#METABOLOMICS WORKBENCH ppjutur_20220622_232801 DATATRACK_ID:3315 STUDY_ID:ST002215 ANALYSIS_ID:AN003624 PROJECT_ID:PR001415 VERSION 1 CREATED_ON July 11, 2022, 9:35 pm #PROJECT PR:PROJECT_TITLE Multi-omics analysis of Aurantiochytrium limacinum PR:PROJECT_TYPE Time course GCMS based untargeted metabolomics in the presence of glucose and PR:PROJECT_TYPE glycerol PR:PROJECT_SUMMARY To evaluate temporal changes in the presence of glycerol, time-course metabolite PR:PROJECT_SUMMARY profiling was done in the presence of glycerol and glucose at three different PR:PROJECT_SUMMARY time-points i.e., 0, 48 and 96 h using GCMS. PR:INSTITUTE International Centre for Genetic Engineering and Biotechnology PR:DEPARTMENT Omics of Algae PR:LAST_NAME Jutur PR:FIRST_NAME Pannaga Pavan PR:ADDRESS Omics of Algae Group, ICGEB campus, Aruna Asaf Ali Marg, New Delhi, Delhi, PR:ADDRESS 110070, India PR:EMAIL jppavan@icgeb.res.in PR:PHONE 26781358 #STUDY ST:STUDY_TITLE Time course GCMS based untargeted metabolomics in the presence of glucose and ST:STUDY_TITLE glycerol ST:STUDY_SUMMARY Our preliminary analysis identifies that glycerol enhanced DHA accumulation in ST:STUDY_SUMMARY native isolate Aurantiochytrium limacinum. To evaluate temporal changes in the ST:STUDY_SUMMARY presence of glycerol, time-course metabolite profiling was done in the presence ST:STUDY_SUMMARY of glycerol and glucose at three different time-points i.e., 0, 48 and 96 h ST:STUDY_SUMMARY using GC-MS. We identified nearly 40 metabolites, among which metabolites ST:STUDY_SUMMARY belonging to pentose phosphate pathway, TCA cycle and amino acid metabolism were ST:STUDY_SUMMARY significantly differentially regulated which suggests its role in glycerol ST:STUDY_SUMMARY induced DHA accumulation. ST:INSTITUTE International Centre for Genetic Engineering and Biotechnology ST:LAST_NAME Jutur ST:FIRST_NAME Pannaga Pavan ST:ADDRESS Omics of Algae Lab, 2nd Floor, New Building, ICGEB, Aruna Asaf Ali Marg, New ST:ADDRESS Delhi ST:EMAIL jppavan@gmail.com ST:PHONE 01126741358 #SUBJECT SU:SUBJECT_TYPE Other organism SU:SUBJECT_SPECIES Aurantiochytrium limacinum #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - D0_1 Treatment:Glucose RAW_FILE_NAME=D0_1; Time point=0 h SUBJECT_SAMPLE_FACTORS - D0_2 Treatment:Glucose RAW_FILE_NAME=D0_2; Time point=0 h SUBJECT_SAMPLE_FACTORS - D0_3 Treatment:Glucose RAW_FILE_NAME=D0_3; Time point=0 h SUBJECT_SAMPLE_FACTORS - D2_Glucose_1 Treatment:Glucose RAW_FILE_NAME=D2_GLU_1; Time point=48 h SUBJECT_SAMPLE_FACTORS - D2_Glucose_2 Treatment:Glucose RAW_FILE_NAME=D2_GLU_2; Time point=48 h SUBJECT_SAMPLE_FACTORS - D2_Glucose_3 Treatment:Glucose RAW_FILE_NAME=D2_GLU_3; Time point=48 h SUBJECT_SAMPLE_FACTORS - D2_Glycerol_1 Treatment:Glycerol RAW_FILE_NAME=D2_GLY_1; Time point=48 h SUBJECT_SAMPLE_FACTORS - D2_Glycerol_2 Treatment:Glycerol RAW_FILE_NAME=D2_GLY_2; Time point=48 h SUBJECT_SAMPLE_FACTORS - D2_Glycerol_3 Treatment:Glycerol RAW_FILE_NAME=D2_GLY_3; Time point=48 h SUBJECT_SAMPLE_FACTORS - D4_Glucose_1 Treatment:Glucose RAW_FILE_NAME=D4_GLU_1; Time