{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001197","ANALYSIS_ID":"AN001993","VERSION":"1","CREATED_ON":"June 18, 2019, 9:44 pm"},

"PROJECT":{"PROJECT_TITLE":"A comprehensive time-course metabolite profiling of the model cyanobacterium Synechocystis sp. PCC 6803 under diurnal light:dark cycles","PROJECT_SUMMARY":"Cyanobacteria are a model photoautotroph and a chassis for the sustainable production of fuels and chemicals. Yet, knowledge of photoautotrophic metabolism in the natural environment of day/night cycles is lacking yet has implications for improved yield from plants, algae, and cyanobacteria. Here, a thorough approach to characterizing diverse metabolites—including carbohydrates, lipids, amino acids, pigments, co-factors, nucleic acids and polysaccharides—in the model cyanobacterium Synechocystis sp. PCC 6803 (S. 6803) under sinusoidal diurnal light-dark cycles was developed and applied. A custom photobioreactor and novel multi-platform mass spectrometry workflow enabled metabolite profiling every 30-120 minutes across a 24-hour diurnal sinusoidal LD (“sinLD”) cycle peaking at 1,600 mol photons m 2 s-1. We report widespread oscillations across the sinLD cycle with 90%, 94%, and 40% of the identified polar/semi-polar, non-polar, and polymeric metabolites displaying statistically significant oscillations, respectively. Microbial growth displayed distinct lag, biomass accumulation, and cell division phases of growth. During the lag phase, amino acids (AA) and nucleic acids (NA) accumulated to high levels per cell followed by decreased levels during the biomass accumulation phase, presumably due to protein and DNA synthesis. Insoluble carbohydrates displayed sharp oscillations per cell at the day-to-night transition. Potential bottlenecks in central carbon metabolism are highlighted. Together, this report provides a comprehensive view of photosynthetic metabolite behavior with high temporal resolution, offering insight into the impact of growth synchronization to light cycles via circadian rhythms. Incorporation into computational modeling and metabolic engineering efforts promises to improve industrially-relevant strain design.","INSTITUTE":"Colorado State University","DEPARTMENT":"Chemical and","LAST_NAME":"Peebles","FIRST_NAME":"Christie","ADDRESS":"700 Meridian Ave, Fort Collins, CO 80523 USA","EMAIL":"wernerajz@gmail.com","PHONE":"2699981811"},

"STUDY":{"STUDY_TITLE":"GC-MS Analysis of Insoluble/Polymeric Amino Acids (part-III)","STUDY_SUMMARY":"Cyanobacteria are a model photoautotroph and a chassis for the sustainable production of fuels and chemicals. Yet, knowledge of photoautotrophic metabolism in the natural environment of day/night cycles is lacking yet has implications for improved yield from plants, algae, and cyanobacteria. Here, a thorough approach to characterizing diverse metabolites—including carbohydrates, lipids, amino acids, pigments, co-factors, nucleic acids and polysaccharides—in the model cyanobacterium Synechocystis sp. PCC 6803 (S. 6803) under sinusoidal diurnal light-dark cycles was developed and applied. A custom photobioreactor and novel multi-platform mass spectrometry workflow enabled metabolite profiling every 30-120 minutes across a 24-hour diurnal sinusoidal LD (“sinLD”) cycle peaking at 1,600 mol photons m 2 s-1. We report widespread oscillations across the sinLD cycle with 90%, 94%, and 40% of the identified polar/semi-polar, non-polar, and polymeric metabolites displaying statistically significant oscillations, respectively. Microbial growth displayed distinct lag, biomass accumulation, and cell division phases of growth. During the lag phase, amino acids (AA) and nucleic acids (NA) accumulated to high levels per cell followed by decreased levels during the biomass accumulation phase, presumably due to protein and DNA synthesis. Insoluble carbohydrates displayed sharp oscillations per cell at the day-to-night transition. Potential bottlenecks in central carbon metabolism are highlighted. Together, this report provides a comprehensive view of photosynthetic metabolite behavior with high temporal resolution, offering insight into the impact of growth synchronization to light cycles via circadian rhythms. Incorporation into computational modeling and metabolic engineering efforts promises to improve industrially-relevant strain design.","INSTITUTE":"Colorado State University","DEPARTMENT":"Chemical and Biological Engineering","LAST_NAME":"Peebles","FIRST_NAME":"Christie","ADDRESS":"700 Meridian Ave, Fort Collins, CO 80523","EMAIL":"christie.peebles@colostate.edu","PHONE":"970-491-6779"},

