Fig. 6

GC-FID chromatograms of secreted lipids from K27(DE3) E. coli strains expressing AtALT4 variants or AtALT3. To create the AtALT4-ABCD chimera, sequence encoding residues 31–36, 64–67, 77–93, and 108–111 of the hot-dog fold thioesterase domain of AtALT4 was exchanged with sequence encoding the identically numbered residues from AtALT3. In addition to the previously listed mutations, residues 94–96 were also replaced with the corresponding sequence from AtALT3 in the AtALT4-ABCDE chimera. β-keto fatty acids produced by ALTs were decarboxylated to methylketones with heat and acid treatment prior to identification and quantification by GC-MS and GC-FID analysis. Chromatograms of authentic fatty acid and methylketone retention standard mixtures, and of secreted lipids from K27(DE3) E. coli harbouring an empty vector, are provided for comparison. Compound identities corresponding to peak numbers are listed below the chromatograms (FA = fully reduced fatty acids, MK = methylketone, 3-OH FA = 3-hydroxy fatty acid)