A Specialized Diacylglycerol Acyltransferase Contributes to the Extreme Medium-Chain Fatty Acid Content of Cuphea Seed Oil.

Seed oils of many Cuphea sp. contain >90% of medium-chain fatty acids, such as decanoic acid (10:0). These seed oils, which are among the most compositionally variant in the plant kingdom, arise from specialized fatty acid biosynthetic enzymes and specialized acyltransferases. These include lysophosphatidic acid acyltransferases (LPAT) and diacylglycerol acyltransferases (DGAT) that are required for successive acylation of medium-chain fatty acids in the sn-2 and sn-3 positions of seed triacylglycerols (TAGs). Here we report the identification of a cDNA for a DGAT1-type enzyme, designated CpuDGAT1, from the transcriptome of C. avigera var pulcherrima developing seeds. Microsomes of camelina (Camelina sativa) seeds engineered for CpuDGAT1 expression displayed DGAT activity with 10:0-CoA and the diacylglycerol didecanoyl, that was approximately 4-fold higher than that in camelina seed microsomes lacking CpuDGAT1. In addition, coexpression in camelina seeds of CpuDGAT1 with a C. viscosissima FatB thioesterase (CvFatB1) that generates 10:0 resulted in TAGs with nearly 15 mol % of 10:0. More strikingly, expression of CpuDGAT1 and CvFatB1 with the previously described CvLPAT2, a 10:0-CoA-specific Cuphea LPAT, increased 10:0 amounts to 25 mol % in camelina seed TAG. These TAGs contained up to 40 mol % 10:0 in the sn-2 position, nearly double the amounts obtained from coexpression of CvFatB1 and CvLPAT2 alone. Although enriched in diacylglycerol, 10:0 was not detected in phosphatidylcholine in these seeds. These findings are consistent with channeling of 10:0 into TAG through the combined activities of specialized LPAT and DGAT activities and demonstrate the biotechnological use of these enzymes to generate 10:0-rich seed oils.

Structurally divergent lysophosphatidic acid acyltransferases with high selectivity for saturated medium chain fatty acids from Cuphea seeds.

Lysophosphatidic acid acyltransferase (LPAT) catalyzes acylation of the sn-2 position on lysophosphatidic acid by an acyl CoA substrate to produce the phosphatidic acid precursor of polar glycerolipids and triacylglycerols (TAGs). In the case of TAGs, this reaction is typically catalyzed by an LPAT2 from microsomal LPAT class A that has high specificity for C18 fatty acids containing Δ9 unsaturation. Because of this specificity, the occurrence of saturated fatty acids in the TAG sn-2 position is infrequent in seed oils. To identify LPATs with variant substrate specificities, deep transcriptomic mining was performed on seeds of two Cuphea species producing TAGs that are highly enriched in saturated C8 and C10 fatty acids. From these analyses, cDNAs for seven previously unreported LPATs were identified, including cDNAs from Cuphea viscosissima (CvLPAT2) and Cuphea avigera var. pulcherrima (CpuLPAT2a) encoding microsomal, seed-specific class A LPAT2s and a cDNA from C. avigera var. pulcherrima (CpuLPATB) encoding a microsomal, seed-specific LPAT from the bacterial-type class B. The activities of these enzymes were characterized in Camelina sativa by seed-specific co-expression with cDNAs for various Cuphea FatB acyl-acyl carrier protein thioesterases (FatB) that produce a variety of saturated medium-chain fatty acids. CvLPAT2 and CpuLPAT2a expression resulted in accumulation of 10:0 fatty acids in the Camelina sativa TAG sn-2 position, indicating a 10:0 CoA specificity that has not been previously described for plant LPATs. CpuLPATB expression generated TAGs with 14:0 at the sn-2 position, but not 10:0. Identification of these LPATs provides tools for understanding the structural basis of LPAT substrate specificity and for generating altered oil functionalities.

A thraustochytrid diacylglycerol acyltransferase 2 with broad substrate specificity strongly increases oleic acid content in engineered Arabidopsis thaliana seeds.

Diacylglycerol acyltransferase (DGAT) catalyses the last step in acyl-CoA-dependent triacylglycerol (TAG) biosynthesis and is an important determinant of cellular oil content and quality. In this study, a gene, designated TaDGAT2, encoding a type 2 DGAT (DGAT2)-related enzyme was identified from the oleaginous marine protist Thraustochytrium aureum. The deduced TaDGAT2 sequence contains a ~460 amino acid domain most closely related to DGAT2s from Dictyostelium sp. (45-50% identity). Recombinant TaDGAT2 restored TAG biosynthesis to the Saccharomyces cerevisiae H1246 TAG-deficient mutant, and microsomes from the complemented mutant displayed DGAT activity with C16 and C18 saturated and unsaturated fatty acyl-CoA and diacylglycerol substrates. To examine its biotechnological potential, TaDGAT2 was expressed under control of a strong seed-specific promoter in wild-type Arabidopsis thaliana and the high linoleic acid fad3fae1 mutant. In both backgrounds, little change was detected in seed oil content, but a striking increase in oleic acid content of seeds was observed. This increase was greatest in fad3fae1 seeds, where relative amounts of oleic acid increased nearly 2-fold to >50% of total fatty acids. In addition, >2-fold increase in oleic acid levels was detected in the triacylglycerol sn-2 position and in the major seed phospholipid phosphatidylcholine. These results suggest that increased seed oleic acid content mediated by TaDGAT2 is influenced in part by the fatty acid composition of host cells and occurs not by enhancing oleic acid content at the TAG sn-3 position directly but by increasing total oleic acid levels in seeds, presumably by limiting flux through phosphatidylcholine-based desaturation reactions.

