#METABOLOMICS WORKBENCH neo_009_20170616_150445 DATATRACK_ID:980 STUDY_ID:ST000622 ANALYSIS_ID:AN000954 PROJECT_ID:PR000454 VERSION 1 CREATED_ON June 20, 2017, 9:58 am #PROJECT PR:PROJECT_TITLE Identification and metabolite profiling of chemical activators of lipid PR:PROJECT_TITLE accumulation in green algae PR:PROJECT_TYPE GC-MS based Metabolite profiles PR:PROJECT_SUMMARY Microalgae are proposed as feedstock organisms useful for producing biofuels and PR:PROJECT_SUMMARY co-products. However, several limitations must be overcome before algae-based PR:PROJECT_SUMMARY production is economically feasible. Among these is the ability to induce lipid PR:PROJECT_SUMMARY accumulation and storage without affecting biomass yield. To overcome this PR:PROJECT_SUMMARY barrier, a chemical genetics approach was employed in which 43,783 compounds PR:PROJECT_SUMMARY were screened against Chlamydomonas reinhardtii and 243 compounds were PR:PROJECT_SUMMARY identified that increase triacylglyceride (TAG) accumulation without terminating PR:PROJECT_SUMMARY growth. Identified compounds were classified by structural similarity and 15 PR:PROJECT_SUMMARY selected for secondary analyses addressing impacts on growth fitness, PR:PROJECT_SUMMARY photosynthetic pigments, and total cellular protein and starch concentrations. PR:PROJECT_SUMMARY TAG accumulation was verified using GC-MS quantification of total fatty acids PR:PROJECT_SUMMARY and targeted TAG and galactolipid (GL) measurements using LC-MRM/MS. These PR:PROJECT_SUMMARY results demonstrated TAG accumulation does not necessarily proceed at the PR:PROJECT_SUMMARY expense of GL. Untargeted metabolite profiling provided important insights into PR:PROJECT_SUMMARY pathway shifts due to 5 different compound treatments and verified the anabolic PR:PROJECT_SUMMARY state of the cells with regard to the oxidative pentose phosphate pathway, PR:PROJECT_SUMMARY Calvin cycle, tricarboxylic acid cycle and amino acid biosynthetic pathways. PR:PROJECT_SUMMARY Metabolite patterns were distinct from nitrogen starvation and other abiotic PR:PROJECT_SUMMARY stresses commonly used to induce oil accumulation in algae. The efficacy of PR:PROJECT_SUMMARY these compounds was also demonstrated in 3 other algal species. These lipid PR:PROJECT_SUMMARY inducing compounds offer a valuable set of tools for delving into the PR:PROJECT_SUMMARY biochemical mechanisms of lipid accumulation in algae and a direct means to PR:PROJECT_SUMMARY improve algal oil content independent of the severe growth limitations PR:PROJECT_SUMMARY associated with nutrient deprivation. PR:INSTITUTE University of Nebraska-Lincoln PR:DEPARTMENT Biochemistry PR:LABORATORY FATTTLab PR:LAST_NAME Wase PR:FIRST_NAME Nishikant PR:ADDRESS 1901 Beadle Center, Vine Street, 1901 VINE STREET, Lincoln, NE, 68588-0664, USA PR:EMAIL nishikant.wase@gmail.com PR:PHONE 4023109931 #STUDY ST:STUDY_TITLE Identification and metabolite profiling of chemical activators of lipid ST:STUDY_TITLE accumulation in green algae ST:STUDY_TYPE GC-MS metabolite profiling of algal lipid activators ST:STUDY_SUMMARY Microalgae are proposed as feedstock organisms useful for producing biofuels and ST:STUDY_SUMMARY co-products. However, several limitations must be overcome before algae-based ST:STUDY_SUMMARY production is economically feasible. Among these is the ability to induce lipid ST:STUDY_SUMMARY accumulation and storage without affecting biomass yield. To overcome this ST:STUDY_SUMMARY barrier, a chemical genetics approach was employed in which 43,783 compounds ST:STUDY_SUMMARY were screened against Chlamydomonas reinhardtii and 243 compounds were ST:STUDY_SUMMARY identified that increase triacylglyceride (TAG) accumulation without terminating ST:STUDY_SUMMARY growth. Identified compounds were classified by structural similarity and 15 ST:STUDY_SUMMARY selected for secondary analyses addressing impacts on growth