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 FM (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. (c) 2012 Published by Elsevier Ltd.