#METABOLOMICS WORKBENCH ppjutur_20210830_043853 DATATRACK_ID:2814 STUDY_ID:ST001965 ANALYSIS_ID:AN003203 PROJECT_ID:PR001251
VERSION             	1
CREATED_ON             	October 27, 2021, 9:52 am
#PROJECT
PR:PROJECT_TITLE                 	Proteomics to Unveil Orchestration of Photorespiration and Central Carbon
PR:PROJECT_TITLE                 	Pathway in Microchloropsis gaditana NIES 2587
PR:PROJECT_TYPE                  	Time Course VLC HC Metabolome
PR:PROJECT_SUMMARY               	Photosynthetic organisms have evolved and adapted strategies to overcome the
PR:PROJECT_SUMMARY               	limiting concentrations of CO2. In this regard, the CO2-concentrating mechanism
PR:PROJECT_SUMMARY               	(CCM) developed by microalgae implies an efficient machinery to acquire CO2 in
PR:PROJECT_SUMMARY               	limiting environment. Inorganic carbon transporters channelize CO2 towards
PR:PROJECT_SUMMARY               	Rubisco, however, there are significant differences in the CCM of some species
PR:PROJECT_SUMMARY               	and it is obscurely understood. In the present study, we performed qualitative
PR:PROJECT_SUMMARY               	metabolomics and proteomics on Microchloropsis gaditana, under the influence of
PR:PROJECT_SUMMARY               	very-low CO2 (VLC; 300 ppm, or 0.03%) and high CO2 (HC; 30,000 ppm, or 3% v/v)
PR:PROJECT_SUMMARY               	at the time intervals of 0, 6, 12 and 24 hrs. Our results demonstrate that HC
PR:PROJECT_SUMMARY               	supplementation channelizes the carbon flux towards enhancing the biomass yield,
PR:PROJECT_SUMMARY               	increasing up to 1.7-fold. Cyclic electron flow driven (CEF) by PSI confers
PR:PROJECT_SUMMARY               	energy to the cells in the case of VLC in the initial acclimatization stage. Our
PR:PROJECT_SUMMARY               	qualitative metabolomic analyses has identified nearly 35 essential metabolites
PR:PROJECT_SUMMARY               	among which significant fold-change was observed as a photorespiratory
PR:PROJECT_SUMMARY               	by-product, glycolate, in VLC resulting in delayed growth and lower biomass.
PR:PROJECT_SUMMARY               	Whole cell proteomics study was performed in M. gaditana in both VLC and HC
PR:PROJECT_SUMMARY               	conditions and a total of 998 proteins were identified. Cells in VLC, undergoes
PR:PROJECT_SUMMARY               	dynamic changes to activate biophysical CCM with the help of bicarbonate
PR:PROJECT_SUMMARY               	transporters. In conclusion, comprehensive changes occur inside the cell that
PR:PROJECT_SUMMARY               	consequently mediate the assimilation and regulation of carbon metabolic loadout
PR:PROJECT_SUMMARY               	such that it favours fatty acid biosynthesis in HC. In conclusion, our emphasis
PR:PROJECT_SUMMARY               	is to delineate carbon assimilation in M. gaditana with the help of advanced
PR:PROJECT_SUMMARY               	multi-omics tools and provide translational approach for the enhanced production
PR:PROJECT_SUMMARY               	of biofuels and biorenewables.
