Production of C6-C14 Medium-Chain Fatty Acids in Seeds and Leaves via Overexpression of Single Hotdog-Fold Acyl-Lipid Thioesterases.

ACYL-LIPID THIOESTERASES (ALT) are a type of plant acyl-acyl carrier protein thioesterase that generate a wide range of medium-chain fatty acids and methylketone (MK) precursors when expressed heterologously in Escherichia coli. While this makes ALT-type thioesterases attractive as metabolic engineering targets to increase production of high-value medium-chain fatty acids and MKs in plant systems, the behavior of ALT enzymes in planta was not well understood before this study. To profile the substrate specificities of ALT-type thioesterases in different plant tissue types, AtALT1-4 from Arabidopsis thaliana, which have widely varied chain length and oxidation state preferences in E. coli, were overexpressed in Arabidopsis seeds, Camelina sativa seeds, and Nicotiana benthamiana leaves. Seed-specific overexpression of ALT enzymes led to medium-chain fatty acid accumulation in Arabidopsis and Camelina seed triacylglycerols, and transient overexpression in N. benthamiana demonstrated that the substrate preferences of ALT-type thioesterases in planta generally agree with those previously determined in E. coli. AtALT1 and AtALT4 overexpression in leaves and seeds resulted in the accumulation of 12-14 carbon-length fatty acids and 6-8 carbon-length fatty acids, respectively. While it was difficult to completely profile the products of ALT-type thioesterases that generate MK precursors (i.e. β-keto fatty acids), our results nonetheless demonstrate that ALT enzymes are catalytically diverse in planta. The knowledge gained from this study is a significant step towards being able to use ALT-type thioesterases as metabolic engineering tools to modify the fatty acid profiles of oilseed crops, other plants, and microorganisms.