#METABOLOMICS WORKBENCH HawkinsSS_20250522_092956 DATATRACK_ID:5938 STUDY_ID:ST003933 ANALYSIS_ID:AN006458 PROJECT_ID:PR002463
VERSION             	1
CREATED_ON             	May 27, 2025, 3:08 pm
#PROJECT
PR:PROJECT_TITLE                 	Lipidomic analysis of genetically engineered PCC 7002 indicate lipid remodeling
PR:PROJECT_TITLE                 	and differential fatty acid production at varied temperatures
PR:PROJECT_SUMMARY               	E. coli and cycanobacteria, including Picosynechococcus sp. PCC 7002 (PCC 7002)
PR:PROJECT_SUMMARY               	are promising hosts for biomanufacturing due to their rapid growth and genetic
PR:PROJECT_SUMMARY               	manipulability. Microbial production of free fatty acids provides a potential
PR:PROJECT_SUMMARY               	renewable source of acyl chains, but genetic engineering for high production
PR:PROJECT_SUMMARY               	strains remains challenging. Using an untargeted lipidomics mass spectrometry
PR:PROJECT_SUMMARY               	workflow, we investigated two strains of PCC 7002 grown at two temperatures in
PR:PROJECT_SUMMARY               	order to probe the alterations in lipid profiles associated with the different
PR:PROJECT_SUMMARY               	genetic and environmental conditions.
PR:INSTITUTE                     	Vanderbilt University
PR:LAST_NAME                     	Shepard
PR:FIRST_NAME                    	Hawkins
PR:ADDRESS                       	7330 Stevenson Center, Station B 351822, Nashville, TN 37235
PR:EMAIL                         	hawkins.s.shepard@vanderbilt.edu
PR:PHONE                         	6153434563
#STUDY
ST:STUDY_TITLE                   	Lipidomic analysis of genetically engineered PCC 7002 indicate lipid remodeling
ST:STUDY_TITLE                   	and differential fatty acid production at varied temperatures
ST:STUDY_SUMMARY                 	An untargeted lipidomics MS workflow was used to investigate the alterations of
ST:STUDY_SUMMARY                 	lipid profiles for two strains of gentetically modified PCC 7002 at two
ST:STUDY_SUMMARY                 	different temperatures. The two strains evaluated were WT and an aas-knockout
ST:STUDY_SUMMARY                 	strain. The temperatures at which both strains were subjected were 30°C and
ST:STUDY_SUMMARY                 	37°C. In the Δaas mutant, which lacks an acyl-activating synthetase (aas) and
ST:STUDY_SUMMARY                 	secretes FFAs, namely C16:1-ACP, C18:1-ACP, C18:2-ACP, and C18:3-ACP, with those
ST:STUDY_SUMMARY                 	FFAs being accumulated at both optimal and cold temperatures. This was
ST:STUDY_SUMMARY                 	unexpected, as cyanobacteria typically do not produce polyunsaturated acyl-ACPs,
ST:STUDY_SUMMARY                 	suggesting compensatory acyltransferase or other enzyme activities that were
ST:STUDY_SUMMARY                 	further explored with proteomic analysis. Several candidates were identified
ST:STUDY_SUMMARY                 	that could potentially reactivate FFAs and would limit secretion that is an
ST:STUDY_SUMMARY                 	important aspect of current biotechnological approaches.