PR:INSTITUTE                     	International Centre for Genetic Engineering and Biotechnology
PR:DEPARTMENT                    	Integrative Biotechnology
PR:LABORATORY                    	Omics of Algae
PR:LAST_NAME                     	Jutur
PR:FIRST_NAME                    	Pannaga Pavan
PR:ADDRESS                       	Omics of Algae Group, ICGEB campus, Aruna Asaf Ali Marg, New Delhi, Delhi,
PR:ADDRESS                       	110070, India
PR:EMAIL                         	jppavan@icgeb.res.in
PR:PHONE                         	26781358
#STUDY
ST:STUDY_TITLE                   	Integration of Metabolomics and Proteomics to Unveil Orchestration of
ST:STUDY_TITLE                   	Photorespiration and Central Carbon Pathway in Microchloropsis gaditana NIES
ST:STUDY_TITLE                   	2587
ST:STUDY_TYPE                    	Time Course VLC HC Metabolome
ST:STUDY_SUMMARY                 	Photosynthetic organisms have evolved and adapted strategies to overcome the
ST:STUDY_SUMMARY                 	limiting concentrations of CO2. In this regard, the CO2-concentrating mechanism
ST:STUDY_SUMMARY                 	(CCM) developed by microalgae implies an efficient machinery to acquire CO2 in
ST:STUDY_SUMMARY                 	limiting environment. Inorganic carbon transporters channelize CO2 towards
ST:STUDY_SUMMARY                 	Rubisco, however, there are significant differences in the CCM of some species
ST:STUDY_SUMMARY                 	and it is obscurely understood. In the present study, we performed qualitative
ST:STUDY_SUMMARY                 	metabolomics and proteomics on Microchloropsis gaditana, under the influence of
ST:STUDY_SUMMARY                 	very-low CO2 (VLC; 300 ppm, or 0.03%) and high CO2 (HC; 30,000 ppm, or 3% v/v)
ST:STUDY_SUMMARY                 	at the time intervals of 0, 6, 12 and 24 hrs. Our results demonstrate that HC
ST:STUDY_SUMMARY                 	supplementation channelizes the carbon flux towards enhancing the biomass yield,
ST:STUDY_SUMMARY                 	increasing up to 1.7-fold. Cyclic electron flow driven (CEF) by PSI confers
ST:STUDY_SUMMARY                 	energy to the cells in the case of VLC in the initial acclimatization stage. Our
ST:STUDY_SUMMARY                 	qualitative metabolomic analyses has identified nearly 35 essential metabolites
ST:STUDY_SUMMARY                 	among which significant fold-change was observed as a photorespiratory
ST:STUDY_SUMMARY                 	by-product, glycolate, in VLC resulting in delayed growth and lower biomass.
ST:STUDY_SUMMARY                 	Whole cell proteomics study was performed in M. gaditana in both VLC and HC
ST:STUDY_SUMMARY                 	conditions and a total of 998 proteins were identified. Cells in VLC, undergoes
ST:STUDY_SUMMARY                 	dynamic changes to activate biophysical CCM with the help of bicarbonate
ST:STUDY_SUMMARY                 	transporters. In conclusion, comprehensive changes occur inside the cell that
ST:STUDY_SUMMARY                 	consequently mediate the assimilation and regulation of carbon metabolic loadout
ST:STUDY_SUMMARY                 	such that it favours fatty acid biosynthesis in HC. In conclusion, our emphasis
ST:STUDY_SUMMARY                 	is to delineate carbon assimilation in M. gaditana with the help of advanced
ST:STUDY_SUMMARY                 	multi-omics tools and provide translational approach for the enhanced production
ST:STUDY_SUMMARY                 	of biofuels and biorenewables.
