Summary of study ST001198

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000807. The data can be accessed directly via it's Project DOI: 10.21228/M8CD73 This work is supported by NIH grant, U2C- DK119886.

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Study IDST001198
Study TitleTargeted LC-MS/MS Analysis of Soluble Metabolites in the MeOH:H2O Phase (part-IV)
Study SummaryCyanobacteria 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
DepartmentChemical and Biological Engineering
Last NamePeebles
First NameChristie
Address700 Meridian Ave, Fort Collins, CO 80523
Emailchristie.peebles@colostate.edu
Phone970-491-6779
Submit Date2019-03-02
Raw Data AvailableYes
Raw Data File Type(s).cdf
Analysis Type DetailLC-MS
Release Date2019-07-17
Release Version1
Christie Peebles Christie Peebles
https://dx.doi.org/10.21228/M8CD73
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000807
Project DOI:doi: 10.21228/M8CD73
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 Biological Engineering
Last Name:Peebles
First Name:Christie
Address:700 Meridian Ave, Fort Collins, CO 80523 USA
Email:wernerajz@gmail.com
Phone:2699981811

Subject:

Subject ID:SU001265
Subject Type:Bacteria
Subject Species:Synechocystis sp. PCC6803
Taxonomy ID:1148
Genotype Strain:NCBI:txid1148
Cell Biosource Or Supplier:ATCC

Factors:

Subject type: Bacteria; Subject species: Synechocystis sp. PCC6803 (Factor headings shown in green)

mb_sample_id local_sample_id time
SA08334612-Synechocystis_6803-cell-7a-3-
SA08334711-Synechocystis_6803-cell-7a-2-
SA08334810-Synechocystis_6803-cell-7a-1-
SA0833559-Synechocystis_6803-cell-630a-3-0.5
SA0833568-Synechocystis_6803-cell-630a-2-0.5
SA0833577-Synechocystis_6803-cell-630a-1-0.5
SA08335813-Synechocystis_6803-cell-730a-10.5
SA08335915-Synechocystis_6803-cell-730a-30.5
SA08336014-Synechocystis_6803-cell-730a-20.5
SA08336118-Synechocystis_6803-cell-8a-31
SA08336216-Synechocystis_6803-cell-8a-11
SA08336317-Synechocystis_6803-cell-8a-21
SA0833526-Synechocystis_6803-cell-6a-3-1
SA0833535-Synechocystis_6803-cell-6a-2-1
SA0833544-Synechocystis_6803-cell-6a-1-1
SA08337633-Synechocystis_6803-cell-5p-310
SA08337732-Synechocystis_6803-cell-5p-210
SA08337831-Synechocystis_6803-cell-5p-110
SA08337934-Synechocystis_6803-cell-6p-111
SA08338036-Synechocystis_6803-cell-6p-311
SA08338135-Synechocystis_6803-cell-6p-211
SA08338237-Synechocystis_6803-cell-630p-111.5
SA08338338-Synechocystis_6803-cell-630p-211.5
SA08338439-Synechocystis_6803-cell-630p-311.5
SA08338541-Synechocystis_6803-cell-7p-212
SA08338642-Synechocystis_6803-cell-7p-312
SA08338740-Synechocystis_6803-cell-7p-112
SA08338844-Synechocystis_6803-cell-730p-212.5
SA08338943-Synechocystis_6803-cell-730p-112.5
SA08339045-Synechocystis_6803-cell-730p-312.5
SA08339148-Synechocystis_6803-cell-8p-313
SA08339247-Synechocystis_6803-cell-8p-213
SA08339346-Synechocystis_6803-cell-8p-113
SA08339449-Synechocystis_6803-cell-9p-114
SA08339551-Synechocystis_6803-cell-9p-314
SA08339650-Synechocystis_6803-cell-9p-214
SA08339753-Synechocystis_6803-cell-11p-216
SA08339852-Synechocystis_6803-cell-11p-116
SA08339954-Synechocystis_6803-cell-11p-316
SA08340057-Synechocystis_6803-cell-1a-318
SA08340155-Synechocystis_6803-cell-1a-118
SA08340256-Synechocystis_6803-cell-1a-218
SA08336420-Synechocystis_6803-cell-9a-22
SA08336519-Synechocystis_6803-cell-9a-12
SA08336621-Synechocystis_6803-cell-9a-32
SA0833491-Synechocystis_6803-cell-5a-1-2
SA0833502-Synechocystis_6803-cell-5a-2-2
SA0833513-Synechocystis_6803-cell-5a-3-2
SA08340360-Synechocystis_6803-cell-3a-320
SA08340458-Synechocystis_6803-cell-3a-120
SA08340559-Synechocystis_6803-cell-3a-220
SA08340661-Synechocystis_6803-cell-5a_day2-122
SA08340762-Synechocystis_6803-cell-5a_day2-222
SA08340863-Synechocystis_6803-cell-5a_day2-322
SA08340964-Synechocystis_6803-cell-6a_day2-123
SA08341065-Synechocystis_6803-cell-6a_day3-223
SA08341166-Synechocystis_6803-cell-6a_day4-323
SA08341267-Synechocystis_6803-cell-630a_day2-123.5
SA08341368-Synechocystis_6803-cell-630a_day3-223.5
SA08341469-Synechocystis_6803-cell-630a_day4-323.5
SA08341571-Synechocystis_6803-cell-7a_day2-224
SA08341672-Synechocystis_6803-cell-7a_day2-324
SA08341770-Synechocystis_6803-cell-7a_day2-124
SA083418QC-1-6426
SA083419QC-1-7126
SA083420QC-1-78b26
SA083421QC-1-826
SA083422QC-1-5726
SA083423QC-1-7826
SA083424QC-1-5026
SA083425QC-1-1526
SA083426QC-1-126
SA083427QC-1-2226
SA083428QC-1-2926
SA083429QC-1-3626
SA083430QC-1-4326
SA08336723-Synechocystis_6803-cell-11a-24
SA08336822-Synechocystis_6803-cell-11a-14
SA08336924-Synechocystis_6803-cell-11a-34
SA08337026-Synechocystis_6803-cell-1p-26
SA08337125-Synechocystis_6803-cell-1p-16
SA08337227-Synechocystis_6803-cell-1p-36
SA08337328-Synechocystis_6803-cell-3p-18
SA08337429-Synechocystis_6803-cell-3p-28
SA08337530-Synechocystis_6803-cell-3p-38
Showing results 1 to 85 of 85

Collection:

Collection ID:CO001259
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 ID:TR001280
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).

Sample Preparation:

Sampleprep ID:SP001273
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.

Combined analysis:

Analysis ID AN001994
Analysis type MS
Chromatography type HILIC
Chromatography system Waters Xevo QS
Column Merck ZIC-pHilic
MS Type ESI
MS instrument type Triple quadrupole
MS instrument name Waters Xevo QS
Ion Mode UNSPECIFIED
Units spectral abundance per cell

Chromatography:

Chromatography ID:CH001442
Instrument Name:Waters Xevo QS
Column Name:Merck ZIC-pHilic
Chromatography Type:HILIC

MS:

MS ID:MS001847
Analysis ID:AN001994
Instrument Name:Waters Xevo QS
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:Positive and negative ion mode switching
Ion Mode:UNSPECIFIED
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