#METABOLOMICS WORKBENCH michaelsa93_20160504_172828 DATATRACK_ID:616 STUDY_ID:ST000394 ANALYSIS_ID:AN000631 PROJECT_ID:PR000308 VERSION 1 CREATED_ON May 10, 2016, 12:30 pm #PROJECT PR:PROJECT_TITLE The circadian oscillator in Synechococcus elongatus controls metabolite PR:PROJECT_TITLE partitioning during diurnal growth PR:PROJECT_SUMMARY Cyanobacteria are increasingly being considered for use in large-scale outdoor PR:PROJECT_SUMMARY production of fuels and industrial chemicals. Cyanobacteria can anticipate daily PR:PROJECT_SUMMARY changes in light availability using an internal circadian clock and rapidly PR:PROJECT_SUMMARY alter their metabolic processes in response to changes light availability. PR:PROJECT_SUMMARY Understanding how signals from the internal circadian clock and external light PR:PROJECT_SUMMARY availability are integrated to control metabolic shifts will be important for PR:PROJECT_SUMMARY engineering cyanobacteria for production in natural outdoor environments. This PR:PROJECT_SUMMARY study has assessed how “knowing” the correct time of day, via the circadian PR:PROJECT_SUMMARY clock, affects metabolic changes when a cyanobacterium goes through a PR:PROJECT_SUMMARY dark-to-light transition. Our data show that the circadian clock plays an PR:PROJECT_SUMMARY important role in inhibiting activation of the oxidative pentose phosphate PR:PROJECT_SUMMARY pathway in the morning. Synechococcus elongatus PCC 7942 is a genetically PR:PROJECT_SUMMARY tractable model cyanobacterium that has been engineered to produce industrially PR:PROJECT_SUMMARY relevant biomolecules and is the best-studied model for a prokaryotic circadian PR:PROJECT_SUMMARY clock. However, the organism is commonly grown in continuous light in the PR:PROJECT_SUMMARY laboratory, and data on metabolic processes under diurnal conditions are PR:PROJECT_SUMMARY lacking. Moreover, the influence of the circadian clock on diurnal metabolism PR:PROJECT_SUMMARY has been investigated only briefly. Here, we demonstrate that the circadian PR:PROJECT_SUMMARY oscillator influences rhythms of metabolism during diurnal growth, even though PR:PROJECT_SUMMARY light–dark cycles can drive metabolic rhythms independently. Moreover, the PR:PROJECT_SUMMARY phenotype associated with loss of the core oscillator protein, KaiC, is distinct PR:PROJECT_SUMMARY from that caused by absence of the circadian output transcriptional regulator, PR:PROJECT_SUMMARY RpaA (regulator of phycobilisome-associated A). Although RpaA activity is PR:PROJECT_SUMMARY important for carbon degradation at night, KaiC is dispensable for those PR:PROJECT_SUMMARY processes. Untargeted metabolomics analysis and glycogen kinetics suggest that PR:PROJECT_SUMMARY functional KaiC is important for metabolite partitioning in the morning. PR:PROJECT_SUMMARY Additionally, output from the oscillator functions to inhibit RpaA activity in PR:PROJECT_SUMMARY the morning, and kaiC-null strains expressing a mutant KaiC phosphomimetic, PR:PROJECT_SUMMARY KaiC-pST, in which the oscillator is locked in the most active output state, PR:PROJECT_SUMMARY phenocopies a ΔrpaA strain. Inhibition of RpaA by the oscillator in the morning PR:PROJECT_SUMMARY suppresses metabolic processes that normally are active at night, and kaiC-null PR:PROJECT_SUMMARY strains show indications of oxidative pentose phosphate pathway activation as PR:PROJECT_SUMMARY