Comparative expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> of the native and hybrid genes for acyl-lipid Δ<Superscript>9</Superscript> desaturase

Expression of the desC gene coding for acyl-lipid Δ⁹ desaturase of thermophilic cyanobacterium Synechocystis sp. PCC6803 was studied in Escherichia coli cells. In a hybrid gene constructed (desC-licBM3), a sequence of the native acyl-lipid Δ⁹ desaturase was fused in frame with the reporter gene coding for thermostable lichenase. Lichenase contained in the hybrid protein simplified selection and analysis of the expression of membrane desaturase in the heterologous host. Comparisons of the expression for the native and hybrid genes in bacterial cells showed that lichenase remained active and thermostable in the hybrid protein, while desaturase retains the capability of introducing a double bound in the corresponding position of fatty acid residues.     

Stearoyl-CoA desaturase and insulin signaling — What is the molecular switch?

Increasing evidence suggests that stearoyl-CoA desaturase (SCD), the rate-limiting enzyme of monounsaturated fatty acid biosynthesis, is an important factor in the pathogenesis of lipid-induced insulin resistance. Mice with a targeted disruption of the SCD1 gene have improved glucose tolerance compared to wild-type mice, despite lower fasting plasma insulin levels. Increased SCD activity has been found in insulin-resistant humans and animals, whereas SCD1 deficiency attenuates both diet- and genetically-induced impairment of insulin action. Phosphorylation of serine and threonine residues on insulin receptor, insulin receptor substrates (IRS1 and IRS2), and on Akt has been shown to be the major step in insulin signaling that is altered due to the lack of SCD1. In this review we discuss perturbations in cell signaling and lipid metabolism cascades in insulin-sensitive tissues due to SCD1 deficiency. In particular, we address the role of cellular signaling molecules including free fatty acids, ceramides, fatty acyl-CoAs, AMP-activated protein kinase, protein tyrosine phosphatase 1B as well as of membrane fluidity. While the precise mechanism of SCD action on insulin signaling remains to be clarified, current findings on SCD point to a very promising novel target for the treatment of insulin resistance.     

Identification and characterization of a novel bifunctional Δ<Superscript>12</Superscript>/Δ<Superscript>15</Superscript>-fatty acid desaturase gene from <Emphasis Type="Italic">Rhodosporidium kratochvilovae</Emphasis>

Objectives

To elucidate the biosynthesis pathway of linoleic acid and α-linolenic acid in Rhodosporidium kratochvilovae YM25235 and investigate the correlation of polyunsaturated fatty acids with its cold adaptation.

Results

A 1341 bp cDNA sequence, designated as RKD12, putatively encoding a Δ¹²-desaturase was isolated from YM25235. Sequence analysis indicated that this sequence comprised a complete ORF encoding 446 amino acids of 50.6 kDa. The encoded amino acid sequence shared higher similarity to known fungal Δ¹²-desaturases that are characteristic of three conserved histidine-rich motifs. RKD12 was further transformed into Saccharomyces cerevisiae INVScl for functional characterization. Fatty acid analysis showed the yeast transformants accumulated two new fatty acids: linoleic acid and α-linolenic acid. Furthermore, mRNA expression level of RKD12 and the content of linoleic acid and α-linolenic acid were increased significantly with the culture temperature downshift from 30 to 15 °C, which might be helpful for the cold adaptation of YM25235.

Conclusion

RKD12 is a novel bifunctional ?¹²/?¹⁵-desaturase gene, and the increased RKD12 mRNA expression level and PUFAs content at low temperature might be helpful for the cold adaptation of YM25235.

     

Modulation of palmitate-induced endoplasmic reticulum stress and apoptosis in pancreatic β-cells by stearoyl-CoA desaturase and Elovl6

Induction of endoplasmic reticulum (ER) stress and apoptosis by elevated exogenous saturated fatty acids (FAs) plays a role in the pathogenesis of β-cell dysfunction and loss of islet mass in type 2 diabetes. Regulation of monounsaturated FA (MUFA) synthesis through FA desaturases and elongases may alter the susceptibility of β-cells to saturated FA-induced ER stress and apoptosis. Herein, stearoyl-CoA desaturase (SCD)1 and SCD2 mRNA expression were shown to be induced in islets from prediabetic hyperinsulinemic Zucker diabetic fatty (ZDF) rats, whereas SCD1, SCD2, and fatty acid elongase 6 (Elovl6) mRNA levels were markedly reduced in diabetic ZDF rat islets. Knockdown of SCD in INS-1 β-cells decreased desaturation of palmitate to MUFA, lowered FA partitioning into complex neutral lipids, and increased palmitate-induced ER stress and apoptosis. Overexpression of SCD2 increased desaturation of palmitate to MUFA and attenuated palmitate-induced ER stress and apoptosis. Knockdown of Elovl6 limited palmitate elongation to stearate, increasing palmitoleate production and attenuating palmitate-induced ER stress and apoptosis, whereas overexpression of Elovl6 increased palmitate elongation to stearate and palmitate-induced ER stress and apoptosis. Overall, these data support the hypothesis that enhanced MUFA synthesis via upregulation of SCD2 activity can protect β-cells from elevated saturated FAs, as occurs in prediabetic states. Overt type 2 diabetes is associated with diminished islet expression of SCD and Elovl6, and this can disrupt desaturation of saturated FAs to MUFAs, rendering β-cells more susceptible to saturated FA-induced ER stress and apoptosis.     