ST:INSTITUTE                     	International Centre for Genetic Engineering and Biotechnology
ST:DEPARTMENT                    	Integrative Biotechnology
ST:LABORATORY                    	Omics of Algae
ST:LAST_NAME                     	Jutur
ST:FIRST_NAME                    	Pavan
ST:ADDRESS                       	Omics of Algae Lab, 2nd Floor, New Building, ICGEB, Aruna Asaf Ali Marg, New
ST:ADDRESS                       	Delhi
ST:EMAIL                         	jppavan@gmail.com
ST:PHONE                         	01126741358
#SUBJECT
SU:SUBJECT_TYPE                  	Other organism
SU:SUBJECT_SPECIES               	Microchloropsis
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	-	VLC_6hr_1	Treatment:VLC	RAW_FILE_NAME=VLC_6hr_1
SUBJECT_SAMPLE_FACTORS           	-	VLC_6hr_2	Treatment:VLC	RAW_FILE_NAME=VLC_6hr_2
SUBJECT_SAMPLE_FACTORS           	-	VLC_6hr_3	Treatment:VLC	RAW_FILE_NAME=VLC_6hr_3
SUBJECT_SAMPLE_FACTORS           	-	VLC_12hr_1	Treatment:VLC	RAW_FILE_NAME=VLC_12hr_1
SUBJECT_SAMPLE_FACTORS           	-	VLC_12hr_2	Treatment:VLC	RAW_FILE_NAME=VLC_12hr_2
SUBJECT_SAMPLE_FACTORS           	-	VLC_12hr_3	Treatment:VLC	RAW_FILE_NAME=VLC_12hr_3
SUBJECT_SAMPLE_FACTORS           	-	VLC_24hr_1	Treatment:VLC	RAW_FILE_NAME=VLC_24hr_1
SUBJECT_SAMPLE_FACTORS           	-	VLC_24hr_2	Treatment:VLC	RAW_FILE_NAME=VLC_24hr_2
SUBJECT_SAMPLE_FACTORS           	-	VLC_24hr_3	Treatment:VLC	RAW_FILE_NAME=VLC_24hr_3
SUBJECT_SAMPLE_FACTORS           	-	HC_6hr_1	Treatment:HC	RAW_FILE_NAME=HC_6hr_1
SUBJECT_SAMPLE_FACTORS           	-	HC_6hr_2	Treatment:HC	RAW_FILE_NAME=HC_6hr_2
SUBJECT_SAMPLE_FACTORS           	-	HC_6hr_3	Treatment:HC	RAW_FILE_NAME=HC_6hr_3
SUBJECT_SAMPLE_FACTORS           	-	HC_12hr_1	Treatment:HC	RAW_FILE_NAME=HC_12hr_1
SUBJECT_SAMPLE_FACTORS           	-	HC_12hr_2	Treatment:HC	RAW_FILE_NAME=HC_12hr_2
SUBJECT_SAMPLE_FACTORS           	-	HC_12hr_3	Treatment:HC	RAW_FILE_NAME=HC_12hr_3
SUBJECT_SAMPLE_FACTORS           	-	HC_24hr_1	Treatment:HC	RAW_FILE_NAME=HC_24hr_1
SUBJECT_SAMPLE_FACTORS           	-	HC_24hr_2	Treatment:HC	RAW_FILE_NAME=HC_24hr_2
SUBJECT_SAMPLE_FACTORS           	-	HC_24hr_3	Treatment:HC	RAW_FILE_NAME=HC_24hr_3
#COLLECTION
CO:COLLECTION_SUMMARY            	Marine microalgae Microchloropsis gaditana NIES 2587 is procured from Microbial
CO:COLLECTION_SUMMARY            	Culture Collection, National Institute for Environmental Studies (NIES),
CO:COLLECTION_SUMMARY            	Tsukuba, Japan. The strain was grown in minimal medium F/2 (Guillard and Ryther,
CO:COLLECTION_SUMMARY            	1962) under a light regime of 16:8 h and an illumination of 150 µmol m−2
CO:COLLECTION_SUMMARY            	s−1 photosynthetically active radiation (PAR) in a multi-cultivator MC 1000-OD
CO:COLLECTION_SUMMARY            	(Photon Systems Instruments, Czech Republic) with a flow rate of 800 mL min-1
CO:COLLECTION_SUMMARY            	with continuous bubbling of air at 24 °C.
CO:SAMPLE_TYPE                   	Algae
#TREATMENT
TR:TREATMENT_SUMMARY             	Microchloropsis gaditana cells were grown in a Multicultivator in the presence
TR:TREATMENT_SUMMARY             	of very-low CO2 (300 ppm) and high CO2 (30,000 ppm) for 24 hours with an
TR:TREATMENT_SUMMARY             	illumination intensity of 150 uE.
