Summary of Study ST000622

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 PR000454. The data can be accessed directly via it's Project DOI: 10.21228/M8CC9H This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Show all samples
Download mwTab file (text) | Download mwTab file(JSON) | Download data files (Contains raw data)

Study ID	ST000622
Study Title	Identification and metabolite profiling of chemical activators of lipid accumulation in green algae
Study Type	GC-MS metabolite profiling of algal lipid activators
Study Summary	Microalgae are proposed as feedstock organisms useful for producing biofuels and co-products. However, several limitations must be overcome before algae-based production is economically feasible. Among these is the ability to induce lipid accumulation and storage without affecting biomass yield. To overcome this barrier, a chemical genetics approach was employed in which 43,783 compounds were screened against Chlamydomonas reinhardtii and 243 compounds were identified that increase triacylglyceride (TAG) accumulation without terminating growth. Identified compounds were classified by structural similarity and 15 selected for secondary analyses addressing impacts on growth fitness, photosynthetic pigments, and total cellular protein and starch concentrations. TAG accumulation was verified using GC-MS quantification of total fatty acids and targeted TAG and galactolipid (GL) measurements using LC-MRM/MS. These results demonstrated TAG accumulation does not necessarily proceed at the expense of GL. Untargeted metabolite profiling provided important insights into pathway shifts due to 5 different compound treatments and verified the anabolic state of the cells with regard to the oxidative pentose phosphate pathway, Calvin cycle, tricarboxylic acid cycle and amino acid biosynthetic pathways. Metabolite patterns were distinct from nitrogen starvation and other abiotic stresses commonly used to induce oil accumulation in algae. The efficacy of these compounds was also demonstrated in 3 other algal species. These lipid inducing compounds offer a valuable set of tools for delving into the biochemical mechanisms of lipid accumulation in algae and a direct means to improve algal oil content independent of the severe growth limitations associated with nutrient deprivation.
Institute	University of Nebraska-Lincoln
Department	Biochemistry
Laboratory	FATTTLab
Last Name	Wase
First Name	Nishikant
Address	1901 Beadle Center, Vine Street, 1901 VINE STREET, Lincoln, NE, 68588-0664, USA
Email	nishikant.wase@gmail.com
Phone	4023109931
Submit Date	2017-06-16
Num Groups	6
Publications	1. Nishikant Wase, Boqiang Tu, James W Allen, Paul N Black, Concetta C DiRusso. Identification and metabolite profiling of chemical activators of lipid accumulation in green algae. Plant Physiology Jun 2017. DOI: 10.1104/pp.17.00433. http://www.plantphysiol.org/content/early/2017/06/26/pp.17.00433; 2. DiRusso, C., & Wase, N. (2016). Compounds for Increasing Lipid Synthesis and Storage. United States. NUtech Ventures (Lincoln, NE, US) http://www.freepatentsonline.com/y2016/0312253.html
Raw Data Available	Yes
Raw Data File Type(s)	cdf
Analysis Type Detail	GC-MS
Release Date	2017-10-03
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR000454
Project DOI:	doi: 10.21228/M8CC9H
Project Title:	Untargeted metabolomic changes in Chlamydomonas reinhardtii treated with lipid inducing small molecules
Project Summary:	A study to investigate the effect of small molecule lipid inducing compounds that leads to hyper accumulation of lipids in N replete cells of Chlamydomonas reinhardtii. These compounds were identified through a high throughput screening designed for that purpose. During that screening, we screened 43,783 compounds and identified 367 primary hits. These 367 hits were further retested using a 8-point dilution series (from 0.25 to 30 uM) and verified the activity of 250 compounds that induce the hyper lipid accumulating phenotype in algae. Once the hit compounds were identified and confirmed, we then performed extensive chemoinformatics analysis to look for common scaffolds and identified several common substructures. We then selected 15 top performing compounds from 5 diverse structural groups and tested biochemical parameters such as growth, lipid accumulating capacity, effect on photosynthetic rates, respiration rates, oxygen consumption rates, analysis of different lipid species to quantify and identify fatty acid species using GC-MS. To understand the global changes in the metabolome, 2 structurally different compounds were selected and compared with cells grown without compounds as control for untargeted metabolomics analysis.
Institute:	University of Nebraska-Lincoln
Department:	Biochemistry
Laboratory:	FATTTLab
Last Name:	Wase
First Name:	Nishikant
Address:	1901 Beadle Center, Vine Street, 1901 VINE STREET, Lincoln, NE, 68588-0664, USA
Email:	nishikant.wase@gmail.com
Phone:	4023109931
Funding Source:	NCESR-704, Nebraska Center for Energy Science Research; EPS-1004094 and 1264409, National Science Foundation ; NSF CBET : 1402896, National Science Foundation
Contributors:	Nishikant Wase, Jiri Adamec, Ron Cerny, Girish Rasineni, Paul N Black, Concetta DiRusso