point=96 h SUBJECT_SAMPLE_FACTORS - D4_Glucose_2 Treatment:Glucose RAW_FILE_NAME=D4_GLU_2; Time point=96 h SUBJECT_SAMPLE_FACTORS - D4_Glucose_3 Treatment:Glucose RAW_FILE_NAME=D4_GLU_3; Time point=96 h SUBJECT_SAMPLE_FACTORS - D4_Glycerol_1 Treatment:Glycerol RAW_FILE_NAME=D4_GLY_1; Time point=96 h SUBJECT_SAMPLE_FACTORS - D4_Glycerol_2 Treatment:Glycerol RAW_FILE_NAME=D4_GLY_2; Time point=96 h SUBJECT_SAMPLE_FACTORS - D4_Glycerol_3 Treatment:Glycerol RAW_FILE_NAME=D4_GLY_3; Time point=96 h #COLLECTION CO:COLLECTION_SUMMARY Marine thraustochytrid Aurantiochytrium limacinum was a native isolate obtained CO:COLLECTION_SUMMARY as a generous gift from ICT-Mumbai, India. It was cultivated in YPD medium with CO:COLLECTION_SUMMARY the optimized concentration of 3% (w/v) glucose and glycerol at 20 C for a CO:COLLECTION_SUMMARY duration of 96 h. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY It was cultivated in YPD medium with the optimized concentration of 3% (w/v) TR:TREATMENT_SUMMARY glucose and glycerol at 20 C for a duration of 96 h. TR:TREATMENT Glucose and Glycerol #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Quenched cells were resuspended in 1 mL of ice-cold SP:SAMPLEPREP_SUMMARY methanol/ethanol/chloroform(2:6:2), followed by sonication of resuspended cells SP:SAMPLEPREP_SUMMARY in sonication bath for 15 min. Later, these samples were centrifuged at SP:SAMPLEPREP_SUMMARY 10,000×g for 15 min at 4 °C to get rid of cell debris. The supernatant was SP:SAMPLEPREP_SUMMARY filtered using a 0.2-µm filter. One hundred microlitres of supernatant was SP:SAMPLEPREP_SUMMARY taken and dried under nitrogen stream. The dried leftover was dissolved in 10 SP:SAMPLEPREP_SUMMARY µL of freshly prepared methoxyamine hydrochloride solution (40 mg mL−1 in SP:SAMPLEPREP_SUMMARY pyridine) and incubated at 30 °C for 90 min with shaking. To the above SP:SAMPLEPREP_SUMMARY solution, 90 µL of N-methyl-N-(trimethylsilyl)trifluoroacetamide was added and SP:SAMPLEPREP_SUMMARY incubated at 37 °C for 30 min. The samples were centrifuged at 14,000×g for 3 SP:SAMPLEPREP_SUMMARY min, and the supernatant was taken for the GC-MS/MS analysis. SP:PROCESSING_STORAGE_CONDITIONS Described in summary SP:SAMPLE_DERIVATIZATION MSTFA SP:SAMPLE_SPIKING Nonadecanoic acid #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 7890A CH:COLUMN_NAME Agilent DB5-MS (30m x 0.25mm, 0.25um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 7890A MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Mass Hunter #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Area MS_METABOLITE_DATA_START Samples D0_1 D0_2 D0_3 D2_Glucose_1 D2_Glucose_2 D2_Glucose_3 D2_Glycerol_1 D2_Glycerol_2 D2_Glycerol_3 D4_Glucose_1 D4_Glucose_2 D4_Glucose_3 D4_Glycerol_1 D4_Glycerol_2 D4_Glycerol_3 Factors Treatment:Glucose Treatment:Glucose Treatment:Glucose Treatment:Glucose Treatment:Glucose Treatment:Glucose Treatment:Glycerol Treatment:Glycerol Treatment:Glycerol Treatment:Glucose Treatment:Glucose Treatment:Glucose Treatment:Glycerol Treatment:Glycerol Treatment:Glycerol L-Alanine 