"SUBJECT":{"SUBJECT_TYPE":"Bacteria","SUBJECT_SPECIES":"Synechocystis sp. PCC 6803","TAXONOMY_ID":"1148","GENOTYPE_STRAIN":"NCBI:txid1148","CELL_BIOSOURCE_OR_SUPPLIER":"ATCC"},
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"COLLECTION":{"COLLECTION_SUMMARY":"For each metabolomics time-point, a 10 mL culture were rapidly sampled via sterile on-reactor syringes into a pre-weighed centrifuge tube, quenched in -4°C 1X PBS, spun at 3,000g for 5 min., decanted, frozen in liquid nitrogen, and lyophilized at -50°C. The workflow from sampling to centrifugation took < 2 minutes; lyophilized samples were stored at -80°C for < 1 month prior to extraction. A biphasic extraction from lyophilized cell pellets was performed via a 2:1:1.6 MTBE:MeOH:H2O biphasic extraction, modified from the protocol developed by Salem et al. (Salem et al., 2016) resulting in a top layer of MTBE with non-polar soluble metabolites, a lower layer of MeOH:H2O with polar and semi-polar soluble metabolites, and an insoluble pellet. Each liquid layer was transferred to a fresh glass vial and dried under nitrogen gas overnight. The MTBE layer was resuspended in 1:1 toluene:MeOH and analyzed via Q-TOF-MS with a UPLC Phenyl Hexyl column (“RP-MS”). The MeOH:H2O layer was resuspended in 1:1 H2O:MeOH, split evenly and subjected to either i) derivatization in methoxyamine HCl and MSTFA followed by GC-MS analysis, or ii) targeted SRM analysis on a tandem quadrupole-MS equipped with a HILIC column. The insoluble pellet was hydrolyzed with a hydrochloric acid (HCl) based on previously published protocols (Fountoulakis and Lahm, 1998) (Huang, Kaiser and Benner, 2012) to analyze individual amino acids, nucleoside, and carbohydrate content of the insoluble polymers utilizing MTBSTFA derivatization for insoluble amino acids. Of the soluble phases, 10 µL were removed from each sample and pooled to create a QC sample, mixed, and aliquoted into thirteen vials. A QC sample was run after every sixth injection.","SAMPLE_TYPE":"Bacterial cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"Synechocystis sp. PCC 6803 [N-1] (ATCC 27184, NCBI Taxonomy ID: 1080229) was utilized for all experiments. A light-emitting diode photobioreactor (LED PBR) was engineered to provide a rectified sinusoidal waveform light profile which (results in the negative half-cycle being set to zero) via two custom 4000K White LED panels (Reliance Laboratories, Port Townsend WA) arranged opposite a water bath facing inwards, 5% CO2 at 200 mL min-1 via in-house gas mixing and custom aerators to provide sufficient mixing, 27-30°C temperature control via a Huber Ministat and custom water bath (Midwest Custom Aquarium, Starbuck MN), and improved light penetration at high volumes via custom flat-panel reactors (FPRs) built in a circular geometry to maximize mixing (Allen Scientific Glass, Boulder CO) (Figure S1). At the peak, 1,600 mol photons m-2s-1 (E) was provided as measured by LightScout Quantum Meter (Model: 3415FXSE). . A single LED-PBR was inoculated and entrained to sinLD cycles for two days; this entrained culture was then use inoculated three biological triplicate FPRs in the LED PBR (Figure S2). Reactors were cultivated under the sinLD cycle profile for an additional day of entrainment prior to sampling (total of 3 days of entrainment)."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Briefly, 6 mL of 75% methanol (MeOH) was added to pellets, vortexed, and transferred to glass vials. 9 mL of 100% methyl tert-butyl ether (MTBE) was added, vortexed for 30 seconds, placed on automatic shaker for 1.5 hours at 4 ºC, and sonicated for 15 minutes. 3.75 mL of water was added, each extraction was vortexed by hand for 1 minute, and centrifuged for 10 minutes at 3,270g at 4ºC. A biphasic solution with a pellet formed: the top, green MTBE layer and the bottom, clear MeOH:H2O layer were separated into separate tubes and dried under N2,gas overnight. The pellet was stored at -80 ºC. After drying, the MTBE layer was resuspended in 100 uL 1:1 toluene:MeOH, transferred to a LC-MS vial insert, and stored at -80C for <1 month prior to MS analysis. The MeOH:H2O layer was resuspended in 1 mL of 1:1 H2O:MeOH, transferred to a 1.7 mL centrifuge tube and spun at 15,000g for 2 minutes at 4 ºC. The supernatant was split into two 465 µL aliquots—one for GCMS and one for LC(HILIC)MS—in glass vials and dried under N2,gas. The protocol outlined above is suitable for filter-quenched cyanobacteria samples and centrifuged cell pellets. The polar methanol/water fraction resulting from the biphasic extraction was processed for analysis by hydrophilic interaction liquid chromatography (HILIC) LC-MS. Dried samples were resuspended in 100 µL 1:1 H2O:MeOH and 10 µL were aliquoted into a pooled QC sample. Samples were stored at -80 ºC until analysis. The pooled QC sample was mixed and aliquoted into twelve vials. A QC injection was run every tenth injection. The dried polar fraction for analysis by GC-MS was stored at -80 ºC until derivatization, immediately prior to MS analysis. Samples were derivatized in 30 uL methoxyamine