Subject:

Subject ID:	SU000645
Subject Type:	Photosynthetic organism
Subject Species:	Chlamydomonas reinhardtii
Taxonomy ID:	3055
Genotype Strain:	Wild Type
Species Group:	Microorganism

Factors:

Subject type: Photosynthetic organism; Subject species: Chlamydomonas reinhardtii (Factor headings shown in green)

mb_sample_id	local_sample_id	Class
SA035018	ContB_1	Control
SA035019	ContA_1	Control
SA035020	ContA_2	Control
SA035021	ContB_2	Control
SA035022	ContA_3	Control
SA035023	ContB_3	Control
SA035024	ContC_3	Control
SA035025	ContC_2	Control
SA035026	ContC_1	Control
SA035027	461_B_2	WD10461
SA035028	461_B_1	WD10461
SA035029	461_B_3	WD10461
SA035030	461_A_3	WD10461
SA035031	461_C_3	WD10461
SA035032	461_A_2	WD10461
SA035033	461_C_2	WD10461
SA035034	461_C_1	WD10461
SA035035	461_A_1	WD10461
SA035036	784_C_1	WD10784
SA035037	784_C_2	WD10784
SA035038	784_A_2	WD10784
SA035039	784_B_3	WD10784
SA035040	784_B_2	WD10784
SA035041	784_A_1	WD10784
SA035042	784_A_3	WD10784
SA035043	784_B_1	WD10784
SA035044	784_C_3	WD10784
SA035045	067_A_2	WD20067
SA035046	067_C_1	WD20067
SA035047	067_C_2	WD20067
SA035048	067_C_3	WD20067
SA035049	067_B_3	WD20067
SA035050	067_B_2	WD20067
SA035051	067_A_3	WD20067
SA035052	067_B_1	WD20067
SA035053	067_A_1	WD20067
SA035054	542_A_1	WD20542
SA035055	542_A_3	WD20542
SA035056	542_A_2	WD20542
SA035057	542_C_3	WD20542
SA035058	542_B_2	WD20542
SA035059	542_B_1	WD20542
SA035060	542_C_1	WD20542
SA035061	542_C_2	WD20542
SA035062	542_B_3	WD20542
SA035063	030_B_1	WD30030
SA035064	030_A_3	WD30030
SA035065	030_A_2	WD30030
SA035066	030_B_2	WD30030
SA035067	030_A_1	WD30030
SA035068	030_C_3	WD30030
SA035069	030_C_2	WD30030
SA035070	030_C_1	WD30030
SA035071	030_B_3	WD30030

Showing results 1 to 54 of 54

Showing results 1 to 54 of 54

Collection:

Collection ID:	CO000639
Collection Summary:	Cells were pre-grown to mid-log phase and treated with 5 selected compounds (final concentration 5 µM) with an initial cell density of 1.0 x 106 cells/mL (100 mL volume; in triplicate) and allowed to grow for 72 h. After 72 h of growth, cells were harvested, media removed and freeze-dried. Accurately measured 50 ± 0.5 mg of freeze dried powder was used for metabolite extraction. Sample powder was pulverized with a single steel ball using TissueLyser LT (Qiagen) at 50 Hz speed for 5 min
Sample Type:	Algae

Treatment:

Treatment ID:	TR000659
Treatment Summary:	Cells were treated either with compounds (5 uM) final concentration in DMSO or DMSO alone (in case of control). Mid-log phase cells were used as starter culture at a initial inoculum density of 1.0E06 cells/mL in 250 mL flasks with 100 mL of TAP media. Cells were allowed to grow in orbital shaker under constant light for 72 hours. After 72 hours, experiment was terminated and cells were harvested via centrifugation.

