Effect of a mutagenized acyl-ACP thioesterase FATA allele from sunflower with improved activity in tobacco leaves and Arabidopsis seeds

Abstract

Acyl-acyl carrier protein (ACP) thioesterases hydrolyze plastid-localised acyl-ACP intermediates and make possible the export of acyl moieties to the cytosol for their incorporation into glycerolipids. The substrate specificity of the acyl-ACP thioesterases importantly determine the type of fatty acids that are exported from plastids. Thus, designing acyl-ACP thioesterases with different substrate specificities or kinetic properties would be of interest for plant lipid biotechnology to produce oils enriched in specialty fatty acids. Although they have not been yet crystallized, their tertiary structures have been modelled by comparison with other enzymes catalyzing similar reactions, suggesting candidate amino acid residues involved in substrate specificity. In the present work, the FatA thioesterase from Helianthus annuus was used to test the impact of changes in the amino acids present in the binding pocket on substrate specificity and catalytic efficiency. Amongst all the mutated enzymes studied, Q215W was especially interesting as it had higher specificity towards saturated acyl-ACP substrates and higher catalytic efficiency compared to wild type H. annuus FatA. Null, wild type and high-efficiency alleles were transiently expressed in tobacco leaves to check their effect on lipid biosynthesis. Expression of active FatA thioesterases altered the composition of leaf triacylglycerols but did not altered total lipid content. However, the expression of the wild type and the high efficiency alleles in Arabidopsis thaliana transgenic seeds resulted in a strong reduction in oil content and an increase in total saturated fatty acid content. The role and influence of acyl-ACP thioesterases in plant metabolism and their possible applications in lipid biotechnology are discussed.

New Insights Into Sunflower (Helianthus annuus L.) FatA and FatB Thioesterases, Their Regulation, Structure and Distribution

“…Highly conserved catalytic amino acids were also present in the C-terminal domain of the protein. These models were applied to both the FatA and Fat B thioesterases, allowing amino acids involved in the enzyme-substrate interactions to be identified and making it possible to design thioesterase alleles with improved catalytic properties ( Mayer and Shanklin, 2007 ; Moreno-Pérez et al, 2014 ). More recent studies determined the crystal structure of FatB1 from Umbellularia californica ( Feng et al, 2017 ), a thioesterase that specifically acts on C12 substrates.…”

Lipid Metabolism in Plants

“…Altered thioesterase expression has therefore been a popular tool for altering the ratio of saturated to unsaturated fatty acids in oilseeds (Moreno-Pérez et al, 2014). However, plants must also release large quantities of fatty acid from ACP to accommodate membrane lipid assembly outside the plastids.…”

“…Plants with unusual acyl-ACP desaturases may also express unusual thioesterases (Moreno-Pérez et al, 2014). For example, macadamia nut and coriander, whose seed oils accumulate Δ 9 -16:1 and Δ 6 -18:1 respectively, have acyl-ACP thioesterases with corresponding activities.…”

Biochemistry of high stearic sunflower, a new source of saturated fats

“…In this regard, a study based on site-directed mutagenesis of different residues located in HaFatA was carried out to try to produce a new allele with higher activity and/or specificity towards stearoyl-ACP [40]. Thus, several amino acids of the active site were substituted with the bulky and hydrophobic amino acid tryptophan.…”