HCl and 30 uL MSTFA, as specified in the following section. Ten microliters were removed from each sample to create a pooled QC sample, mixed, and aliquoted into thirteen vials. A QC sample was run after every sixth injection. The non-polar MTBE phase was processed for non-targeted LC-MS analysis. Twenty microliters from each sample were pooled, mixed, and aliquoted into thirteen pooled QC samples. QC injections were placed after every sixth injection. An acid hydrolysis protocol was developed for identification of amino acids, nucleosides, and carbohydrates bound in insoluble pellet of protein, DNA/RNA, and polysaccharides, respectively. Pellets remaining from the biphasic extraction were removed from storage at -80 degrees C and residual solvent was evaporated under nitrogen gas. Pellets were re-suspended in 3 mL of 6 M hydrochloric acid (HCl) using vigorous vortexing and pipette re-suspension. The resulting suspension was a bright teal. The suspension was transferred equally two three separate glass vials for hydrolysis of the separate polymer constituents. Hydrolysis of proteins to amino acids was completed with a hydrochloric acid (HCl) hydrolysis, based on previously published protocols (Fountoulakis and Lahm 1998). Briefly, vials were incubated at 110 degrees C for with a loose cap seal. After 4 hours, the acid in each vial was entirely evaporated; 1 mL of 6 M HCl was added to each vial, vortexed, sealed tightly, and returned to 110 degrees C. After a total of 24 hours, vials were removed, and remaining acid was evaporated under nitrogen gas. Samples were resuspended in 150 µL of 1:1 MeOH:H2O, 20 uL was removed to create a pooled QC sample, the pooled QC was aliquoted into fourteen vials, and the solvent was evaporated under nitrogen gas. Amino acid samples were derivatized in 30 uL of methoxyamine HCl in pyridine and 30 uL of MTBSTFSA. The peak integration of each amino acid in each sample was manually checked and curated in the software Chromeleon™. Aspartic acid with 2- and 3-derivitization agent modifications were detected and summed for the total spectral abundance. Threonine with 2- and 3-derivitization agent modifications were detected and summed for the total spectral abundance. Polysaccharides and nucleic acid polymers were hydrolyzed to nucleosides using a modified protocol from Huang et al. (2012) (Huang, Kaiser, and Benner 2012). Briefly, vials were incubated at 130 degrees C for 10 minutes, removed and allowed to cool, and 100 uL was transferred to a glass teardrop vial. The remaining pellet was incubated at 160 degrees C for 40 minutes, removed and allowed to cool, re-suspended in 100 uL LC-MS grade water, vortexed for 15 seconds, centrifuged at 1,500g for 2 minutes, and the supernatant was transferred to the glass teardrop vial which contained purines from the 130 degree C incubation. The acid was evaporated under nitrogen gas. Samples were removed after the 10 minute 130ºC incubation to preserve purines (guanine and adenine) from degradation during the 40 minute 160ºC incubation."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"For non-targeted GC-MS experiments, metabolites were detected using a Trace 1310 GC coupled to a Thermo ISQ mass spectrometer. Samples (1 µL) were injected at a 10:1 split ratio to a 30 m TG-5MS column (Thermo Scientific, 0.25 mm i.d., 0.25 μm film thickness) with a 1.2 mL/min helium gas flow rate. GC inlet was held at 285°C. The oven program started at 140°C for 1 min, followed by a ramp of 15°C/min to 330°C, and 5 min hold. Masses between 50-650 m/z were scanned at 5 scans/sec under electron impact ionization. Transfer line and ion source were held at 300 and 260°C, respectively. Pooled QC samples were injected after every 6 actual samples.","CHROMATOGRAPHY_TYPE":"GC","INSTRUMENT_NAME":"Thermo ISQ","COLUMN_NAME":"Trace 1310 GC"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo ISQ","INSTRUMENT_TYPE":"GC-TOF","MS_TYPE":"EI","ION_MODE":"POSITIVE","MS_COMMENTS":"Raw data was converted to *.CSV with Waters® Databridge. For idMS/MS (RP-LC-MS runs), a file was converted for low-collision, high-collision, and LockSpray for each sample. Peaks were detected within the XCMS workflow using the Centwave algorithm (Smith et al. 2006)."},

"MS_METABOLITE_DATA":{
"Units":"spectral abundance per cell",

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"Metabolites":[{"metabolite_name":"alanine"},{"metabolite_name":"Glycine"},{"metabolite_name":"X2 aminobutyric acid"},{"metabolite_name":"valine"},{"metabolite_name":"leucine"},{"metabolite_name":"isoleucine"},{"metabolite_name":"Threonine_R1"},{"metabolite_name":"Proline"},{"metabolite_name":"aspartic acid_R1"},{"metabolite_name":"Glutamine pyroglutamic acid"},{"metabolite_name":"Methionine"},{"metabolite_name":"serine"},{"metabolite_name":"Threonine_R2"},{"metabolite_name":"Phenylalanine"},{"metabolite_name":"aspartic acid_R2"},{"metabolite_name":"glutamic acid"},{"metabolite_name":"lysine"},{"metabolite_name":"arginine"},{"metabolite_name":"histidine"},{"metabolite_name":"tyrosine"},{"metabolite_name":"cystine"}]
}

}