Treatment ID:

TR000659

Treatment Summary:

Cells were treated either with compounds (5 uM) final concentration in DMSO or DMSO alone (in case of control). Mid-log phase cells were used as starter culture at a initial inoculum density of 1.0E06 cells/mL in 250 mL flasks with 100 mL of TAP media. Cells were allowed to grow in orbital shaker under constant light for 72 hours. After 72 hours, experiment was terminated and cells were harvested via centrifugation.

Sample Preparation:

Sampleprep ID:	SP000652
Sampleprep Summary:	Harvested cells were flash frozen in liquid N and freeze dried. Accurately measured 50 ± 0.5 mg of freeze dried powder was used for metabolite extraction. Sample powder was pulverized with a single steel ball using TissueLyser LT (Qiagen) at 50 Hz speed for 5 min. One milliliter of extraction buffer containing MeOH:CHCl3:H2O (5:2:2; v/v/v; pre-cooled at -20 °C) was added and vortexed for 5 min. Ribitol (0.2 mg/mL in water; 10 µL) was spiked in the extraction buffer as internal standard in order to identify potential chromatographic errors. The homogenized material was centrifuged at 14000 rpm for 5 min and the supernatant was transferred to new tubes. 400 µL of pure water was added to the supernatant, samples were vortexed and centrifuged at 14,000 rpm for 5 min. The upper polar phase was transferred to new tubes for GC-MS analysis. An aliquot of 300 µL was dried out in vacuum concentrator without heating. To the dried material, 10 µL methoxyamine HCL in 100% pyridine (40 mg/mL) was added and shaken at 30 °C for 90 minutes and subsequently 90 µL of MSTFA 1% TMCS was added for trimethylsilylation of acidic protons and shaken at 37 °C for 30 minutes. The reaction mixture was transferred to GCvials with glass microinserts and closed by crimp caps. GC-MS data acquisition was performed as per previously published report (Wase et al., 2014)

Sampleprep ID:

SP000652

Sampleprep Summary:

Harvested cells were flash frozen in liquid N and freeze dried. Accurately measured 50 ± 0.5 mg of freeze dried powder was used for metabolite extraction. Sample powder was pulverized with a single steel ball using TissueLyser LT (Qiagen) at 50 Hz speed for 5 min. One milliliter of extraction buffer containing MeOH:CHCl3:H2O (5:2:2; v/v/v; pre-cooled at -20 °C) was added and vortexed for 5 min. Ribitol (0.2 mg/mL in water; 10 µL) was spiked in the extraction buffer as internal standard in order to identify potential chromatographic errors. The homogenized material was centrifuged at 14000 rpm for 5 min and the supernatant was transferred to new tubes. 400 µL of pure water was added to the supernatant, samples were vortexed and centrifuged at 14,000 rpm for 5 min. The upper polar phase was transferred to new tubes for GC-MS analysis. An aliquot of 300 µL was dried out in vacuum concentrator without heating. To the dried material, 10 µL methoxyamine HCL in 100% pyridine (40 mg/mL) was added and shaken at 30 °C for 90 minutes and subsequently 90 µL of MSTFA 1% TMCS was added for trimethylsilylation of acidic protons and shaken at 37 °C for 30 minutes. The reaction mixture was transferred to GCvials with glass microinserts and closed by crimp caps. GC-MS data acquisition was performed as per previously published report (Wase et al., 2014)

Combined analysis:

Analysis ID	AN000954
Analysis type	MS
Chromatography type	GC
Chromatography system	Agilent 6890N
Column	Agilent DB-5MS UI Capillary column
MS Type	EI
MS instrument type	Single quadrupole
MS instrument name	Agilent 5973
Ion Mode	POSITIVE
Units	peak area

Chromatography:

Chromatography ID:	CH000679
Instrument Name:	Agilent 6890N
Column Name:	Agilent DB-5MS UI Capillary column
Internal Standard:	Ribitol
Sample Injection:	1 uL
Chromatography Type:	GC

MS:

MS ID:	MS000849
Analysis ID:	AN000954
Instrument Name:	Agilent 5973
Instrument Type:	Single quadrupole
MS Type:	EI
Ion Mode:	POSITIVE