Optimized extraction of microalgae’s microalgae’s metabolites: a crucial step in metabolites: a crucial step in Optimized extraction of High- High -Throughput Screening programs dedicated to Throughput Screening programs dedicated to phytoplankton chemodiversity chemodiversity phytoplankton Track 12-1 Marine natural products and biomolecules Serive B., Kaas R., La Barre S., Bach S., Cadoret J-P.
Bioresources exploration Microalgaes: a high potential of biodiversity Van de Peer et al , 2000 - Laboratory of Physiology and Biotechnology of Algae 2
Microalgae biodiversity Dunaliella salina Source : IFREMER - Laboratory of Physiology and Biotechnology of Algae
Microalgae biodiversity Rhodomonas salina Source : IFREMER - Laboratory of Physiology and Biotechnology of Algae
Microalgae biodiversity Tetraselmis sp. Source : IFREMER - Laboratory of Physiology and Biotechnology of Algae
Microalgae biodiversity Closterium baillyanum Source : IFREMER - Laboratory of Physiology and Biotechnology of Algae
Microalgae biodiversity Euglena proxima Source : IFREMER - Laboratory of Physiology and Biotechnology of Algae
Microalgae biodiversity Porphyridium purpureum Source : IFREMER - Laboratory of Physiology and Biotechnology of Algae
Microalgae biodiversity Unknown cyanobacteria Source : IFREMER - Laboratory of Physiology and Biotechnology of Algae
Microalgae biodiversity Arthrospira platensis Source : IFREMER - Laboratory of Physiology and Biotechnology of Algae
Microalgae biodiversity Staurastrum vestitum - Laboratory of Physiology and Biotechnology of Algae
Microalgae biodiversity Chaetoceros muelleri Source : gt-online.pl - Laboratory of Physiology and Biotechnology of Algae
Microalgae biodiversity Skeletonema costatum Source : IFREMER - Laboratory of Physiology and Biotechnology of Algae
Microalgae biodiversity Asterionella formosa Source : planktonforum.eu - Laboratory of Physiology and Biotechnology of Algae
Microalgae biodiversity Alexandrium minutum Source : Gert Hansen - Laboratory of Physiology and Biotechnology of Algae
Microalgae biodiversity - Laboratory of Physiology and Biotechnology of Algae
Biodiversity 12 phyla eucaryota 1 phylum procaryota = Chemodiversity CH CH 3 3 N O O O CH 3 Polyketides, terpenes, alkaloides, isoprenoids, CH O 3 CH 3 O oxylipins, peptides, macrolides, microlides, H CH H 3 O lectines, glycolipids, polysaccharides, PUFAs, O CH 3 mycosporine-like AA, pigments = Pharmacodiversity Potential against cancers, Parkinson, Alzheimer, neurodegenerative diseases, bacterial and viral diseases, as immunostimulant, as fluorescent probes - Laboratory of Physiology and Biotechnology of Algae 17
Microalgae chemodiversity and pharmacodiversity Anti Age-related Macular Degeneration Lutein Zeaxanthin Landrum & Bone, 2001 - Laboratory of Physiology and Biotechnology of Algae 18
Microalgae chemodiversity and pharmacodiversity Moreau et al , 2006 Fucoxanthine O. aurita Violaxanthine D. tertiolecta Pasquet V., Morisset P., Ihammouine S., Chepied A., Aumailley L., Berard J-B., Serive B. , Kaas R., Lanneluc I., Thiery V., Lafferriere M., Piot J-M., Patrice T., Cadoret J-P., Picot L., 2011. Antiproliferative activity of Violaxanthin isolated from bioguided fractionation of Dunaliella tertiolecta extracts . Marine Drugs , 9, 819-831. - Laboratory of Physiology and Biotechnology of Algae 19
Microalgae chemodiversity and pharmacodiversity Usijirene Palythene Asterina-330 Mycosporine-glycine Photoprotectant Photoprotectant Antiproliferative Antiproliferative activity activity Antioxidant Antioxidant Palythine Palythenic acid Porphyra-334 Shinorine Mimouni et al, 2012. The Potential of Microalgae for the Production of Bioactive Molecules of Pharmaceutical Interest. Current Pharmaceutical Biotechnology 13(15): 2733-2750. - Laboratory of Physiology and Biotechnology of Algae 20