well as increased abundance of primary metabolites. Inhibitory clock output may PR:PROJECT_SUMMARY serve to allow secondary metabolite biosynthesis in the morning, and some PR:PROJECT_SUMMARY metabolites resulting from these processes may feed back to reinforce clock PR:PROJECT_SUMMARY timing. PR:INSTITUTE University of California, Davis PR:DEPARTMENT Genome and Biomedical Sciences Facility PR:LABORATORY WCMC Metabolomics Core PR:LAST_NAME Fiehn PR:FIRST_NAME Oliver PR:ADDRESS 1315 Genome and Biomedical Sciences Facility, 451 Health Sciences Drive, Davis, PR:ADDRESS CA 95616 PR:EMAIL ofiehn@ucdavis.edu PR:PHONE (530) 754-8258 PR:FUNDING_SOURCE NIH U24DK097154 PR:PUBLICATIONS doi: 10.1073/pnas.1504576112 #STUDY ST:STUDY_TITLE The circadian oscillator in Synechococcus elongatus controls metabolite ST:STUDY_TITLE partitioning during diurnal growth ST:STUDY_SUMMARY Cyanobacteria are increasingly being considered for use in large-scale outdoor ST:STUDY_SUMMARY production of fuels and industrial chemicals. Cyanobacteria can anticipate daily ST:STUDY_SUMMARY changes in light availability using an internal circadian clock and rapidly ST:STUDY_SUMMARY alter their metabolic processes in response to changes light availability. ST:STUDY_SUMMARY Understanding how signals from the internal circadian clock and external light ST:STUDY_SUMMARY availability are integrated to control metabolic shifts will be important for ST:STUDY_SUMMARY engineering cyanobacteria for production in natural outdoor environments. This ST:STUDY_SUMMARY study has assessed how “knowing” the correct time of day, via the circadian ST:STUDY_SUMMARY clock, affects metabolic changes when a cyanobacterium goes through a ST:STUDY_SUMMARY dark-to-light transition. Our data show that the circadian clock plays an ST:STUDY_SUMMARY important role in inhibiting activation of the oxidative pentose phosphate ST:STUDY_SUMMARY pathway in the morning. Synechococcus elongatus PCC 7942 is a genetically ST:STUDY_SUMMARY tractable model cyanobacterium that has been engineered to produce industrially ST:STUDY_SUMMARY relevant biomolecules and is the best-studied model for a prokaryotic circadian ST:STUDY_SUMMARY clock. However, the organism is commonly grown in continuous light in the ST:STUDY_SUMMARY laboratory, and data on metabolic processes under diurnal conditions are ST:STUDY_SUMMARY lacking. Moreover, the influence of the circadian clock on diurnal metabolism ST:STUDY_SUMMARY has been investigated only briefly. Here, we demonstrate that the circadian ST:STUDY_SUMMARY oscillator influences rhythms of metabolism during diurnal growth, even though ST:STUDY_SUMMARY light–dark cycles can drive metabolic rhythms independently. Moreover, the ST:STUDY_SUMMARY phenotype associated with loss of the core oscillator protein, KaiC, is distinct ST:STUDY_SUMMARY from that caused by absence of the circadian output transcriptional regulator, ST:STUDY_SUMMARY RpaA (regulator of phycobilisome-associated A). Although RpaA activity is ST:STUDY_SUMMARY important for carbon degradation at night, KaiC is dispensable for those ST:STUDY_SUMMARY processes. Untargeted metabolomics analysis and glycogen kinetics suggest that ST:STUDY_SUMMARY functional KaiC is important for metabolite partitioning in the morning. ST:STUDY_SUMMARY Additionally, output from the oscillator functions to inhibit RpaA activity in ST:STUDY_SUMMARY