ST:INSTITUTE                     	Vanderbilt University
ST:LAST_NAME                     	Shepard
ST:FIRST_NAME                    	Hawkins
ST:ADDRESS                       	7330 Stevenson Center, Station B 351822, Nashville, TN 37235
ST:EMAIL                         	hawkins.s.shepard@vanderbilt.edu
ST:PHONE                         	6153434563
#SUBJECT
SU:SUBJECT_TYPE                  	Bacteria
SU:SUBJECT_SPECIES               	Picosynechococcus sp. PCC 7002
SU:TAXONOMY_ID                   	32049
#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           	-	aas37a	Sample source:Cyanobacteria | Genotype:aas-knockout | Treatment:control	RAW_FILE_NAME(Raw file name)=20241008_DOE_HSS_0008_neg_FS_aas37a_r1.d
SUBJECT_SAMPLE_FACTORS           	-	aas37b	Sample source:Cyanobacteria | Genotype:aas-knockout | Treatment:control	RAW_FILE_NAME(Raw file name)=20241008_DOE_HSS_00018_neg_FS_aas37b_r1.d
SUBJECT_SAMPLE_FACTORS           	-	aas37c	Sample source:Cyanobacteria | Genotype:aas-knockout | Treatment:control	RAW_FILE_NAME(Raw file name)=20241008_DOE_HSS_0026_neg_FS_aas37c_r1.d
SUBJECT_SAMPLE_FACTORS           	-	aas37d	Sample source:Cyanobacteria | Genotype:aas-knockout | Treatment:control	RAW_FILE_NAME(Raw file name)=20241008_DOE_HSS_0034_neg_FS_aas37d_r1.d
SUBJECT_SAMPLE_FACTORS           	-	aas30a	Sample source:Cyanobacteria | Genotype:aas-knockout | Treatment:Low-tempeature	RAW_FILE_NAME(Raw file name)=20241008_DOE_HSS_0009_neg_FS_aas30a_r1.d
SUBJECT_SAMPLE_FACTORS           	-	aas30b	Sample source:Cyanobacteria | Genotype:aas-knockout | Treatment:Low-tempeature	RAW_FILE_NAME(Raw file name)=20241008_DOE_HSS_0014_neg_FS_aas30b_r1.d
SUBJECT_SAMPLE_FACTORS           	-	aas30c	Sample source:Cyanobacteria | Genotype:aas-knockout | Treatment:Low-tempeature	RAW_FILE_NAME(Raw file name)=20241008_DOE_HSS_0028_neg_FS_aas30c_r1.d
SUBJECT_SAMPLE_FACTORS           	-	aas30d	Sample source:Cyanobacteria | Genotype:aas-knockout | Treatment:Low-tempeature	RAW_FILE_NAME(Raw file name)=20241008_DOE_HSS_0033_neg_FS_aas30d_r1.d
SUBJECT_SAMPLE_FACTORS           	-	WT37a	Sample source:Cyanobacteria | Genotype:Wild-type | Treatment:control	RAW_FILE_NAME(Raw file name)=20241008_DOE_HSS_0011_neg_FS_WT37a_r1.d
SUBJECT_SAMPLE_FACTORS           	-	WT37b	Sample source:Cyanobacteria | Genotype:Wild-type | Treatment:control	RAW_FILE_NAME(Raw file name)=20241008_DOE_HSS_0017_neg_FS_WT37b_r1.d
SUBJECT_SAMPLE_FACTORS           	-	WT37c	Sample source:Cyanobacteria | Genotype:Wild-type | Treatment:control	RAW_FILE_NAME(Raw file name)=20241008_DOE_HSS_0027_neg_FS_WT37c_r1.d
SUBJECT_SAMPLE_FACTORS           	-	WT37d	Sample source:Cyanobacteria | Genotype:Wild-type | Treatment:control	RAW_FILE_NAME(Raw file name)=20241008_DOE_HSS_0035_neg_FS_WT37d_r1.d
SUBJECT_SAMPLE_FACTORS           	-	WT30a	Sample source:Cyanobacteria | Genotype:Wild-type | Treatment:Low-tempeature	RAW_FILE_NAME(Raw file name)=20241008_DOE_HSS_0010_neg_FS_WT30a_r1.d
SUBJECT_SAMPLE_FACTORS           	-	WT30b	Sample source:Cyanobacteria | Genotype:Wild-type | Treatment:Low-tempeature	RAW_FILE_NAME(Raw file name)=20241008_DOE_HSS_0015_neg_FS_WT30b_r1.d
SUBJECT_SAMPLE_FACTORS           	-	WT30c	Sample source:Cyanobacteria | Genotype:Wild-type | Treatment:Low-tempeature	RAW_FILE_NAME(Raw file name)=20241008_DOE_HSS_0029_neg_FS_WT30c_r1.d
SUBJECT_SAMPLE_FACTORS           	-	WT30d	Sample source:Cyanobacteria | Genotype:Wild-type | Treatment:Low-tempeature	RAW_FILE_NAME(Raw file name)=20241008_DOE_HSS_0032_neg_FS_WT30d_r1.d
#COLLECTION
CO:COLLECTION_SUMMARY            	The wild-type and aas-knockout strains of Picosynechococcus sp. PCC 7002 were
CO:COLLECTION_SUMMARY            	cultivated in A+ media at 30°C and 37°C with shaking at 130 rpm, 1% carbon
CO:COLLECTION_SUMMARY            	dioxide, 300 micromolar/square meter/second until reaching mid-log phase (OD730
CO:COLLECTION_SUMMARY            	of 0.6-0.7). 60 mL cultures were quenched with 90 mL of cold 30% MeOH/water
