Cellular factors mediating the production of astaxanthin by Haematococcus pluvialis

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

3. 40 Lag enhanced productivity 40 Cell (x105/ml) 10 Cell (x105/ml) 10 Chlorophyll (µg/ml) Chlorophyll (µg/ml) 1 1 0 20 40 60 80 100 0 10 20 30 40 50 60 70 80 Time(h) Time(h) Synchronized cultures and Life Cycle Normal Light High Light CA Chlamy06

4. Light intensity (mE m-2 s-1) 25 100 500 10000 100 1000 10 Biomass dry weight (g/L) Cell density (Mc/L) Chl (mg/L) 100 1 10 0.1 Cells Chl Biomass Chl 0.01 1 0 7 14 21 Continuous illumination (day) 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). CA Chlamy06

5. A NL HL - N Cells – 105/mL - P - S 30 10 Final yield – mg/ml C 100 B 41 8 10 39 6 30 TCar:Chl – w/w Chlorophyll – mg/mL 4 10 41 2 53 13 3 0 0 1 2 3 4 5 0 1 2 3 4 5 Time - day 3 Time - day Effect of different stresses on growth and astaxanthin accumulation 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. CA Chlamy06

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

7. Low light • High light 0 time 5 min 10 min 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. The mechanism of chloroplast-crossing remains unresolved. CA Chlamy06

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

9. 10 NL HL Control DCMU DBMIB Cells – 105/mL 3 DBMIB Fdx PQ Cyt b6f PS II PS I PQH2 PC DCMU 1 20 4 3 Chlorophyll – mg/mL 10 TCar:Chl – w/w 2 6 1 4 0 0 12 24 36 0 12 24 36 Time - hour Time - hour Effect of electron flow inhibitors 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. CA Chlamy06

10. SOD 30 NL HL 20 Activity - U/mg prot 10 0 0 1 2 3 4 5 Time - day 100 80 60 60 Activity - mU/mg prot 40 Activity - mU/mg prot 40 GR 20 DHAR 20 0 APX 0 1 2 3 4 5 0 Time - day 0 1 2 3 4 5 Time - day 1.5 Activity - U/mg prot 1.0 0.5 0 0 1 2 3 4 5 Time - day Antioxidative enzymes activities in soluble extracts 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. CA Chlamy06

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

12. 20 A – +MB HL 15 NL H12NL Chlorophyll – mg/mL 10 5 B 4 TCar:Chl – w/w Cl- N N S+ 2 N 0 0 12 24 36 48 Time - hour Effect of singlet oxygen generator 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. 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 • 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 Change in cell anabolism (lipids) Astaxanthin accumulation Motile cell Palmelloid Red cyst CA Chlamy06

14. Thank you ☺ CA Chlamy06