TR:TREATMENT_COMPOUND            	CO2
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Quenched cells were resuspended in 1 mL of ice-cold
SP:SAMPLEPREP_SUMMARY            	methanol/ethanol/chloroform(2:6:2), followed by sonication of resuspended cells
SP:SAMPLEPREP_SUMMARY            	in sonication bath for 15 min. Later, these samples were centrifuged at
SP:SAMPLEPREP_SUMMARY            	10,000×g for 15 min at 4 °C to get rid of cell debris. The supernatant was
SP:SAMPLEPREP_SUMMARY            	filtered using a 0.2-µm filter. One hundred microlitres of supernatant was
SP:SAMPLEPREP_SUMMARY            	taken and dried under nitrogen stream. The dried leftover was dissolved in 10
SP:SAMPLEPREP_SUMMARY            	µL of freshly prepared methoxyamine hydrochloride solution (40 mg mL−1 in
SP:SAMPLEPREP_SUMMARY            	pyridine) and incubated at 30 °C for 90 min with shaking. To the above
SP:SAMPLEPREP_SUMMARY            	solution, 90 µL of N-methyl-N-(trimethylsilyl)trifluoroacetamide was added and
SP:SAMPLEPREP_SUMMARY            	incubated at 37 °C for 30 min. The samples were centrifuged at 14,000×g for 3
SP:SAMPLEPREP_SUMMARY            	min, and the supernatant was taken for the GC-MS/MS analysis.
SP:PROCESSING_STORAGE_CONDITIONS 	Described in summary
SP:EXTRACTION_METHOD             	Sonication
SP:SAMPLE_DERIVATIZATION         	MSTFA
SP:SAMPLE_SPIKING                	Ribitol (Internal Standard)
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	GC-triple quadrupole analysis was performed on an HP-5 gas chromatograph with
CH:CHROMATOGRAPHY_SUMMARY        	standard liners containing glass wool in split mode (1:5) at 250°C injector
CH:CHROMATOGRAPHY_SUMMARY        	temperature. The GC was operated at constant flow of 1 ml/min helium on a 30-m,
CH:CHROMATOGRAPHY_SUMMARY        	0.25-mm i.d., 0.25-μm HP-5 column, a start temperature of 60°C, 3 min
CH:CHROMATOGRAPHY_SUMMARY        	isothermal, temperature ramping by 5°C/min to 180°C, 3 min isothermal and
CH:CHROMATOGRAPHY_SUMMARY        	finally temperature ramping of 10°C/min to 310°C.
CH:CHROMATOGRAPHY_TYPE           	GC
CH:INSTRUMENT_NAME               	Agilent 7890A
CH:COLUMN_NAME                   	Agilent HP5-MS (30m x 0.25mm, 0.25 um)
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Agilent 7890A
MS:INSTRUMENT_TYPE               	Triple quadrupole
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	Mass Hunter
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	Area
MS_METABOLITE_DATA_START
Samples	VLC_6hr_1	VLC_6hr_2	VLC_6hr_3	VLC_12hr_1	VLC_12hr_2	VLC_12hr_3	VLC_24hr_1	VLC_24hr_2	VLC_24hr_3	HC_6hr_1	HC_6hr_2	HC_6hr_3	HC_12hr_1	HC_12hr_2	HC_12hr_3	HC_24hr_1	HC_24hr_2	HC_24hr_3
Factors	Treatment:VLC	Treatment:VLC	Treatment:VLC	Treatment:VLC	Treatment:VLC	Treatment:VLC	Treatment:VLC	Treatment:VLC	Treatment:VLC	Treatment:HC	Treatment:HC	Treatment:HC	Treatment:HC	Treatment:HC	Treatment:HC	Treatment:HC	Treatment:HC	Treatment:HC
Alanine	3244387	1746127	3273323	1181743	9725067	26019342	2164276	699854	2625316	15864880	5150896	28353606	4777717	10964164	9319152	64136868	12459556	578467