125584230.9 91734745.32 57885259.73 1382455.341 2626638.464 1244952.062 3228913.217 2236932.639 4188827.146 4188827.146 4094929.239 1589219.155 L-Glycine 88090719.42 59750586.54 31410453.66 605770.5 2038907.639 702385.6813 498136.4816 600261.0814 1742410.382 188200.8927 1044998.1 598763.5661 L-Valine 75521276.58 50716358.89 25911441.19 8075194.273 15305042.26 3240149.909 27329693.73 15284921.82 989737.614 989737.614 86405.66364 224068.6007 L-Leucine 78449500.5 52778231.47 27106962.45 579610.2955 2490625.663 395243.8687 3472645.97 1933944.919 1049757.27 1049757.27 L-Proline 401234072.8 279133220.9 157032369 15597033.27 34996398.67 7712811.738 29865089.86 18788950.8 1805109.995 1805109.995 27034357.43 11650607.82 L-Isoleucine 27207072.54 18304945.44 9402818.335 L-Serine 15133808.11 9973457.471 4813106.833 214328.4091 136933.3183 131706.819 L-Threonine 71316512.99 47669495.77 24022478.56 793121.6136 999846.9987 L-Methionine 1204881.242 41925.36364 73848.11165 533386.3947 303617.2532 L-Phenylalanine 6524012.076 122660.7273 501949.1534 125646.9449 L-Aspartic acid 49213.11364 119115.656 102639.543 394479.2061 248559.3745 444292.4996 L-Tyrosine 816629.4091 559006.7837 L-Tryptophan 18619.59805 GABA 4276471.794 35748 59566.32511 253693.6292 156629.9772 Propanoic acid 1378130.731 36096.29545 985772.6609 41519.65162 185605.2758 Acetic acid 2347838.8 2522998.347 2698157.893 133275.8896 386441.7768 94569.56495 94569.56495 Succinic acid 213616367.6 147621691.3 81627015.08 3330210.114 7158739.936 4071620.658 270516.2307 2171068.444 20215670.19 681323.8504 20364444.45 Malic acid 7023505.771 4441126.162 1858746.552 87664.77273 Glutamic acid 1147070.969 70758.08197 Phosphoric acid 2020629.832 1241195.636 461761.4395 111098.0682 5197164.124 209145.2268 478980.0588 344062.6428 3690062.257 3690062.257 Glycerol 6215130.805 4329392.734 2443654.663 703080.5893 155244.7933 Propanedioic acid 3192922.592 2087485.279 982047.9656 95412.45403 60306.3317 60306.3317 D-Galactose 6374801.778 156406.4091 1737018.902 800493.3561 1432489.841 1116491.599 222796.8555 990900.4319 Myo-Inositol 269628304.9 135993296.2 2358287.439 33391.31818 2626638.464 6334581.729 163831.8529 552956.7093 358394.2811 163199.7199 32406188.83 1428086.717 1589219.155 D-Ribose 4956714.856 139308.7955 400883.9878 4618616.888 11666172.71 11666172.71 1963440.106 D-deoxyribose 347778.4045 248906.5911 187244.8064 1047844.261 199393.0138 GLUTAMINIC ACID 186337.3638 3256712.792 1721525.078 413920.959 D-Glucose 277287927.4 288918397.3 300548867.3 6018231.148 775598.3288 4904167.529 2839882.929 19947505.09 1796684.391 1796684.391 8538346.958 57010149.12 Pentadecanoic acid 20837919.64 13237890.28 5637860.912 264213.3636 642911.7276 202156.4253 388870.0879 295513.2566 420384.4101 Hexadecanoic acid 301627500 185224076.5 68820653.01 1791062.273 3937129.713 362020.051 1666370.763 3787840.843 2727105.803 1125114.228 1125114.228 1565815.275 1942517.808 543347.729 Heptadecanoic acid 37223299.98 21953919.28 6684538.585 152350.6364 264966.6676 70502.46591 376406.5278 Octadecanedioic acid 2053103.48 Trehalose 