{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST000622","ANALYSIS_ID":"AN000954","VERSION":"1","CREATED_ON":"June 20, 2017, 9:58 am"},

"PROJECT":{"PROJECT_TITLE":"Identification and metabolite profiling of chemical activators of lipid accumulation in green algae","PROJECT_TYPE":"GC-MS based Metabolite profiles","PROJECT_SUMMARY":"Microalgae are proposed as feedstock organisms useful for producing biofuels and co-products. However, several limitations must be overcome before algae-based production is economically feasible. Among these is the ability to induce lipid accumulation and storage without affecting biomass yield. To overcome this barrier, a chemical genetics approach was employed in which 43,783 compounds were screened against Chlamydomonas reinhardtii and 243 compounds were identified that increase triacylglyceride (TAG) accumulation without terminating growth. Identified compounds were classified by structural similarity and 15 selected for secondary analyses addressing impacts on growth fitness, photosynthetic pigments, and total cellular protein and starch concentrations. TAG accumulation was verified using GC-MS quantification of total fatty acids and targeted TAG and galactolipid (GL) measurements using LC-MRM/MS. These results demonstrated TAG accumulation does not necessarily proceed at the expense of GL. Untargeted metabolite profiling provided important insights into pathway shifts due to 5 different compound treatments and verified the anabolic state of the cells with regard to the oxidative pentose phosphate pathway, Calvin cycle, tricarboxylic acid cycle and amino acid biosynthetic pathways. Metabolite patterns were distinct from nitrogen starvation and other abiotic stresses commonly used to induce oil accumulation in algae. The efficacy of these compounds was also demonstrated in 3 other algal species. These lipid inducing compounds offer a valuable set of tools for delving into the biochemical mechanisms of lipid accumulation in algae and a direct means to improve algal oil content independent of the severe growth limitations associated with nutrient deprivation.","INSTITUTE":"University of Nebraska-Lincoln","DEPARTMENT":"Biochemistry","LABORATORY":"FATTTLab","LAST_NAME":"Wase","FIRST_NAME":"Nishikant","ADDRESS":"1901 Beadle Center, Vine Street, 1901 VINE STREET, Lincoln, NE, 68588-0664, USA","EMAIL":"nishikant.wase@gmail.com","PHONE":"4023109931"},

"STUDY":{"STUDY_TITLE":"Identification and metabolite profiling of chemical activators of lipid accumulation in green algae","STUDY_TYPE":"GC-MS metabolite profiling of algal lipid activators","STUDY_SUMMARY":"Microalgae are proposed as feedstock organisms useful for producing biofuels and co-products. However, several limitations must be overcome before algae-based production is economically feasible. Among these is the ability to induce lipid accumulation and storage without affecting biomass yield. To overcome this barrier, a chemical genetics approach was employed in which 43,783 compounds were screened against Chlamydomonas reinhardtii and 243 compounds were identified that increase triacylglyceride (TAG) accumulation without terminating growth. Identified compounds were classified by structural similarity and 15 selected for secondary analyses addressing impacts on growth fitness, photosynthetic pigments, and total cellular protein and starch concentrations. TAG accumulation was verified using GC-MS quantification of total fatty acids and targeted TAG and galactolipid (GL) measurements using LC-MRM/MS. These results demonstrated TAG accumulation does not necessarily proceed at the expense of GL. Untargeted metabolite profiling provided important insights into pathway shifts due to 5 different compound treatments and verified the anabolic state of the cells with regard to the oxidative pentose phosphate pathway, Calvin cycle, tricarboxylic acid cycle and amino acid biosynthetic pathways. Metabolite patterns were distinct from nitrogen starvation and other abiotic stresses commonly used to induce oil accumulation in algae. The efficacy of these compounds was also demonstrated in 3 other algal species. These lipid inducing compounds offer a valuable set of tools for delving into the biochemical mechanisms of lipid accumulation in algae and a direct means to improve algal oil content independent of the severe growth limitations associated with nutrient deprivation.","INSTITUTE":"Univ of Nebraska-Lincoln","DEPARTMENT":"Biochemistry","LABORATORY":"FATTTLab","LAST_NAME":"Wase","FIRST_NAME":"Nishikant","ADDRESS":"1901 Beadle Center, Vine Street, 1901 VINE STREET, Lincoln, NE, 68588-0664, USA","EMAIL":"nishikant.wase@gmail.com","PHONE":"4023109931","NUM_GROUPS":"6"},