Glutamine	110463.2	110463.2	110463.2	110463.2	110463.2	110463.2	110463.2	110463.2	110463.2	110463.2	110463.2	110463.2	110463.2	110463.2	110463.2	552316	110463.2	110463.2
Glycine	2391243	10508067	570653	7083560	699441	330813	625713	714878	371842	7567633	66162.6	823216	66162.6	569479	66162.6	25052594	1757040	4643990
Histidine	105537.8	105537.8	105537.8	105537.8	105537.8	105537.8	105537.8	105537.8	105537.8	105537.8	105537.8	105537.8	105537.8	105537.8	105537.8	527689	105537.8	105537.8
Leucine	74303	1654095	74303	74303	74303	608053	74303	371515	74303	41397018	74303	74303	1804657	469832	74303	74303	74303	2738516
Lysine	529861.4	529861.4	529861.4	529861.4	529861.4	529861.4	529861.4	529861.4	529861.4	529861.4	529861.4	529861.4	529861.4	529861.4	529861.4	2649307	529861.4	529861.4
Proline	20509465	16241432	11449789	8091030	9501006	9152727	20766175	18148565	7783481	27385525	16012782	16139087	16080179	8182184	29752313	8158507	60703051	17676351
Valine	102214.8	102214.8	102214.8	102214.8	102214.8	102214.8	102214.8	102214.8	102214.8	1410017	102214.8	102214.8	102214.8	102214.8	1847273	1055414	553859	511074
Galactose	158232	791160	158232	158232	158232	158232	158232	158232	158232	1879705	2589335	158232	158232	46977004	158232	2306522	1087734	1209505
Glucose	126876	1419256	1006619	126876	126876	126876	943361	126876	634380	5269071	4043480	44232477	126876	2405102	1660080	1075098	60328823	14870736
Glycerol	745008	871613	750859	1007172	1007172	1282731	1709879	1874981	516369	885973	3108184	655707	1306483	586422	549440	2172715	1208948	1320905
Maltose	3444494	89724.4	89724.4	89724.4	89724.4	89724.4	89724.4	89724.4	89724.4	89724.4	2345229	497986	1934196	89724.4	89724.4	89724.4	448622	875292
Acetic acid	4462784	4462784	782991	153860.8	153860.8	153860.8	8125303	8681611	7618974	769304	1453309	1475354	3190188	1301083	8031965	60828001	8256980	9193593
Carbonic acid	461738	1099601	1623589	92347.6	1971034	843992	1176226	553231	92347.6	92347.6	565156	92347.6	92347.6	92347.6	92347.6	547746	92347.6	92347.6
Fumaric acid	4688012	96786.2	96786.2	96786.2	96786.2	96786.2	96786.2	96786.2	96786.2	96786.2	1062453	96786.2	483931	96786.2	96786.2	4973896	96786.2	21085272
Glycolic acid	1459950	1395142	3797136	1338589	1338589	1275177	1267217	4057096	1122408	224481.6	224481.6	224481.6	224481.6	224481.6	224481.6	224481.6	224481.6	224481.6
Lactic acid	781349	714332	90069.2	1053267	90069.2	2145235	2394128	733093	450346	1008956	90069.2	688446	3230608	2673536	2308500	4692327	3627775	1693333
Malonic acid	65830.8	65830.8	757696	65830.8	65830.8	1251250	65830.8	329154	1329371	65830.8	1376083	65830.8	65830.8	65830.8	65830.8	65830.8	439845	65830.8
Oxalic acid	2521220	2852884	272544.8	2480696	2480696	2469891	2397039	272544.8	4384701	4099165	2470513	2819058	2751527	272544.8	2691746	3136111	2123615	1362724
Succinic acid	3908088	898527	84755.8	84755.8	84755.8	5327993	478109	84755.8	562563	84755.8	423779	480662	953411	84755.8	1503036	1507483	84755.8	1626719