fitness, ST:STUDY_SUMMARY photosynthetic pigments, and total cellular protein and starch concentrations. ST:STUDY_SUMMARY TAG accumulation was verified using GC-MS quantification of total fatty acids ST:STUDY_SUMMARY and targeted TAG and galactolipid (GL) measurements using LC-MRM/MS. These ST:STUDY_SUMMARY results demonstrated TAG accumulation does not necessarily proceed at the ST:STUDY_SUMMARY expense of GL. Untargeted metabolite profiling provided important insights into ST:STUDY_SUMMARY pathway shifts due to 5 different compound treatments and verified the anabolic ST:STUDY_SUMMARY state of the cells with regard to the oxidative pentose phosphate pathway, ST:STUDY_SUMMARY Calvin cycle, tricarboxylic acid cycle and amino acid biosynthetic pathways. ST:STUDY_SUMMARY Metabolite patterns were distinct from nitrogen starvation and other abiotic ST:STUDY_SUMMARY stresses commonly used to induce oil accumulation in algae. The efficacy of ST:STUDY_SUMMARY these compounds was also demonstrated in 3 other algal species. These lipid ST:STUDY_SUMMARY inducing compounds offer a valuable set of tools for delving into the ST:STUDY_SUMMARY biochemical mechanisms of lipid accumulation in algae and a direct means to ST:STUDY_SUMMARY improve algal oil content independent of the severe growth limitations ST:STUDY_SUMMARY associated with nutrient deprivation. ST:INSTITUTE Univ of Nebraska-Lincoln ST:DEPARTMENT Biochemistry ST:LABORATORY FATTTLab ST:LAST_NAME Wase ST:FIRST_NAME Nishikant ST:ADDRESS 1901 Beadle Center, Vine Street, 1901 VINE STREET, Lincoln, NE, 68588-0664, USA ST:EMAIL nishikant.wase@gmail.com ST:PHONE 4023109931 ST:NUM_GROUPS 6 #SUBJECT SU:SUBJECT_TYPE Photosynthetic organism SU:SUBJECT_SPECIES Chlamydomonas reinhardtii SU:TAXONOMY_ID 3055 SU:GENOTYPE_STRAIN Wild Type #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - 030_A_1 Class:WD30030 SUBJECT_SAMPLE_FACTORS - 030_A_2 Class:WD30030 SUBJECT_SAMPLE_FACTORS - 030_A_3 Class:WD30030 SUBJECT_SAMPLE_FACTORS - 030_B_1 Class:WD30030 SUBJECT_SAMPLE_FACTORS - 030_B_2 Class:WD30030 SUBJECT_SAMPLE_FACTORS - 030_B_3 Class:WD30030 SUBJECT_SAMPLE_FACTORS - 030_C_1 Class:WD30030 SUBJECT_SAMPLE_FACTORS - 030_C_2 Class:WD30030 SUBJECT_SAMPLE_FACTORS - 030_C_3 Class:WD30030 SUBJECT_SAMPLE_FACTORS - 067_A_1 Class:WD20067 SUBJECT_SAMPLE_FACTORS - 067_A_2 Class:WD20067 SUBJECT_SAMPLE_FACTORS - 067_A_3 Class:WD20067 SUBJECT_SAMPLE_FACTORS - 067_B_1 Class:WD20067 SUBJECT_SAMPLE_FACTORS - 067_B_2 Class:WD20067 SUBJECT_SAMPLE_FACTORS - 067_B_3 Class:WD20067 SUBJECT_SAMPLE_FACTORS - 067_C_1 Class:WD20067 SUBJECT_SAMPLE_FACTORS - 067_C_2 Class:WD20067 SUBJECT_SAMPLE_FACTORS - 067_C_3 Class:WD20067 SUBJECT_SAMPLE_FACTORS - 461_A_1 Class:WD10461 SUBJECT_SAMPLE_FACTORS - 461_A_2 Class:WD10461 SUBJECT_SAMPLE_FACTORS - 461_A_3 Class:WD10461 SUBJECT_SAMPLE_FACTORS - 461_B_1 Class:WD10461 SUBJECT_SAMPLE_FACTORS - 461_B_2 Class:WD10461 SUBJECT_SAMPLE_FACTORS - 461_B_3 Class:WD10461 SUBJECT_SAMPLE_FACTORS - 461_C_1 Class:WD10461 SUBJECT_SAMPLE_FACTORS - 461_C_2 Class:WD10461 SUBJECT_SAMPLE_FACTORS - 461_C_3 Class:WD10461 SUBJECT_SAMPLE_FACTORS - 542_A_1 Class:WD20542 SUBJECT_SAMPLE_FACTORS - 542_A_2 Class:WD20542 SUBJECT_SAMPLE_FACTORS - 542_A_3 Class:WD20542 SUBJECT_SAMPLE_FACTORS - 542_B_1 Class:WD20542 SUBJECT_SAMPLE_FACTORS - 542_B_2 Class:WD20542 SUBJECT_SAMPLE_FACTORS - 542_B_3 Class:WD20542 SUBJECT_SAMPLE_FACTORS - 542_C_1 Class:WD20542 SUBJECT_SAMPLE_FACTORS - 542_C_2 Class:WD20542 SUBJECT_SAMPLE_FACTORS - 542_C_3 Class:WD20542 SUBJECT_SAMPLE_FACTORS - 784_A_1 Class:WD10784 SUBJECT_SAMPLE_FACTORS - 784_A_2 Class:WD10784 SUBJECT_SAMPLE_FACTORS - 784_A_3 Class:WD10784 SUBJECT_SAMPLE_FACTORS - 784_B_1 Class:WD10784 SUBJECT_SAMPLE_FACTORS - 784_B_2 Class:WD10784 SUBJECT_SAMPLE_FACTORS - 784_B_3 Class:WD10784 SUBJECT_SAMPLE_FACTORS - 784_C_1 Class:WD10784 SUBJECT_SAMPLE_FACTORS - 784_C_2 Class:WD10784 SUBJECT_SAMPLE_FACTORS - 784_C_3 