the morning, and kaiC-null strains expressing a mutant KaiC phosphomimetic, ST:STUDY_SUMMARY KaiC-pST, in which the oscillator is locked in the most active output state, ST:STUDY_SUMMARY phenocopies a ΔrpaA strain. Inhibition of RpaA by the oscillator in the morning ST:STUDY_SUMMARY suppresses metabolic processes that normally are active at night, and kaiC-null ST:STUDY_SUMMARY strains show indications of oxidative pentose phosphate pathway activation as ST:STUDY_SUMMARY well as increased abundance of primary metabolites. Inhibitory clock output may ST:STUDY_SUMMARY serve to allow secondary metabolite biosynthesis in the morning, and some ST:STUDY_SUMMARY metabolites resulting from these processes may feed back to reinforce clock ST:STUDY_SUMMARY timing. ST:INSTITUTE University of California, Davis ST:DEPARTMENT Genome and Biomedical Sciences Facility ST:LABORATORY WCMC Metabolomics Core ST:LAST_NAME Fiehn ST:FIRST_NAME Oliver ST:ADDRESS 1315 Genome and Biomedical Sciences Facility, 451 Health Sciences Drive, Davis, ST:ADDRESS CA 95616 ST:EMAIL ofiehn@ucdavis.edu ST:PHONE (530) 754-8258 ST:STUDY_COMMENTS The first 4 samples were a test run to see how efficient the analysis was and ST:STUDY_COMMENTS were run on a lipidomics platform. The next 12 samples were the used in the ST:STUDY_COMMENTS paper and were the same as the original 4 samples, but they were split into 3 ST:STUDY_COMMENTS biological replicates and run on the GC platform. ST:PUBLICATIONS doi: 10.1073/pnas.1504576112 #SUBJECT SU:SUBJECT_TYPE Cells SU:SUBJECT_SPECIES Synechococcus elongatus PCC 7942 SU:TAXONOMY_ID 1140 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS WT T0 SDiamInj04_WT T0_CSH.d Genotype:WT | Time Point:- SUBJECT_SAMPLE_FACTORS WT T4 SDiamInj05_WT T4_CSH.d Genotype:WT | Time Point:4 SUBJECT_SAMPLE_FACTORS KaiC T0 SDiamInj03_KaiC T0_CSH.d Genotype:KaiC mutant | Time Point:- SUBJECT_SAMPLE_FACTORS KaiC T4 SDiamInj02_KaiC T4_CSH.d Genotype:KaiC mutant | Time Point:4 #COLLECTION CO:COLLECTION_SUMMARY Bacteria were grown in a turbidostat/bioreactor at equal cell density (measured CO:COLLECTION_SUMMARY by optical density at 750nm), under a 12:12h Light/Dark cycle. After collection CO:COLLECTION_SUMMARY samples were immediately placed on ice and then centrifuged at 5000RPM for 10min CO:COLLECTION_SUMMARY at 4 degrees Celsius. After centrifugation supernatant was decanted and cell CO:COLLECTION_SUMMARY pellets were immediately frozen in liquid N2. CO:COLLECTION_PROTOCOL_FILENAME StudyDesign-SpencerDiamond-10814.pdf CO:SAMPLE_TYPE Cell CO:COLLECTION_TIME Samples were collected at T0 (beginning of day) and T4 (4h into day). CO:VOLUMEORAMOUNT_COLLECTED 40ml of sample was collected at each time point CO:STORAGE_CONDITIONS Samples were put into a 50mL conical tube containing ice up to the 30ml mark. #TREATMENT TR:TREATMENT_SUMMARY 2: WT bacteria and KaiC mutant The phenotype associated with loss of the core TR:TREATMENT_SUMMARY oscillator protein, KaiC, is distinct from that caused by absence of the TR:TREATMENT_SUMMARY circadian output transcriptional regulator, RpaA (regulator of TR:TREATMENT_SUMMARY phycobilisome-associated A). Untargeted metabolomics analysis and glycogen TR:TREATMENT_SUMMARY kinetics suggest that functional KaiC is important for metabolite partitioning TR:TREATMENT_SUMMARY in the morning. Additionally, output from the oscillator functions to inhibit