Acyl-ACP thioesterases from Camelina sativa: Cloning, enzymatic characterization and implication in seed oil fatty acid composition

Abstract

Acyl-acyl carrier protein (ACP) thioesterases are intraplastidial enzymes that terminate de novo fatty acid biosynthesis in the plastids of higher plants by hydrolyzing the thioester bond between ACP and the fatty acid synthesized. Free fatty acids are then esterified with coenzyme A prior to being incorporated into glycerolipids. Acyl-ACP thioesterases are classified into two families, FatA and FatB, which differ in their amino acid sequence and substrate specificity. Here, the FatA and FatB thioesterases from Camelina sativa seeds, a crop of interest in plant biotechnology, were cloned, sequenced and characterized. The mature proteins encoded by these genes were characterized biochemically after they were heterologously expressed in Escherichia coli and purified. Camelina sativa contained three different alleles of both the FatA and FatB genes. These genes were expressed most strongly in expanding tissues in which lipids are very actively synthesized, such as developing seed endosperm. The CsFatA enzyme displayed high catalytic efficiency on oleoyl-ACP and CsFatB acted efficiently on palmitoyl-ACP. The contribution of these two enzymes to the synthesis of Camelina sativa oil was discussed in the light of these results.

Genome-Wide Association Study of Genetic Control of Seed Fatty Acid Biosynthesis in Brassica napus

“…The analysis of natural genetic variation in FA biosynthesis in B. oleracea revealed that the activity of the FATB enzyme was associated with a QTL on chromosome C5 (Barker et al, 2007). As a major determinant of fatty acid composition in seeds, FAT enzymes were also extensively studied and genetically modified in various oil crops, including Ricinus communis, Macadamia tetraphylla , and Camelina sativa (Sánchez-García et al, 2010; Moreno-Pérez et al, 2011; Rodríguez-Rodríguez et al, 2014; Kim et al, 2015). A search of the B. napus Darmor reference genome using the nucleotide sequence of our GWAS associated FATB gene identified three homolog, which share 100% nucleotide sequence identity with FATB on chromosomes A03 (BnaA03g47660D), C09 (BnaC09g30860D), and 99.87% identity on chromosome A07 (BnaA07g08340D).…”

Thioesterase overexpression in Nicotiana benthamiana leaf increases the fatty acid flux into triacylgycerol

“…Overexpression of specific thioesterases has been demonstrated to be effective for modifying the profile of bioengineered oil [6]. The specificity of the acyl-ACP thioesterases is important in defining the FA profile, and thus, these enzymes are relevant targets to manipulate the FA composition of seed oils [10]. The composition of FAs exported from the plastid in different plants, or different tissues within a plant, is dependent on the relative activities of the acyl-ACP thioesterases and acyl-ACP desaturases [31,32] to produce various saturated and monounsaturated FAs [33].…”

“…FATA orthologues possess similar substrate specificities among different species, with high activity upon 18 : 1 D9 -ACP substrate [8,9]. FATB enzymes are further classified into two subclasses: FATB1, with preference towards saturated acyl-ACPs, especially 16 : 0-ACP, and FATB2 with preference towards short/medium-chain saturated acyl-ACPs [10]. Therefore, acyl-ACP thioesterases are key enzymes in determining which FAs are exported to the cytosol and subsequently incorporated into glycerolipids synthesized in the ER, including triacylglycerol (TAG) [11].…”

Metabolic Engineering a Model Oilseed Camelina sativa for the Sustainable Production of High-Value Designed Oils

“…It is hypothesized that the coexpression of these three enzyme genes with RcFAH12 in camelina seed will allow the amount of hydroxylated TAG to accumulate to levels appropriate for commercial use. Since fatty acid thioesterase A (FatA) and fatty acid thioesterase B (FatB) in camelina plastid were identified to be specific for oleoyl-ACP and palmitoleic acid-ACP, respectively (Rodriguez-Rodriguez et al, 2014), in the future, the molecular manipulation of these two enzymes as targets will allow cells to selectively accumulate oleic acid (18:1D9) or palmitoleic acid (16:1D9) in TAGs and increase the resistance to oxidation of camelina seed oil.…”