Class:WD10784 SUBJECT_SAMPLE_FACTORS - ContA_1 Class:Control SUBJECT_SAMPLE_FACTORS - ContA_2 Class:Control SUBJECT_SAMPLE_FACTORS - ContA_3 Class:Control SUBJECT_SAMPLE_FACTORS - ContB_1 Class:Control SUBJECT_SAMPLE_FACTORS - ContB_2 Class:Control SUBJECT_SAMPLE_FACTORS - ContB_3 Class:Control SUBJECT_SAMPLE_FACTORS - ContC_1 Class:Control SUBJECT_SAMPLE_FACTORS - ContC_2 Class:Control SUBJECT_SAMPLE_FACTORS - ContC_3 Class:Control #COLLECTION CO:COLLECTION_SUMMARY Cells were pre-grown to mid-log phase and treated with 5 selected compounds CO:COLLECTION_SUMMARY (final concentration 5 µM) with an initial cell density of 1.0 x 106 cells/mL CO:COLLECTION_SUMMARY (100 mL volume; in triplicate) and allowed to grow for 72 h. After 72 h of CO:COLLECTION_SUMMARY growth, cells were harvested, media removed and freeze-dried. Accurately CO:COLLECTION_SUMMARY measured 50 ± 0.5 mg of freeze dried powder was used for metabolite extraction. CO:COLLECTION_SUMMARY Sample powder was pulverized with a single steel ball using TissueLyser LT CO:COLLECTION_SUMMARY (Qiagen) at 50 Hz speed for 5 min #TREATMENT TR:TREATMENT_SUMMARY Cells were treated either with compounds (5 uM) final concentration in DMSO or TR:TREATMENT_SUMMARY DMSO alone (in case of control). Mid-log phase cells were used as starter TR:TREATMENT_SUMMARY culture at a initial inoculum density of 1.0E06 cells/mL in 250 mL flasks with TR:TREATMENT_SUMMARY 100 mL of TAP media. Cells were allowed to grow in orbital shaker under constant TR:TREATMENT_SUMMARY light for 72 hours. After 72 hours, experiment was terminated and cells were TR:TREATMENT_SUMMARY harvested via centrifugation. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Harvested cells were flash frozen in liquid N and freeze dried. Accurately SP:SAMPLEPREP_SUMMARY measured 50 ± 0.5 mg of freeze dried powder was used for metabolite extraction. SP:SAMPLEPREP_SUMMARY Sample powder was pulverized with a single steel ball using TissueLyser LT SP:SAMPLEPREP_SUMMARY (Qiagen) at 50 Hz speed for 5 min. One milliliter of extraction buffer SP:SAMPLEPREP_SUMMARY containing MeOH:CHCl3:H2O (5:2:2; v/v/v; pre-cooled at -20 °C) was added and SP:SAMPLEPREP_SUMMARY vortexed for 5 min. Ribitol (0.2 mg/mL in water; 10 µL) was spiked in the SP:SAMPLEPREP_SUMMARY extraction buffer as internal standard in order to identify potential SP:SAMPLEPREP_SUMMARY chromatographic errors. The homogenized material was centrifuged at 14000 rpm SP:SAMPLEPREP_SUMMARY for 5 min and the supernatant was transferred to new tubes. 400 µL of pure SP:SAMPLEPREP_SUMMARY water was added to the supernatant, samples were vortexed and centrifuged at SP:SAMPLEPREP_SUMMARY 14,000 rpm for 5 min. The upper polar phase was transferred to new tubes for SP:SAMPLEPREP_SUMMARY GC-MS analysis. An aliquot of 300 µL was dried out in vacuum concentrator SP:SAMPLEPREP_SUMMARY without heating. To the dried material, 10 µL methoxyamine HCL in 100% pyridine SP:SAMPLEPREP_SUMMARY (40 mg/mL) was added and shaken at 30 °C for 90 minutes and subsequently 90 µL SP:SAMPLEPREP_SUMMARY of MSTFA 1% TMCS was added for trimethylsilylation of acidic protons and shaken SP:SAMPLEPREP_SUMMARY at 37 °C for 30 minutes. The reaction mixture was transferred to GCvials with SP:SAMPLEPREP_SUMMARY glass microinserts and closed by crimp caps. GC-MS data acquisition was SP:SAMPLEPREP_SUMMARY performed as per previously published report (Wase et al., 2014) #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 6890N CH:COLUMN_NAME Agilent DB-5MS UI Capillary column CH:INTERNAL_STANDARD Ribitol CH:SAMPLE_INJECTION 1 uL #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME FATTTLab AN:DETECTOR_TYPE MSD AN:DATA_FORMAT Agilent .d #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Agilent 5973 MS:INSTRUMENT_TYPE Single quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_RESULTS_FILE ST000622_AN000954_Results.txt UNITS:peak area #END