TR:TREATMENT_SUMMARY RpaA activity in the morning, and kaiC-null strains expressing a mutant KaiC TR:TREATMENT_SUMMARY phosphomimetic, KaiC-pST, in which the oscillator is locked in the most active TR:TREATMENT_SUMMARY output state, phenocopies a ΔrpaA strain. KaiC-null strains show indications of TR:TREATMENT_SUMMARY oxidative pentose phosphate pathway activation as well as increased abundance of TR:TREATMENT_SUMMARY primary metabolites. Inhibitory clock output may serve to allow secondary TR:TREATMENT_SUMMARY metabolite biosynthesis in the morning, and some metabolites resulting from TR:TREATMENT_SUMMARY these processes may feed back to reinforce clock timing. TR:TREATMENT_PROTOCOL_FILENAME StudyDesign-SpencerDiamond-10814.pdf #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 1. Add 0.5mL of extraction solvent to tube, gently pipet to remove all cells, SP:SAMPLEPREP_SUMMARY transfer cells to 2mL eppendorf tube. Repeat for a total of 1mL extraction SP:SAMPLEPREP_SUMMARY solvent + cells in 2mL eppendorf tube. 2. Add 2 small stainless steel grinding SP:SAMPLEPREP_SUMMARY beads to eppendorf tube 3. Use the GenoGrinder to grind for 3 minutes at 1,250 SP:SAMPLEPREP_SUMMARY rpm. 4. Centrifuge at 14,000xg for 5 minutes. 5. Transfer supernatant to a fresh SP:SAMPLEPREP_SUMMARY 2mL eppendorf tube. 6. Add 1mL of extraction solvent to tube containing cell SP:SAMPLEPREP_SUMMARY pellet + beads, and repeat steps 3 and 4. 7. Collect supernatant, and combine SP:SAMPLEPREP_SUMMARY with supernatant collected in step 5. Total volume of extracted sample will be SP:SAMPLEPREP_SUMMARY approximately 2mL. 8. Dry down 50uL of extracted sample in 1.5mL eppendorf tube SP:SAMPLEPREP_SUMMARY for GC-TOF analysis. 9. Store backups in -20 or -80C. SP:SAMPLEPREP_PROTOCOL_FILENAME SOP_Extraction_of_Yeast_Cells.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 6550 CH:COLUMN_NAME Waters Acquity CSH C18 (100 x 2.1mm, 1.7um) CH:COLUMN_NAME 1.7um Pre-Column CH:FLOW_GRADIENT 15% B to 99%B CH:FLOW_RATE 0.6 mL/min CH:COLUMN_TEMPERATURE 65 C CH:METHODS_FILENAME Data_Dictionary_Fiehn_laboratory_CSH_QTOF_lipidomics_05-29-2014.pdf CH:SOLVENT_A 60:40 Acetonitrile:Water +10mM Ammonium Acetate +10mM Acetic Acid CH:SOLVENT_B 9:1 Isopropanol:Acetonitrile +10mM Ammonium Acetate +10mM Acetic Acid CH:COLUMN_PRESSURE 450-850 bar CH:INTERNAL_STANDARD See data dictionary CH:RETENTION_TIME See data dictionary CH:SAMPLE_INJECTION 5 uL CH:ANALYTICAL_TIME 13 min CH:CAPILLARY_VOLTAGE 3500 V CH:TIME_PROGRAM 15 min CH:WEAK_WASH_SOLVENT_NAME Isopropanol CH:STRONG_WASH_SOLVENT_NAME Isopropanol CH:TARGET_SAMPLE_TEMPERATURE Autosampler temp 4 C CH:RANDOMIZATION_ORDER Excel generated #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME WCMC Metabolomics Core AN:SOFTWARE_VERSION MassHunter AN:DATA_FORMAT .d #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Agilent 6550 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:CAPILLARY_VOLTAGE 3500 V MS:COLLISION_GAS Nitrogen MS:DRY_GAS_FLOW 13 L/min MS:DRY_GAS_TEMP 200 C MS:FRAGMENT_VOLTAGE 175 V MS:FRAGMENTATION_METHOD Auto MS/MS MS:ION_SOURCE_TEMPERATURE 325 C MS:ION_SPRAY_VOLTAGE 1000 V MS:IONIZATION Neg MS:PRECURSOR_TYPE Intact Molecule MS:REAGENT_GAS Nitrogen MS:SOURCE_TEMPERATURE 325 C MS:DATAFORMAT .d MS:DESOLVATION_GAS_FLOW 11 L/min MS:DESOLVATION_TEMPERATURE 350 C MS:NEBULIZER 35 psig MS:OCTPOLE_VOLTAGE 750 V MS:RESOLUTION_SETTING extended