"SUBJECT":{"SUBJECT_TYPE":"Photosynthetic organism","SUBJECT_SPECIES":"Chlamydomonas reinhardtii","TAXONOMY_ID":"3055","GENOTYPE_STRAIN":"Wild Type"},
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"COLLECTION":{"COLLECTION_SUMMARY":"Cells were pre-grown to mid-log phase and treated with 5 selected compounds (final concentration 5 µM) with an initial cell density of 1.0 x 106 cells/mL (100 mL volume; in triplicate) and allowed to grow for 72 h. After 72 h of growth, cells were harvested, media removed and freeze-dried. Accurately measured 50 ± 0.5 mg of freeze dried powder was used for metabolite extraction. Sample powder was pulverized with a single steel ball using TissueLyser LT (Qiagen) at 50 Hz speed for 5 min"},

"TREATMENT":{"TREATMENT_SUMMARY":"Cells were treated either with compounds (5 uM) final concentration in DMSO or DMSO alone (in case of control). Mid-log phase cells were used as starter culture at a initial inoculum density of 1.0E06 cells/mL in 250 mL flasks with 100 mL of TAP media. Cells were allowed to grow in orbital shaker under constant light for 72 hours. After 72 hours, experiment was terminated and cells were harvested via centrifugation."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Harvested cells were flash frozen in liquid N and freeze dried. Accurately measured 50 ± 0.5 mg of freeze dried powder was used for metabolite extraction. Sample powder was pulverized with a single steel ball using TissueLyser LT (Qiagen) at 50 Hz speed for 5 min. One milliliter of extraction buffer containing MeOH:CHCl3:H2O (5:2:2; v/v/v; pre-cooled at -20 °C) was added and vortexed for 5 min. Ribitol (0.2 mg/mL in water; 10 µL) was spiked in the extraction buffer as internal standard in order to identify potential chromatographic errors. The homogenized material was centrifuged at 14000 rpm for 5 min and the supernatant was transferred to new tubes. 400 µL of pure water was added to the supernatant, samples were vortexed and centrifuged at 14,000 rpm for 5 min. The upper polar phase was transferred to new tubes for GC-MS analysis. An aliquot of 300 µL was dried out in vacuum concentrator without heating. To the dried material, 10 µL methoxyamine HCL in 100% pyridine (40 mg/mL) was added and shaken at 30 °C for 90 minutes and subsequently 90 µL of MSTFA 1% TMCS was added for trimethylsilylation of acidic protons and shaken at 37 °C for 30 minutes. The reaction mixture was transferred to GCvials with glass microinserts and closed by crimp caps. GC-MS data acquisition was performed as per previously published report (Wase et al., 2014)"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"GC","INSTRUMENT_NAME":"Agilent 6890N","COLUMN_NAME":"Agilent DB-5MS UI Capillary column","INTERNAL_STANDARD":"Ribitol","SAMPLE_INJECTION":"1 uL"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","LABORATORY_NAME":"FATTTLab","DETECTOR_TYPE":"MSD","DATA_FORMAT":"Agilent .d"},

"MS":{"MS_COMMENTS":"-","INSTRUMENT_NAME":"Agilent 5973","INSTRUMENT_TYPE":"Single quadrupole","MS_TYPE":"EI","ION_MODE":"POSITIVE","MS_RESULTS_FILE":"ST000622_AN000954_Results.txt UNITS:peak area"}

}