Toward production of jet fuel functionality in oilseeds: identification of FatB acyl-acyl carrier protein thioesterases and evaluation of combinatorial expression strategies in Camelina seeds

Abstract

Seeds of members of the genus Cuphea accumulate medium-chain fatty acids (MCFAs; 8:0-14:0). MCFA- and palmitic acid- (16:0) rich vegetable oils have received attention for jet fuel production, given their similarity in chain length to Jet A fuel hydrocarbons. Studies were conducted to test genes, including those from Cuphea, for their ability to confer jet fuel-type fatty acid accumulation in seed oil of the emerging biofuel crop Camelina sativa. Transcriptomes from Cuphea viscosissima and Cuphea pulcherrima developing seeds that accumulate >90% of C8 and C10 fatty acids revealed three FatB cDNAs (CpuFatB3, CvFatB1, and CpuFatB4) expressed predominantly in seeds and structurally divergent from typical FatB thioesterases that release 16:0 from acyl carrier protein (ACP). Expression of CpuFatB3 and CvFatB1 resulted in Camelina oil with capric acid (10:0), and CpuFatB4 expression conferred myristic acid (14:0) production and increased 16:0. Co-expression of combinations of previously characterized Cuphea and California bay FatBs produced Camelina oils with mixtures of C8-C16 fatty acids, but amounts of each fatty acid were less than obtained by expression of individual FatB cDNAs. Increases in lauric acid (12:0) and 14:0, but not 10:0, in Camelina oil and at the sn-2 position of triacylglycerols resulted from inclusion of a coconut lysophosphatidic acid acyltransferase specialized for MCFAs. RNA interference (RNAi) suppression of Camelina β-ketoacyl-ACP synthase II, however, reduced 12:0 in seeds expressing a 12:0-ACP-specific FatB. Camelina lines presented here provide platforms for additional metabolic engineering targeting fatty acid synthase and specialized acyltransferases for achieving oils with high levels of jet fuel-type fatty acids.

Keywords: Camelina; Cuphea; FatB acyl-ACP thioesterase; jet fuel oilseed; medium-chain fatty acid..

Fig. 1.

Fatty acid composition of seeds and leaves in Cuphea pulcherrima and Cuphea viscosissima. Fatty acids were extracted from leaves and seeds of C. pulcherrima and C. viscosissima and analysed using gas chromatography. Values are the means ±SD from five biological replicates.

Fig. 2.

Spatial expression of FatB acyl-ACP thioesterases in C. pulcherrima and C. viscosissima. Total RNA was isolated from individual tissues and converted into cDNAs for RT-PCR analyses for evaluation of FatB gene expression in different tissues of C. pulcherrima and C.viscossisima. Cuphea eIF4-a1 and actin genes were used as an internal control for RT-PCR.

Fig. 3.

Phylogenetic tree of Cuphea FatB amino acid sequence clusters. Amino acid sequences of Cuphea FatBs were obtained from the protein database of the National Center for Biotechnology Information (NCBI). A phylogenic tree was built with the MEGA6 software, using the minimum-evolution method with 1000 bootstrap replications. Cuphea calophylla (CcFatB1, ABB71580; CcFatB2, ABB71581), Cuphea wrightii (CwFatB1, AAC49783; CwFatB2, AAC49784), Cuphea viscosissima (CvFatB1, AEM72522; CvFatB2, AEM72523; CvFatB3, AEM72524), Cuphea palustris (CpFatB1, 588563; CpFatB2, 1588564), Cuphea hookeriana (ChFatB2, AAC49269). Based on the literature and results presented herein, the relative substrate preferences of FatBs are indicated (e.g. 10:0 >8:0).

Fig. 4.

Alignment of deduced Cuphea FatB acyl-ACP thioesterases. Identical amino acids are shaded in black; conserved residues are shaded in grey. The triangle indicates the predicted site of the transit peptide.

Fig. 5.

Fatty acid composition of the sn-2 position of seed oil TAG. Fatty acid composition of the sn-2 position of seed oil TAG in plants expressing FatB genes alone or in combination with CnLPAT as determined by lipase digestion-based analyses. The data represent averages of four biological replicates ±SD. (A) CpFatB2 and CpFatB2 with CnLPAT. (B) UcFatB1 and UcFatB1 with CnLPAT. (C) ChFatB2 and ChFatB2 with CnLPAT.

Fig. 6.

Total fatty acid contents of engineered Camelina lines. Total fatty acid contents of seeds from FatB with or without CnLPAT expression were analysed by gas chromatography. The data represents means ±SD with five biological replicates. Asterisks indicate statistical differences compared with the wild type (*P<0.05).

Fig. 7.

Fatty acid composition (weight%) of seed lipids in CsKASII-RNAi Camelina expressing UcFatB1. Fatty acids were extracted from transgenic Camelina seeds and analysed using gas chromatography. Values are the means ±SD of five biological replicates.

Fig. 8.

Acyl-ACP analysis of developing seeds of Cuphea viscosissima and wild-type and transgenic Camelina seeds. Shown in (A) are acyl-ACP pools in developing seeds of C. viscosissima. Shown in (B) are acyl-ACP profiles in wild-type Camelina (WT) and transgenic Camelina seeds expressing UcFATB1, CpuFATB1, and ChFatB2 at 15 DAF. The data are means ±SD of five biological replicates.