#METABOLOMICS WORKBENCH michaelsa93_20160504_172828 DATATRACK_ID:616 STUDY_ID:ST000394 ANALYSIS_ID:AN000630 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 6530 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 Formate +10mM Formic Acid CH:SOLVENT_B 9:1 Isopropanol:Acetonitrile +10mM Ammonium Formate +10mM Formic Acid CH:COLUMN_PRESSURE 450-850 bar CH:INTERNAL_STANDARD See data dictionary CH:RETENTION_TIME See data dictionary CH:SAMPLE_INJECTION 1.67 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 6530 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:CAPILLARY_VOLTAGE 3500 V MS:COLLISION_GAS Nitrogen MS:DRY_GAS_FLOW 8 L/min MS:DRY_GAS_TEMP 325 C MS:FRAGMENT_VOLTAGE 120 V MS:FRAGMENTATION_METHOD Auto MS/MS MS:ION_SOURCE_TEMPERATURE 325 C MS:ION_SPRAY_VOLTAGE 1000 V MS:IONIZATION Pos 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 9.71_693.56 _9.70_688.60 1028 985 727 847 6.85_617.51 _6.85_612.56 1185 1204 150 1331 6.40_641.51 _6.40_636.56 1834 1886 1935 2469 4.80_884.61 9779 9245 7416 8663 6.96_810.68 _6.96_792.676.96_832.66 6.69_972.73 _6.69_994.72 192 306 763 986 1.68_480.35 664 562 463 645 4.79_706.54 1107 917 1239 1280 4.33_704.52 178 4104 7879 8383 5.12_720.56 116682 96552 65908 71792 5.40_734.57 904 4206 685 734 4.86_732.55 41240 37373 20863 23604 4.43_730.54 3764 3501 4515 4302 4.79_728.52 749 1421 1979 4918 5.63_748.59 940901 898686 587961 656747 5.16_746.57 9346085 8918413 6176024 6810107 4.70_744.55 1556514 1412675 2261754 2290695 6.05_762.60 5653 5495 513 701 5.52_760.59 167509 150112 93630 113013 5.04_758.58 1997 1098 2423 36101 5.80_774.61 975399 887335 1512692 1833086 5.22_772.59 58811 52873 664385 750674 4.86_770.57 1239 1277 24667 29635 6.13_788.62 1145 1246 1706 1840 5.64_786.60 889 1066 11000 12485 4.91_782.57 396 419 1505 2003 4.54_780.56 2519 2442 1811 2203 5.92_800.62 2838 2700 1963 1982 5.48_798.60 102 38 1405 3283 5.22_796.59 179 193 1702 2697 4.97_808.58 1325 215 1088 1467 5.28_768.59 7955 441 7157 9220 5.19_766.58 162858 156257 117438 151517 5.12_792.59 83 199 10166 394 6.32_746.57 827 2309 802 4467 5.70_768.56 6158 5823 3950 4548 4.91_764.52 12120 11683 29461 30278 6.01_752.56 7186 3022 20759 2729 5.41_750.54 907 712 937 57924 10.36_796.74 1446 1252 1317 1415 10.81_829.73 _10.81_824.77 18277 18054 18104 18156 10.38_827.71 _10.38_822.75 1768 1443 1624 1118 9.98_825.69 _9.98_820.74 1135 1027 1147 981 11.20_857.76 _11.20_852.80 17615 14920 15891 17073 10.80_855.7 _410.80_850.79 2154 1773 1976 1166 10.41_853.73 _10.41_848.77 2039 1507 1680 1264 10.01_851.71 _10.01_846.76 1001 805 803 787 11.56_885.79 _11.56_880.83 5310 8812 5212 8386 10.88_881.76 _10.88_876.80 4467 3406 3190 2133 10.46_879.74 _10.46_874.79 2374 2139 1677 2375 10.09_877.73 _10.09_872.77 1632 1372 1707 1619 10.84_907.77 _10.84_902.82 2750 2693 2717 2576 10.10_903.74 _10.10_898.79 1492 1358 1860 1413 3.44_279.16 3384 2740 2255 3742 3.44_391.29 79092 66745 51927 85394 5.03_865.53 8789 8251 6156 6862 4.86_766.57 162882 178000 39 169838 5.06_1475.16 8489 140 4445 4346 3.45_435.32 4660 4029 3350 4827 3.45_803.55 19114 15463 12442 24136 3.15_381.30 16578 14101 11929 15437 4.92_887.56 49163 45490 32811 37298 4.87_577.52 629855 600700 489544 603014 4.97_603.53 1091 977 25620 33369 5.55_1533.11 570 493 51451 59986 5.53_792.57 57 216 8487 10263 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention Time Quantified m/z KEGG ID Pubchem ID 9.71_693.56 _9.70_688.60 9.72 693.5581 _688.6027 6.85_617.51 _6.85_612.56 617.5116612.5562 6.40_641.51 _6.40_636.56 6.42 641.5115 _636.5562 4.80_884.61 4.778 884.6069 6.96_810.68 _6.96_792.676.96_832.66 6.959 810.6817 _792.6711 _832.6636 6.69_972.73 _6.69_994.72 6.693 972.7346 _994.7165 1.68_480.35 1.694 480.3448 4.79_706.54 4.811 706.5378 4.33_704.52 4.322 704.5225 5.12_720.56 5.11 720.5538 5.40_734.57 5.425 734.5691 4.86_732.55 4.878 732.5535 4.43_730.54 4.438 730.5378 4.79_728.52 4.811 728.5222 5.63_748.59 5.64 748.5851 5.16_746.57 5.185 746.57 4.70_744.55 4.721 744.5541 6.05_762.60 6.072 762.6004 5.52_760.59 5.499 760.5904 5.04_758.58 5.019 758.5751 5.80_774.61 5.806 774.6007 5.22_772.59 5.3 772.5854 4.86_770.57 4.861 770.5694 6.13_788.62 6.146 788.6161 5.64_786.60 5.649 786.6004 4.91_782.57 4.927 782.5721 4.54_780.56 4.546 780.5538 5.92_800.62 5.972 800.6161 5.48_798.60 5.425 798.6004 5.22_796.59 5.234 796.5848 4.97_808.58 4.969 808.5848 5.28_768.59 5.284 768.5902 5.19_766.58 5.184 766.5759 5.12_792.59 5.234 792.5902 6.32_746.57 6.337 746.57 5.70_768.56 5.723 768.556 4.91_764.52 4.927 764.5235 6.01_752.56 6.03 752.559 5.41_750.54 5.433 750.5437 10.36_796.74 10.383 796.7389 10.81_829.73 _10.81_824.77 10.806 829.7256 _824.7702 10.38_827.71 _10.38_822.75 10.4 827.7099 _822.7545 9.98_825.69 _9.98_820.74 9.993 825.6943 _820.7389 11.20_857.76 _11.20_852.80 11.212 857.7569 _852.8015 10.80_855.7 _410.80_850.79 10.822 855.7412 _850.7858 10.41_853.73 _10.41_848.77 10.424 853.7256 _848.7702 10.01_851.71 _10.01_846.76 10.018 851.7099 _846.7545 11.56_885.79 _11.56_880.83 11.577 885.7882 _880.8328 10.88_881.76 _10.88_876.80 10.831 881.7569 _876.8015 10.46_879.74 _10.46_874.79 10.474 879.7412 _874.7858 10.09_877.73 _10.09_872.77 10.101 877.7256 _872.7702 10.84_907.77 _10.84_902.82 10.857 907.7725 _902.8171 10.10_903.74 _10.10_898.79 10.126 903.7412 _898.7858 3.44_279.16 3.443 279.1618 3.44_391.29 3.443 391.2899 5.03_865.53 5.035 865.5291 4.86_766.57 4.869 766.5705 5.06_1475.16 5.035 1475.1545 3.45_435.32 3.443 435.3227 3.45_803.55 3.443 803.5461 3.15_381.30 3.161 381.2989 4.92_887.56 4.902 887.5628 4.87_577.52 4.853 577.5168 4.97_603.53 4.994 603.5326 5.55_1533.11 5.533 1533.1061 5.53_792.57 5.549 792.568 METABOLITES_END #END