CO:COLLECTION_SUMMARY            	(-20°C). Then the cell pellets were collected by centrifugation at 4,000g for
CO:COLLECTION_SUMMARY            	10 min at -4°C, the supernatant was decanted, and the pellets were snap frozen
CO:COLLECTION_SUMMARY            	with liquid Nitrogen, lyophilized, and transferred into 2mL tubes and stored at
CO:COLLECTION_SUMMARY            	-80°C until undergoing sample preparation
CO:SAMPLE_TYPE                   	Bacterial cells
#TREATMENT
TR:TREATMENT_SUMMARY             	The two treatments/factors involved with this study were genotype and growth
TR:TREATMENT_SUMMARY             	temperature. The two strains investigated were WT PCC 7002 and aas-knockout PCC
TR:TREATMENT_SUMMARY             	7002. Each strain was cultured at 30°C and 37°C.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	The lyophilized Synechococcus sp. PCC 7002 pellets were resuspended in 2 mL of
SP:SAMPLEPREP_SUMMARY            	-20°C methanol. Portions of each sample were normalized based on OD730
SP:SAMPLEPREP_SUMMARY            	measurements to a volume of 100 µL before adding 800 µL of cold MeOH. The
SP:SAMPLEPREP_SUMMARY            	samples were then incubated at -80°C overnight to facilitate protein
SP:SAMPLEPREP_SUMMARY            	precipitation. These samples were centrifuged for 10 minutes at 1000 x g (4 ˚C)
SP:SAMPLEPREP_SUMMARY            	before transferring the supernatant and drying it in vacuo. Dried samples were
SP:SAMPLEPREP_SUMMARY            	resuspended in 100 µL water, vortexed thoroughly, followed by 100 µL MeOH, and
SP:SAMPLEPREP_SUMMARY            	incubated at room temperature for 10 minutes. An isotopically labeled lipid
SP:SAMPLEPREP_SUMMARY            	mixture (SPLASH LIPIDOMIX, Avanti) was added for QA/QC purposes. A liquid-liquid
SP:SAMPLEPREP_SUMMARY            	extraction was performed by adding 800 µL MTBE, vortexing, and subsequently
SP:SAMPLEPREP_SUMMARY            	incubating on ice for 10 mins. After incubation, samples were centrifuged for 10
SP:SAMPLEPREP_SUMMARY            	minutes at 1000 x g and 4°C. The nonpolar, MTBE layer containing the
SP:SAMPLEPREP_SUMMARY            	lipophilic components was removed and dried under vacuum. These fractions were
SP:SAMPLEPREP_SUMMARY            	resuspended for HPLC-MS analysis in 100 µL 50:50 IPA:MeOH containing 40 µg/mL
SP:SAMPLEPREP_SUMMARY            	heptadecanoic acid and nonadecanoic acid, as well as 10 µg/mL glucosyl(β)
SP:SAMPLEPREP_SUMMARY            	sphingosine and N-heptadecanoyl-D-erythrosphingosine.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Agilent 1290 Infinity
CH:COLUMN_NAME                   	Thermo Hypersil GOLD AQ (100 x 2.1 mm, 1.9 µm)
CH:SOLVENT_A                     	90% Water/10% Methanol; 10 mM Ammonium acetate
CH:SOLVENT_B                     	50% Isopropyl alcohol/30% Methanol/20% Acetonitrile 
CH:FLOW_GRADIENT                 	70-30% B in 0.5 min, 30-70% B in 1.5 min, 70-100% B in 13 min, 100% B for 6 min,
CH:FLOW_GRADIENT                 	100-10% B in 1 min, 10% B for 2 min, 10-30% B in 1 min, and 30-70% B in 5 mins,
CH:FLOW_GRADIENT                 	followed by a 4-minute post-injection period for column equilibration
CH:FLOW_RATE                     	0.25 mL/min
CH:COLUMN_TEMPERATURE            	40°C
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Agilent 6560 Ion Mobility
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	Mass spectrometer was operated in negative ion polarity with the following
MS:MS_COMMENTS                   	parameters: gas temperature, 280°C; drying gas, 5 L/min; nebulizer, 20 psi;
MS:MS_COMMENTS                   	sheath gas temperature, 300°C; sheath gas flow, 11.8 L/min; capillary voltage
MS:MS_COMMENTS                   	(VCap), 3500 V; nozzle voltage, 2000 V; fragmentor, 320 V; and octopole RF Vpp,
MS:MS_COMMENTS                   	750 V
MS:MS_RESULTS_FILE               	ST003933_AN006458_Results.txt	UNITS:m/z	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END