Aims The challenge is therefore to identify molecules issued from this biodiversity which are often poorly known in terms of chemodiversity. For high-throughput screening of such metabolites, it is essential to reach the inner content of the cell. A part of our project was the research and development of a technique enabling a high extraction yield of any metabolite, taking into account the difficulty of extracting bound and or inaccessible molecules with a wide variety of polarities. The final aim is to compare without extraction bias the potential of microalgae. - Laboratory of Physiology and Biotechnology of Algae 21
Choice of recalcitrant biological models For the study of the chemodiversity extraction 15 µm Porphyridium purpureum Phaeodactylum tricornutum Length : 5-8 µm Length : 3-4 x 15-20 µm Phylum : Rhodophyta Phylum : Ochrophyta Features : Features : - Exopolysaccharides - Silica wall - Thick wall - Laboratory of Physiology and Biotechnology of Algae 22
Chemodiversity extraction Comparison of 9 disruption techniques Main studied parameters: Effectiveness (1-(recalcitrant cells/total)) Reproducibility Operating cost Temperature stability Easy to handling Method Effectiveness Reproductibility Other considerations → Soaking - - Long processing → Cryogrinding - - Liquid nitrogen handling require → Potter homogeniser - - n.r. → Homogeniser + - Blade blunted by diatoms → Bead-beater + - Particles release → Planetary micro mill +++ +++ Particles release → Sonication ++ + ROS release, hot spots → Mixer mill – stainless steel jars + + n.r. Mixer mill – plastic grinding tube +++ +++ n.r. - Laboratory of Physiology and Biotechnology of Algae 23
Grinding optimisation on Retsch MM-400 Image analysis to evaluate acurately the particle size distribution Isoresponses overlay 1 + Control Optimized grinding A 0.9 P. Purpureum P. tricornutum Optimum P. purpureum P. tricornutum 0.8 0.7 Weight (g) Grinding time : 27 min 0.6 0.5 Beads weight : 0.92 g 0.4 0.3 50 50 0.2 µm µm 0 5 10 15 20 25 30 35 0.8 Time (min) B Control Optimized grinding 0.7 Optimum 0.6 Weight (g) Grinding time : 30 min 0.5 0.4 Beads weight : 0.24 g 0.3 + 0.2 50 50 0 5 10 15 20 25 30 35 µm µm Time (min) Size classes: ( ) Fragments; ( ) Whole cells ; ( ) Agregates. Serive et al, 2012. Bioresource Technology 124 Optimisation of 3 studied variables by image analysis: creation of an index of desirability. - Laboratory of Physiology and Biotechnology of Algae 24
Total extract, chemodiversity and dereplication → In a total extract, we have different kind of molecules: Known by the scientific community and known by us Known somewhere in the world but unknown by our database Unknown by everyone → Isoplot absorbance � metabolic snapshot because of the microalgae cellular plasticity Isoplot absorbance Bioactivity for a same strain and different Wavelenght (nm) environmental conditions 13 12 11 10 Score ROS 9 8 7 6 Time (min) SK(1,2,3) SK-R(1,2,3) L(1,2,3,4) - Laboratory of Physiology and Biotechnology of Algae 25
Methodology in the work flow Total extract OMICs studies Efficient metabolite extraction Microalgae chemodiversity ■ Genomic ■ Transcriptomic Metabolic snapshot ■ Proteomic ■ Metabolomic ■ Pigmentomic Serive et al, 2012. Bioresource Technology 124 - Laboratory of Physiology and Biotechnology of Algae 26
Photomer project Identify new photosensitizers from microalgae for PhotoDynamic Therapy 1 2 3 4 - Laboratory of Physiology and Biotechnology of Algae 27
Pigment analysis and spectral screening 45 microalgae strains composition → Spectral screening of interest in pharmacognosy. → High interest for oceanography applications. A B C D Examples of original spectra from the following strains: A- Alexandrium minutum B- Alexandrium tamarense C- Chlorarachnion reptans D- Phaeodactylum tricornutum - Laboratory of Physiology and Biotechnology of Algae 28
OCEAN CHemodiversity Against Cell cycle Targets Research of new marine inhibitors targeted against desease-relevant proteins kinases - Laboratory of Physiology and Biotechnology of Algae 29
J-B Charcot A B Bruker SolariX 12.0 Tesla C FTMS - Laboratory of Physiology and Biotechnology of Algae 30
Thank you for your attention Contact: Benoit.Serive@live.fr - Laboratory of Physiology and Biotechnology of Algae 31
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