α-Ketoglutaric acid	146684.4	146684.4	146684.4	146684.4	146684.4	146684.4	146684.4	146684.4	146684.4	146684.4	146684.4	146684.4	146684.4	146684.4	146684.4	1038194	786227	733422
Ethanolamine	256435.8	256435.8	256435.8	256435.8	256435.8	256435.8	256435.8	256435.8	256435.8	256435.8	1282179	256435.8	256435.8	256435.8	256435.8	1423818	256435.8	256435.8
Myo-inositol	73027.4	73027.4	73027.4	73027.4	73027.4	365137	832424	1036318	635416	1977009	2114060	1612916	765865	569666	795959	3044554	3957299	2602442
Phosphoric acid	1500565	1500565	1235441	1152303	1152303	984853	1118752	1578122	867981	1414042	1623446	1825218	173596.2	1181982	1711619	1687590	2619138	173596.2
Phytol	1224688	1965148	1675163	3879982	643226	693487	128645.2	2219793	1359176	4590567	1737246	5709150	1920050	2267145	1540912	9753473	6538897	948597
Trehalose	80037.6	80037.6	80037.6	80037.6	80037.6	80037.6	400188	722329	80037.6	939610	1144189	793395	80037.6	80037.6	80037.6	6392031	5454392	2869033
Eicosanoic acid	118335.8	1289853	118335.8	118335.8	118335.8	118335.8	118335.8	118335.8	118335.8	118335.8	118335.8	118335.8	981601	1031608	118335.8	591679	118335.8	1431629
Eicosatrienoic acid	614626.8	614626.8	614626.8	614626.8	614626.8	614626.8	614626.8	614626.8	614626.8	614626.8	614626.8	5376156	614626.8	614626.8	614626.8	3073134	614626.8	614626.8
EPA	3738423	4294600	3613940	1585031	1585031	1357623	2317849	2499010	1946588	13576630	12299915	6023957	4983740	4850821	5636854	7178920	11703883	17281155
Heptadecanoic acid	5388795	3856771	3495418	1530696	4767452	3422655	4008819	3640047	2839867	3968369	4016083	3562594	3640851	3729061	2224093	2662821	3058054	4332881
Hexadecanoic acid	2101303	4177954	1962388	90015.4	1545883	450077	477967	969130	90015.4	600105	612422	550622	2565741	2825304	2307097	521906	1341636	9167624
α-Linolenic acid	242783.4	3551841	242783.4	242783.4	242783.4	242783.4	1672664	3129278	1362551	3097129	3941378	242783.4	242783.4	242783.4	1213917	242783.4	242783.4	5210969
Octadecanoic acid	4006060	2811838	2470334	3561139	3180630	911786	1343472	281349	56269.8	1790635	56269.8	56269.8	4755592	1963404	3699239	6862406	1163276	1059364
Ergosterol	363149.8	2473963	3875860	363149.8	1990758	363149.8	363149.8	363149.8	363149.8	363149.8	1815749	363149.8	2471012	363149.8	363149.8	363149.8	4835148	4779419
Stigmasterol	207764.8	207764.8	207764.8	207764.8	207764.8	207764.8	1501631	207764.8	207764.8	207764.8	207764.8	1038824	1443004	207764.8	2038512	207764.8	207764.8	207764.8
α-Tocopherol	90516.4	90516.4	90516.4	90516.4	90516.4	90516.4	90516.4	452582	90516.4	90516.4	794206	819109	90516.4	90516.4	90516.4	691840	868152	984062
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name
Alanine
Glutamine
Glycine
Histidine
Leucine
Lysine
Proline
Valine
Galactose
Glucose
Glycerol
Maltose
Acetic acid
Carbonic acid
Fumaric acid
Glycolic acid
Lactic acid
Malonic acid
Oxalic acid
Succinic acid
α-Ketoglutaric acid
Ethanolamine
Myo-inositol
Phosphoric acid
Phytol
Trehalose
Eicosanoic acid
Eicosatrienoic acid
EPA
Heptadecanoic acid
Hexadecanoic acid
α-Linolenic acid
Octadecanoic acid
Ergosterol
Stigmasterol
α-Tocopherol
METABOLITES_END
#END