dynamic range MS:SCAN_RANGE_MOVERZ 60-1700 Da MS:SCANNING_CYCLE 2 Hz MS:SCANNING_RANGE 60-1700 Da MS:SKIMMER_VOLTAGE 65 V #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS counts MS_METABOLITE_DATA_START Samples SDiamInj04_WT T0_CSH.d SDiamInj05_WT T4_CSH.d SDiamInj03_KaiC T0_CSH.d SDiamInj02_KaiC T4_CSH.d Factors Genotype:WT | Time Point:- Genotype:WT | Time Point:4 Genotype:KaiC mutant | Time Point:- Genotype:KaiC mutant | Time Point:4 6.36_600.51 _6.55_610.54 1168 1696 138 166 7.06_628.54 482 1400 511 1899 7.74_656.58 _7.91_666.6 683 2919 290 411 7.08_654.56 26944 28181 12103 26095 8.59_686.62 _8.59_696.65 627 281 548 592 8.37_684.61 _8.52_694.64 507 413 567 860 7.70_682.59 1643 132 1272 1123 7.82_682.59 1643 1306 1272 983 3.25_255.23 905119 772285 781081 926266 2.71_253.22 1257187 751318 750167 1088699 3.82_283.26 1621523 1347303 1765982 1487258 3.34_281.25 710786 639535 478504 768694 2.94_279.23 73813 81909 52901 85818 6.39_790.60 114421 134355 161828 239565 7.04_844.65 88 87 9706 115766 4.97_750.53 71594 66869 69011 102671 1.01_512.3 56 70 48 1.46_530.30 774 860 494 739 1.21_554.30 344 4360 312 6940 1.22_524.28 2872 2352 831 2265 5.4_790.56 83 1933 59 2185 4.87_788.55 1514 1364 1569 1170 6.29_806.59 417 440 554 574 5.54_794.55 _5.73_804.58 3176 2848 3366 3060 5.22_792.54 _5.22_802.56 _5.22_742.54 287 697 2372 471 5.72_820.56 _5.89_830.59 1210 1331 1567 1875 4.95_850.56 7268 6290 5578 7526 5.43_868.56 880 827 754 694 5.12_878.59 14157 13425 16131 23070 5.55_878.59 18554 17273 20819 30533 4.88_876.58 160753 143844 653 136053 4.59_874.56 29555 27376 30126 37369 6.05_788.58 1288 44298 45695 1359 5.51_786.56 18483 17130 40033 47836 5.23_800.84 648 398 1100 763 5.4_714.51 1704 1091 215 920 6.03_742.54 5667 5163 3510 4694 5.3_738.51 37307 35175 28138 27605 5.11_766.54 1591 1345 1584 1811 5.93_766.54 36699 38177 42457 48163 5.11_762.51 452389 419547 369032 459132 4.96_790.54 80307 71697 67318 78700 5.72_698.51 42 180 923 1415 5.13_720.5 864 733 684 522 5.65_748.53 3370 2669 303 2648 6.3_776.56 1372 1470 2180 2094 5.48_772.53 1862 15649 339 18848 4.47_807.5 36398 35423 11355 13988 5.06_835.53 485 79 512 1124 4.6_833.52 1053 697 519 1054 5.16_861.55 18713 17507 14788 16994 4.75_859.53 655 745 472 643 3.13_367.27 3802 2500 4768 3741 5.08_962.53 2234 2907 468 2023 5.33_964.55 659 1433 502 216 5.56_843.58 112 43 2052 187 5.81_834.61 54 32 1390 1813 0.62_329.23 886 784 733 804 4.91_1019.51 2987 3008 146 343 5.37_1032.537 3023 2566 2578 3775 5.61_1047.55 817 699 446 563 5.88_960.55 628 540 350 509 5.88_1096.52 1085 1058 479 1031 1.27_632.32 1293 1491 201 1914 4.25_794.51 552347 509892 480493 602667 5.73_872.57 1313 1312 1232 1318 6.06_1060.57 1691 1719 511 466 6.08_924.6 86 1390 2105 5.81_990.57 533 555 351 624 4.17_709.51 446 405 650 514 4.17_787.52 1472 701 700 681 4.26_685.53 259 1157 362 2276 4.26_881.52 276 155 275 159 4.26_813.54 107 102 326 255 4.26_793.48 4.26_803.51 466 5993 440 595 4.27_722.50 111437 119729 174450 221360 4.27_735.52 14130 13232 1900 2402 4.34_898.55 12663 723 10171 11748 4.47_774.53 135 3476 220 310 4.53_836.54 571 501 384 566 4.58_763.56 159 2353 70 1265 4.66_790.52 822 126 398 1298 4.65_840.53 987 363 662 350 4.58_892.53 1975 1576 411 924 4.76_737.54 1906 2287 3266 3039 4.76_815.55 126 2787 395 743 4.76_805.52 819 590 7609 6059 4.82_986.53 661 900 747 672 4.87_713.56 2211 1642 2256 2401 4.93_953.53 943 624 457 824 