958735.6435 775020.3869 591305.1302 38703.63636 92869.7567 52608.10371 Squalene 45259037.14 31220800.03 17182562.92 286762.2045 1098571.692 703701.9887 2653553.686 1678627.837 EPA 2627189.064 86703.29545 1211891.901 DHA 11091028.28 13076398.77 15061769.27 1347952.932 6243739.346 3257139.363 5133315.206 4195227.284 2263074.384 2382764.5 5-Methyluridine 3052333.584 3946286.664 4840239.744 133721.7273 271684.5144 814396.9788 668693.144 127251.36 5,6-Dimethyluracil 545211.8323 37257.93182 106736.3296 59962.38155 Butanoic acid 87497493.23 1794506.023 3536288.294 1047614.823 1203444.52 1822845.434 1513144.977 46711.83922 2422315.231 Cholesterol 45550968.73 32064163.51 18577358.29 227284.7727 889077.5314 4224346.723 874390.2302 4233996.068 2554193.149 370912.8392 579801.4118 579801.4118 940036.6743 4089498.473 1104113.245 D-Lactic acid 7833258.945 5541778.44 3250297.934 64358.20455 83365.48271 90229.32285 97872.20111 94050.76198 13065032.46 168262.5451 9-Octadecanoic acid 4073901.914 129080.6591 764037.1627 Octadecanoic acid 97560604.75 1874310.114 1919258.233 348116.6921 241456.6334 294786.6628 729412.8165 Eicosanoic acid 822314.1801 Stigmasterol 57885259.73 75159.72727 1278792.157 283891.9523 636715.5227 460303.7375 1578315.667 411672.8714 D-Allose 96791100.9 3702039.66 13584990.21 19343870.32 19343870.32 3446879.167 O,O''-Trimethylsilyl-O'-stearoylglycerol 6939435.106 3649640.609 359846.1121 28789.75 155921.4314 265422.5591 D-Erythrose 1355214.432 145481.4091 685459.982 1780625.493 280686.1978 1472989.752 800857.4521 Citric acid 96215.63636 75126.21706 322692.4167 768546.7203 545619.5685 D-Tagatofuranose 268494.1364 361026.0117 482190.9556 3398727.791 D-Mannose 1029219.836 2298475.107 979972.531 4652816.492 206569.6525 2195488.55 Ribitol 221240.1951 109977.583 284992.7418 284992.7418 431310.071 D-sorbitol 928639.1479 3072077.617 404463.6062 404463.6062 1906660.183 Fumaric acid 295922.5371 163445.9093 XYLITOL 70725.78407 247170.5839 306353.1604 D-Arabinose 1479964.419 2343427.659 6356361.377 5507166.859 252707.5738 396261.3595 Docosanoic acid 4687210.421 D-Fructose 88679.62648 130574.7131 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name HMDB PubChem KEGG SMILES Comment L-Alanine HMDB0000161 5950 C00041 C[C@@H](C(=O)O)N 1 L-Glycine HMDB0000123 750 C00037 C(C(=O)O)N 1 L-Valine HMDB0000883 6287 C00183 CC(C)[C@@H](C(=O)O)N 1 L-Leucine HMDB0000687 6106 C00123 CC(C)C[C@@H](C(=O)O)N 1 L-Proline HMDB0000162 145742 C00148 C1C[C@H](NC1)C(=O)O 1 L-Isoleucine HMDB0000172 6306 C00407 CC[C@H](C)[C@@H](C(=O)O)N 1 L-Serine HMDB0000187 5951 C00065 C([C@@H](C(=O)O)N)O 1 L-Threonine HMDB0000167 6288 C00188 C[C@H]([C@@H](C(=O)O)N)O 1 L-Methionine HMDB0000696 6137 C00073 CSCC[C@@H](C(=O)O)N 1 L-Phenylalanine HMDB0000159 6140 C00079 C1=CC=C(C=C1)C[C@@H](C(=O)O)N 1 L-Aspartic acid HMDB0000191 5960 C00049 C([C@@H](C(=O)O)N)C(=O)O 1 L-Tyrosine HMDB0000158 6057 C00082 C1=CC(=CC=C1C[C@@H](C(=O)O)N)O 1 L-Tryptophan HMDB0000929 6305 C00078 C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)N 1 GABA HMDB0000112 223130 C00334 C(CC(=O)O)CN 1 