TY - JOUR
T1 - Metabolic phenotyping of the cyanobacterium Synechocystis 6803 engineered for production of alkanes and free fatty acids
AU - Hu, Ping
AU - Borglin, Sharon
AU - Kamennaya, Nina A.
AU - Chen, Liang
AU - Park, Hanwool
AU - Mahoney, Laura
AU - Kijac, Aleksandra
AU - Shan, George
AU - Chavarría, Krystle L.
AU - Zhang, Chunmin
AU - Quinn, Nigel W.T.
AU - Wemmer, David
AU - Holman, Hoi Ying
AU - Jansson, Christer
N1 - Funding Information: This work was supported by the US Department of Energy Office of Biological and Environmental Research’s Structural Biology (BSISB) Program through contract DE-AC02-05CH11231 with Lawrence Berkeley National Laboratory. The SR-FTIR spectromicroscopy work was conducted at the infrared beamline at the Advanced Light Source, which is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the US Department of Energy. Funding from a Laboratory Directed Research and Development (LDRD) grant (CyanoAlkanes) to C.J. is acknowledged.
PY - 2013/1/1
Y1 - 2013/1/1
N2 - We demonstrate a simple high-throughput single-cell approach that exploits the ultrahigh brightness and non-invasive nature of synchrotron infrared beam to characterize strains of the cyanobacterium Synechocystis 6803 (S. 6803) constructed with altered metabolic traits affecting the acyl-CoA pool. Their metabolic responses to the modified traits were phenotyped by single-cell synchrotron radiation Fourier transform infrared (SR-FTIR) spectromicroscopy and multivariate analysis. SR-FTIR difference spectra and cluster vector plots segregated the strains as phenotypic populations based on signals in the hydrocarbon and biomolecular fingerprint regions, although each population incorporated a stochastic distribution of cells with different metabolic properties. All engineered strains exhibited an increase in FTIR features attributed to functional groups in hydrocarbon, fatty acid (FA), and/or FA ester chains, and a decrease in polysaccharide features. The metabolic signatures obtained by SR-FTIR were consistent with detailed qualitative and quantitative metabolic information provided in GC/MS/NMR data. A strain with extra copies of the FAR and FAD genes, encoding, respectively, the fatty acyl-ACP reductase and fatty aldehyde decarbonylase enzymes in the alkane biosynthesis pathway, showed up to a fivefold increase in the intracellular levels of heptadecane, a threefold increase in 9-heptadecene, and a significant increase in secreted 16:0 and 18:0 free FAs (FFAs). Inactivation of the AAS gene, encoding acyl-ACP synthetase, prevented re-thioesterification of FFAs generated from membrane lipid recycling and led to elevated levels and of intracellular FFAs of an altered composition, and a decrease in heptadecane and secreted FFAs. Introduction of a FatB gene, encoding a thioesterase (TE), which catalyzes the liberation of FFAs from acyl-ACP, yielded little effect in itself. However, the activity of the TE enzyme was clearly manifested in combination with AAS inactivation; A TE-containing train lacking AAS showed a dramatic (30-fold) increase in intracellular FFAs (with the majority being 16:0) and increases in heptadecane and secreted FFAs.
AB - We demonstrate a simple high-throughput single-cell approach that exploits the ultrahigh brightness and non-invasive nature of synchrotron infrared beam to characterize strains of the cyanobacterium Synechocystis 6803 (S. 6803) constructed with altered metabolic traits affecting the acyl-CoA pool. Their metabolic responses to the modified traits were phenotyped by single-cell synchrotron radiation Fourier transform infrared (SR-FTIR) spectromicroscopy and multivariate analysis. SR-FTIR difference spectra and cluster vector plots segregated the strains as phenotypic populations based on signals in the hydrocarbon and biomolecular fingerprint regions, although each population incorporated a stochastic distribution of cells with different metabolic properties. All engineered strains exhibited an increase in FTIR features attributed to functional groups in hydrocarbon, fatty acid (FA), and/or FA ester chains, and a decrease in polysaccharide features. The metabolic signatures obtained by SR-FTIR were consistent with detailed qualitative and quantitative metabolic information provided in GC/MS/NMR data. A strain with extra copies of the FAR and FAD genes, encoding, respectively, the fatty acyl-ACP reductase and fatty aldehyde decarbonylase enzymes in the alkane biosynthesis pathway, showed up to a fivefold increase in the intracellular levels of heptadecane, a threefold increase in 9-heptadecene, and a significant increase in secreted 16:0 and 18:0 free FAs (FFAs). Inactivation of the AAS gene, encoding acyl-ACP synthetase, prevented re-thioesterification of FFAs generated from membrane lipid recycling and led to elevated levels and of intracellular FFAs of an altered composition, and a decrease in heptadecane and secreted FFAs. Introduction of a FatB gene, encoding a thioesterase (TE), which catalyzes the liberation of FFAs from acyl-ACP, yielded little effect in itself. However, the activity of the TE enzyme was clearly manifested in combination with AAS inactivation; A TE-containing train lacking AAS showed a dramatic (30-fold) increase in intracellular FFAs (with the majority being 16:0) and increases in heptadecane and secreted FFAs.
KW - Alkanes
KW - Cyanobacteria
KW - FTIR
KW - Fatty acids
KW - Metabolic engineering
KW - Metabolic phenotyping
KW - Synechocystis 6803
UR - http://www.scopus.com/inward/record.url?scp=84870742918&partnerID=8YFLogxK
U2 - https://doi.org/10.1016/j.apenergy.2012.08.047
DO - https://doi.org/10.1016/j.apenergy.2012.08.047
M3 - Article
SN - 0306-2619
VL - 102
SP - 850
EP - 859
JO - Applied Energy
JF - Applied Energy
ER -