4.96_1667.11 92 1478 5840 11012 4.98_911.56 286 80 624 1105 5.05_834.57 229 400 1205 4039 5.07_870.55 894 1028 1188 1098 5.07_746.51 11321 11469 10948 557 5.05_788.52 637070 593566 564968 655123 5.42_833.56 527 474 1591 3735 5.43_946.58 518 509 560 786 5.43_936.55 1519 249 1682 2366 5.73_956.54 2123 2033 644 1454 6.00_814.60 474 169 237 1264 6.21_900.60 608 701 821 900 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Batch RT Batch m/z KEGG ID Pubchem ID 6.36_600.51 _6.55_610.54 6.369 600.5129 _624.5567 7.06_628.54 7.093 628.5441 7.74_656.58 _7.91_666.6 7.784 656.5756 _680.6193 7.08_654.56 7.176 654.5597 8.59_686.62 _8.59_696.65 8.641 686.6212 _710.6667 8.37_684.61 _8.52_694.64 8.424 684.61 _708.6507 7.70_682.59 7.742 682.591 7.82_682.59 8.036 682.591 3.25_255.23 2.447 255.233 2.71_253.22 1.801 253.2173 3.82_283.26 3.215 283.2643 3.34_281.25 2.663 281.2486 2.94_279.23 2.033 279.2331 6.39_790.60 6.411 790.5969 7.04_844.65 7.085 844.6453 4.97_750.53 5 750.5285 1.01_512.3 0.95 526.3151 1.46_530.30 1.753 530.3016 1.21_554.30 1.288 554.301 1.22_524.28 1.238 524.2785 5.4_790.56 5.218 804.5761 4.87_788.55 4.698 802.5599 6.29_806.59 6.225 820.6068 5.54_794.55 _5.73_804.58 5.531 794.547 _818.5917 5.22_792.54 _5.22_802.56 _5.22_742.54 5.027 792.5396 _816.5761 _742.5392 5.72_820.56 _5.89_830.59 5.706 820.5603 _844.6074 4.95_850.56 4.85 864.5761 5.43_868.56 5.396 868.5622 5.12_878.59 5.5 892.6076 5.55_878.59 5.406 892.6076 4.88_876.58 4.788 890.5905 4.59_874.56 4.5 888.5756 6.05_788.58 5.909 802.5968 5.51_786.56 5.326 800.5804 5.23_800.84 5.038 814.554 5.4_714.51 5.218 714.5079 6.03_742.54 5.967 742.5392 5.3_738.51 5.108 738.5079 5.11_766.54 4.918 766.5392 5.93_766.54 5.789 766.5392 5.11_762.51 4.927 762.5079 4.96_790.54 5 790.5392 5.72_698.51 5.539 698.5127 5.13_720.5 4.969 720.4974 5.65_748.53 5.473 748.5287 6.3_776.56 6.192 776.5605 5.48_772.53 5.293 772.5281 4.47_807.5 4.278 807.5028 5.06_835.53 4.861 835.5342 4.6_833.52 4.1 833.5186 5.16_861.55 4.6 861.5499 4.75_859.53 4.558 859.5332 3.13_367.27 3.117 367.2651 5.08_962.53 4.894 962.5318 5.33_964.55 5.208 964.5477 5.56_843.58 5.416 843.5818 5.81_834.61 5.696 834.605 0.62_329.23 0.521 329.2335 4.91_1019.51 4.694 1019.5142 5.37_1032.537 5.208 1032.537 5.61_1047.55 5.446 1047.5458 5.88_960.55 5.714 960.5485 5.88_1096.52 5.714 1096.5231 1.27_632.32 1.288 632.3182 4.25_794.51 4.257 794.5094 5.73_872.57 5.522 872.5658 6.06_1060.57 5.942 1060.5676 6.08_924.6 5.938 924.5971 5.81_990.57 5.597 990.5658 4.17_709.51 4.291 709.5054 4.17_787.52 4.291 787.5207 4.26_685.53 4.399 685.5282 4.26_881.52 4.399 881.519 4.26_813.54 4.399 813.5371 4.26_793.48 4.407 793.4793 4.26_803.51 4.39 803.507 4.27_722.50 4.39 722.4968 4.27_735.52 4.399 735.5206 4.34_898.55 4.237 898.5527 4.47_774.53 4.386 774.5279 4.53_836.54 4.428 836.5433 4.58_763.56 4.528 763.5617 4.66_790.52 4.608 790.5178 4.65_840.53 4.508 840.5323 4.58_892.53 4.511 892.5322 4.76_737.54 4.719 737.5368 4.76_815.55 4.719 815.5531 4.76_805.52 4.708 805.524 4.82_986.53 4.777 986.5317 4.87_713.56 4.844 713.5584 4.93_953.53 4.944 953.533 4.96_1667.11 4.935 1667.1055 4.98_911.56 4.96 911.562 5.05_834.57 5.013 834.5648 5.07_870.55 5.035 870.5464 5.07_746.51 5.227 746.5129 5.05_788.52 5.002 788.5232 5.42_833.56 5.431 833.5551 5.43_946.58 5.396 946.5771 5.43_936.55 5.396 936.548 5.73_956.54 5.73 956.5369 6.00_814.60 6.075 814.5959 6.21_900.60 6.22 900.5948 METABOLITES_END #END