Propanoic acid HMDB0000237 1032 C00163 CCC(=O)O 1 Acetic acid HMDB0000042 176 C00033 CC(=O)O 1 Succinic acid HMDB0000254 1110 C00042 C(CC(=O)O)C(=O)O 1 Malic acid HMDB0000744 525 C03668 C(C(C(=O)O)O)C(=O)O 1 Glutamic acid HMDB0000148 33032 C00025 C(CC(=O)O)[C@@H](C(=O)O)N 1 Phosphoric acid HMDB0002142 57424078 C00009 OP(=O)(O)O 1 Glycerol HMDB0000131 753 C00116 C(C(CO)O)O 1 Propanedioic acid HMDB0000691 867 C04025 C(C(=O)O)C(=O)O 1 D-Galactose HMDB0000143 439357 C00984 C([C@@H]1[C@@H]([C@@H]([C@H]([C@H](O1)O)O)O)O)O 1 Myo-Inositol HMDB0000211 NA C00137 O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O 1 D-Ribose HMDB0000283 5779 C00121 C([C@@H]1[C@H]([C@H](C(O1)O)O)O)O 1 D-deoxyribose HMDB0003224 22833604 C01801 C1[C@H]([C@@H](O[C@@H]1O)CO)O 1 GLUTAMINIC ACID HMDB0000148 33032 C00025 C(CC(=O)O)[C@@H](C(=O)O)N 1 D-Glucose HMDB0000122 5793 C00221 C([C@@H]1[C@H]([C@@H]([C@H](C(O1)O)O)O)O)O 1 Pentadecanoic acid HMDB0000826 13849 C16537 CCCCCCCCCCCCCCC(=O)O 1 Hexadecanoic acid HMDB0000220 985 C00249 CCCCCCCCCCCCCCCC(=O)O 1 Heptadecanoic acid HMDB0002259 10465 NA CCCCCCCCCCCCCCCCC(=O)O 1 Octadecanedioic acid HMDB0000782 70095 NA C(CCCCCCCCC(=O)O)CCCCCCCC(=O)O 1 Trehalose HMDB0000975 7427 C01083 C([C@@H]1[C@H]([C@@H]([C@H]([C@H](O1)O[C@@H]2[C@@H]([C@H]([C@@H]([C@H](O2)CO)O)O)O)O)O)O)O 1 Squalene HMDB0000256 11975273 C00751 CC(=CCC/C(=C/CC/C(=C/CC/C=C(C)/CC/C=C(/C)CCC=C(C)C)/C)/C)C 1 EPA HMDB0001999 446284 C06428 CC/C=CC/C=CC/C=CC/C=CC/C=CCCCC(=O)O 1 DHA HMDB0002183 445580 C06429 CC/C=CC/C=CC/C=CC/C=CC/C=CC/C=CCCC(=O)O 1 5-Methyluridine HMDB0000884 445408 NA CC1=CN(C(=O)NC1=O)[C@H]2[C@@H]([C@@H]([C@H](O2)CO)O)O 1 5,6-Dimethyluracil NA NA NA NA 0 Butanoic acid HMDB0000039 264 C00246 CCCC(=O)O 1 Cholesterol HMDB0000067 5997 C00187 C[C@H](CCCC(C)C)[C@H]1CC[C@@H]2[C@@]1(CC[C@H]3[C@H]2CC=C4[C@@]3(CCC(C4)O)C)C 1 D-Lactic acid HMDB0001311 61503 C00256 C[C@H](C(=O)O)O 1 9-Octadecanoic acid NA NA NA NA 0 Octadecanoic acid HMDB0000827 5281 C01530 CCCCCCCCCCCCCCCCCC(=O)O 1 Eicosanoic acid HMDB0002212 10467 C06425 CCCCCCCCCCCCCCCCCCCC(=O)O 1 Stigmasterol HMDB0000937 5281330 C05442 CC[C@H](/C=C/[C@@H](C)[C@H]1CC[C@@H]2[C@@]1(CC[C@H]3[C@H]2CC=C4[C@@]3(CC[C@@H](C4)O)C)C)C(C)C 1 D-Allose HMDB0001151 12285879 C01487 C([C@H]1[C@@H]([C@@H]([C@@H](C(O1)O)O)O)O)O 1 O,O''-Trimethylsilyl-O'-stearoylglycerol NA NA NA NA 0 D-Erythrose HMDB0002649 5460672 C01796 C1[C@H]([C@H](C(O1)O)O)O 1 Citric acid HMDB0000094 311 C00158 C(C(=O)O)C(CC(=O)O)(C(=O)O)O 1 D-Tagatofuranose NA NA NA NA 0 D-Mannose HMDB0000169 18950 C00936 C([C@@H]1[C@H]([C@@H]([C@@H](C(O1)O)O)O)O)O 1 Ribitol HMDB0000508 NA C00474 OC[C@H](O)[C@H](O)[C@H](O)CO 1 D-sorbitol HMDB0000247 5780 C00794 C([C@H]([C@H]([C@@H]([C@H](CO)O)O)O)O)O 1 Fumaric acid HMDB0000134 444972 C00122 C(=C/C(=O)O)C(=O)O 1 XYLITOL HMDB0002917 6912 C00379 C([C@H](C([C@H](CO)O)O)O)O 1 D-Arabinose HMDB0029942 66308 NA C([C@H]([C@H]([C@@H](C=O)O)O)O)O 1 Docosanoic acid HMDB0000944 8215 C08281 CCCCCCCCCCCCCCCCCCCCCC(=O)O 1 D-Fructose HMDB0000660 439709 C02336 C([C@@H]1[C@H]([C@@H]([C@](O1)(CO)O)O)O)O 1 METABOLITES_END #END