 2 DMAPP Activation (ATP) Condensation Reduction (NADPH) Phytoene Lycopene Oxidation b carotene a carotene Cyclization Lutein Zeaxanthin Chloroplast Xanthophylls Cyt-P450 Net oxidation Canthaxanthin Astaxanthin Lipid globule O OH HO Export ? Cyt-P450 Net oxidation CA Chlamy06

7 Lipid globules traffic in the cytosol
Low light High light 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 The mechanism of chloroplast-crossing remains unresolved. CA Chlamy06

8 Electron flow in the thylakoid membrane
H2O PQ PS I PS II NADP+ NADPH + H+ PC 2H+ 2H+ +1/2 O2 Cyt b6f PQH2 Fdx DCMU DBMIB CA Chlamy06

9 Effect of electron flow inhibitors
HL 3 Cells – 105/mL 1 10 NL DCMU DBMIB Control PQ Fdx PS I PS II PC Cyt b6f PQH2 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. 4 20 Chlorophyll – mg/mL 6 10 Time - hour 12 24 36 Time - hour 1 2 3 4 12 24 36 TCar:Chl – w/w CA Chlamy06

10 Antioxidative enzymes activities in soluble extracts
Time - day 10 20 30 1 2 3 4 5 Activity - U/mg prot NL SOD HL 0.5 1.0 1.5 1 2 3 4 5 Activity - U/mg prot APX Time - day 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. 20 40 60 1 2 3 4 5 Time - day Activity - mU/mg prot DHAR 1 2 3 4 5 Time - day Activity - mU/mg prot 20 40 60 80 100 GR CA Chlamy06

11 Chloroplast antioxidative enzymes activities
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. Fdr Fdox O2- DHAR GSH GSSG GR NADP+ NADPH PSI FdR H2O2 SOD H2O APX Asc DHA O2 Thylakoid Stroma CA Chlamy06

12 Effect of singlet oxygen generator
Cl- N S+ Effect of singlet oxygen generator 5 10 15 20 Chlorophyll – mg/mL A NL HL +MB H12NL Methylene blue generates reactive singlet oxygen upon illumination. The dye is taken up by algae. The inhibitory effect of MB on growth, while mimicking high irradiance can be prevented by a pulse of high light intensity. TCar:Chl – w/w 2 4 12 24 36 48 Time - hour B Nevertheless, the action of the dye promotes sustained astaxanthin build-up in both stationary and growing cultures. Finally, it is important to note that astaxanthin accumulation under stress is not prevented by ROS scavengers added to the medium. CA Chlamy06

13 Cell response to stress in the green alga Haematococcus Mode of action
+ LIGHT Environmental Stresses: Nutrient deprivation Salt stress High light Low temperature Drought Aging Slowdown of cell division Excess of light Generation of ROS Cellular sensing, mediators Activation of cell response Encystment 1. xanthophyll cycle 2. ROS quenching enzymes 3. antioxidants Change in cell anabolism (lipids) Red cyst Palmelloid Motile cell Astaxanthin accumulation CA Chlamy06

14 Thank you CA Chlamy06

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