Presentation on theme: "Cellular factors mediating the production of astaxanthin by Haematococcus pluvialis Claude Aflalo & Sammy Boussiba Microalgal Biotechnology Laboratory."— Presentation transcript:
Cellular factors mediating the production of astaxanthin by Haematococcus pluvialis Claude Aflalo & Sammy Boussiba Microalgal Biotechnology Laboratory Blaustein Institutes for Desert Research Ben Gurion University, Sde Boker, Israel Thanks to: Bing Wang, Yuval Meshulem, Aliza Zarka, Ben Friehoff
CA Chlamy062 Haematococcus pluvialis Haematococcus Green algae under stress 5 m Red snow Chlamydomonas nivalis Chloromonas nivalis 5 m
CA Chlamy063 Synchronized cultures and Life Cycle 01020304050607080 Time (h) 1 10 40 Cell (x10 5 /ml) Chlorophyll (µg/ml) 1 10 40 Cell (x10 5 /ml) Chlorophyll (µg/ml) 020406080100 Time (h) High LightNormal Light Lag enhanced productivity
CA Chlamy064 500 25 Light intensity ( E m -2 s -1 ) 1 10 100 1000 10000 071421 Continuous illumination (day) Cell density (Mc/L) Chl (mg/L) 0.01 0.1 1 10 100 Biomass dry weight (g/L) CellsChlBiomass 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).
CA Chlamy065 Cells – 10 5 /mL 10 3 30 NL HL - N - P - S A 10 100 30 3 Chlorophyll – g/mL B Time - day 012345 Effect of different stresses on growth and astaxanthin accumulation Time - day 0 2 4 6 8 10 012345 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. 13 53 41 39 41 Final yield – g/ml
CA Chlamy066 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
CA Chlamy067 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 time5 min10 min Low light High light The mechanism of chloroplast-crossing remains unresolved.
CA Chlamy068 H2OH2O PQ PS I PS II NADP + NADPH + H + PC 2H + 2H + +1/2 O 2 2H + Cyt b6f PQH 2 Fdx Electron flow in the thylakoid membrane DBMIB DCMU
CA Chlamy069 Time - hour 0 1 2 3 4 0122436 TCar:Chl – w/w 4 20 Chlorophyll – g/mL 6 10 Time - hour 0122436 HL 3 Cells – 10 5 /mL 1 10 NL DCMU DBMIB Control PQ Fdx PS I PS II PC DCMU DBMIB Cyt b6f PQH 2 Effect of electron flow inhibitors While both inhibitors effectively stop growth, the action of DBMIB that promotes accumulation of reduced plastoquinone (PQH 2 ), results in substantial astaxanthin accumulation, at high – but not at normal – light intensity.
CA Chlamy0610 Antioxidative enzymes activities in soluble extracts 0 20 40 60 012345 Time - day Activity - mU/mg prot DHAR 012345 Time - day Activity - mU/mg prot 0 20 40 60 80 100 GR 0 0.5 1.0 1.5 012345 Activity - U/mg prot APX Time - day 0 10 20 30 012345 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.
CA Chlamy0611 Chloroplast antioxidative enzymes activities Fd r Fd ox O2-O2- O2-O2- DHAR GSH GSSG GR NADP + NADPH PSI FdR H2O2H2O2 SOD H2OH2O APX Asc DHA PSI O2O2 H2O2H2O2 SOD H2OH2O APX 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.
CA Chlamy0612 Cl - N N N S+S+ TCar:Chl – w/w 0 2 4 012243648 Time - hour B 5 10 15 20 Chlorophyll – g/mL A NL HL +MB H 12 NL – 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.
CA Chlamy0613 Excess of light Generation of ROS Cellular sensing, mediators Activation of cell response + LIGHT Environmental Stresses: l Nutrient deprivation l Salt stress l High light l Low temperature l Drought l Aging Cell response to stress in the green alga Haematococcus Mode of action Slowdown of cell division 1. xanthophyll cycle 2. ROS quenching enzymes 3. antioxidants Change in cell anabolism(lipids) Palmelloid Motile cell Astaxanthin accumulation Encystment Red cyst