LC-PUFA from photosynthetic microalgae: occurrence, biosynthesis, and prospects in biotechnology.

Microalgae offer potential for numerous commercial applications, among them the production of long-chain polyunsaturated fatty acids (LC-PUFAs). These valuable fatty acids are important for a variety of nutraceutical and pharmaceutical purposes, and the market for these products is continually growing. An appropriate ratio of LC-PUFA of the ω-3 and ω-6 groups is vital for "healthy" nutrition, and adequate dietary intake has strong health benefits in humans. Microalgae of diverse classes are primary natural producers of LC-PUFA. This mini-review presents an introductory overview of LC-PUFA-related health benefits in humans, describes LC-PUFA occurrence in diverse microalgal classes, depicts the major pathways of their biosynthesis in microalgae, and discusses the prospects for microalgal LC-PUFA production.

Selection of a DGLA-producing mutant of the microalga Parietochloris incisa: I. Identification of mutation site and expression of VLC-PUFA biosynthesis genes.

Chemical mutagenesis of the phototrophic green microalga Parietochloris incisa, producing high amounts of arachidonic acid (ARA), resulted in selection of a mutant deficient in ARA and rich in dihomo-γ-linolenic acid (DGLA) and thus ∆5 desaturase defective. The mutagenesis produced a nonsense mutation in the ∆5 desaturase gene, resulting in alteration of the 62nd codon TGG into a stop codon. Thus, the polypeptide encoded by the mutant ∆5 desaturase gene is severely truncated and biochemically inactive, as was confirmed by heterologous expression in Saccharomyces cerevisiae. The mutation did not affect the oleogenic nature of the strain, and the total fatty acid content in the mutant biomass reached 39%, in comparison to 34% in the wild type, after 14 days of nitrogen starvation; biomass yields amounted to 5.1 and 3.6 g/l, respectively. While in the wild type, DGLA and ARA comprised about 1% and 58% of total fatty acids, respectively; the mutation annulled ARA almost totally but increased the DGLA proportion to 32% only with a corresponding increase in the proportion of oleic acid. Consequently, DGLA comprised 12.3% of dry weight, in comparison to 19.4% ARA in the wild type. The expression profiles of the genes coding enzymes involved in VLC-PUFA biosynthesis, ∆12, ∆6, ∆5 desaturases and ∆6 PUFA elongase, during nitrogen starvation, were compared. The transcript levels of all four genes, which were coordinately up-regulated in the wild type, appeared to be drastically reduced in the mutant, indicating their co-regulated transcription.

Identification and characterization of Delta12, Delta6, and Delta5 Desaturases from the green microalga Parietochloris incisa.

The freshwater microalga Parietochloris incisa accumulates, under nitrogen starvation, large amounts of triacylglycerols containing approximately 60% of the omega6 very long-chain polyunsaturated fatty acid (VLC-PUFA), arachidonic acid. Based on sequence homology, we isolated three cDNA sequences from P. incisa, designated PiDesD12, PiDesD6, PiDesD5. The deduced amino acid sequences of the three genes contained three conserved histidine motifs; the front-end desaturases, PiDes6 and PiDes5, contained a fused N-terminal cytochrome b5 domain. By functional characterization in the yeast Saccharomyces cerevisiae, we confirmed that PiDesD6, PiDesD5 cDNA encode membrane bound desaturases with Delta6, and Delta5 activity, respectively. Both PiDes6 and PiDes5 can indiscriminately desaturate both omega6 and omega3 substrates. A phylogenetic analysis showed that the three genes were homologous to the corresponding desaturases from green microalgae and lower plants that were functionally characterized. Quantitative real-time PCR revealed the concerted expression pattern of all three genes in P. incisa cells subjected to nitrogen starvation, featuring maximum expression level on day 3 of starvation, corresponding to the sharpest increase in the share of arachidonic acid.

Cloning and characterization of the 6 polyunsaturated fatty acid elongase from the green microalga Parietochloris incisa.

The very-long-chain polyunsaturated fatty acid (VLC-PUFA), arachidonic acid (ARA, 20:4omega-6) is a component of neuron tissues such as brain and retina cells and a primary substrate for the biosynthesis of biologically active eicosanoids. The green freshwater microalga Parietochloris incisa (Trebouxiophyceae) has been shown to accumulate an extraordinary high content of ARA-rich triacylglycerols. It was thus interesting to characterize the genes involved in lipid biosynthesis in this alga. We report here the identification of a cDNA encoding for a P. incisa PUFA elongase (PiELO1) and demonstrate that the expression of PiELO1 in yeast Saccharomyces cereviseae confers its elongase activity on C18 6 PUFA. Phylogenetic analysis indicated that PiELO1 is highly similar to functionally characterized 6 PUFA elongase genes from other green algae and lower plants. Quantitative real-time PCR expression studies showed that PiELO1 is upregulated under nitrogen starvation, the condition triggering and enhancing storage oil and ARA accumulation in P. incisa.