QTL and candidate genes phytoene synthase and ζ-carotene desaturase associated with the accumulation of carotenoids in maize

Carotenoids are a class of fat-soluble antioxidant vitamin compounds present in maize (Zea mays L.) that may provide health benefits to animals or humans. Four carotenoid compounds are predominant in maize grain: -carotene, -cryptoxanthin, zeaxanthin, and lutein. Although -carotene has the highest pro-vitamin A activity, it is present in a relatively low concentration in maize kernels. We set out to identify quantitative trait loci (QTL) affecting carotenoid accumulation in maize kernels. Two sets of segregating families were evaluated—a set of F_2:3 lines derived from a cross of W64a x A632, and their testcross progeny with AE335. Molecular markers were evaluated on the F_2:3 lines and a genetic linkage map created. High-performance liquid chromatography was performed to measure -carotene, -cryptoxanthin, zeaxanthin, and lutein on both sets of materials. Composite interval mapping identified chromosome regions with QTL for one or more individual carotenoids in the per se and testcross progenies. Notably QTL in the per se population map to regions with candidate genes, yellow 1 and viviparous 9, which may be responsible for quantitative variation in carotenoids. The yellow 1 gene maps to chromosome six and is associated with phytoene synthase, the enzyme catalyzing the first dedicated step in the carotenoid biosynthetic pathway. The viviparous 9 gene maps to chromosome seven and is associated with -carotene desaturase, an enzyme catalyzing an early step in the carotenoid biosynthetic pathway. If the QTL identified in this study are confirmed, particularly those associated with candidates genes, they could be used in an efficient marker-assisted selection program to facilitate increasing levels of carotenoids in maize grain.Communicated by P. Langridge     

Co-expression of the borage Δ6 desaturase and the Arabidopsis Δ15 desaturase results in high accumulation of stearidonic acid in the seeds of transgenic soybean

Two relatively rare fatty acids, γ-linolenic acid (GLA) and stearidonic acid (STA), have attracted much interest due to their nutraceutical and pharmaceutical potential. STA, in particular, has been considered a valuable alternative source for omega-3 fatty acids due to its enhanced conversion efficiency in animals to eicosapentaenoic acid when compared with the more widely consumed omega-3 fatty acid, α-linolenic acid (ALA), present in most vegetable oils. Exploiting the wealth of information currently available on in planta oil biosynthesis and coupling this information with the tool of genetic engineering it is now feasible to deliberately perturb fatty acid pools to generate unique oils in commodity crops. In an attempt to maximize the STA content of soybean oil, a borage Δ⁶ desaturase and an Arabidopsis Δ¹⁵ desaturase were pyramided by either sexual crossing of transgenic events, re-transformation of a Δ⁶ desaturase event with the Δ¹⁵ desaturase or co-transformation of both desaturases. Expression of both desaturases in this study was under the control of the seed-specific soybean β-conglycinin promoter. Soybean events that carried only the Δ¹⁵ desaturase possessed a significant elevation of ALA content, while events with both desaturases displayed a relative STA abundance greater than 29%, creating a soybean with omega-3 fatty acids representing over 60% of the fatty acid profile. Analyses of the membrane lipids in a subset of the transgenic events suggest that soybean seeds compensate for enhanced production of polyunsaturated fatty acids by increasing the relative content of palmitic acid in phosphatidylcholine and other phospholipids.     

Effect of the <Emphasis Type="Italic">desA</Emphasis> gene encoding Δ12 acyl-lipid desaturase on the chloroplast structure and tolerance to hypothermia of potato plants

Effects of the desA gene from the cyanobacterium Synechocystis sp. encoding Δ12 acyl-lipid desaturase and increasing the level of unsaturated fatty acids (linoleic acid (18:2) primarily) in membrane lipids, which was inserted into potato (Solanum tuberosum L., cv. Desnitsa) plants, on chloroplast ultrastructure and plant tolerance to low temperatures were studied. The main attention was focused on modifications in the chloroplast structure and their possible relation to potato plant tolerance to oxidative and low-temperature stresses under the influence to their transformation with the Δ12 acyl-lipid desaturase gene from cyanobacterium (desA-licBM3-plants). Morphometric analysis showed that, in comparison with wild-type (WT) plants, in desA-licBM3-plants the number of grana in chloroplasts increased substantially. The total number of thylakoids in transformant chloroplasts was almost twice higher than in WT plants. The number of plastoglobules per chloroplast of transformed plants increased by 25%. A marked increase in the number of grana, total number of thylakoids, and the number of plastoglobules in chloroplasts of desA-licBM3-plants indicates their more intense lipid metabolism, as compared with WT plants, and this resulted in the conservation of some part of lipids in plastoglobules. In addition, the expression of heterological desA gene encoding Δ12 acyl-lipid desaturase positively influenced stabilization of not only structure but also functioning of chloroplast membranes, thus preventing a transfer of electrons from the ETR to oxygen and subsequent ROS generation at hypothermia. This was confirmed by the analysis of the rate of superoxide anion generation in tested genotypes.     

A modified <Emphasis Type="Italic">n</Emphasis>–3 fatty acid desaturase gene from <Emphasis Type="Italic">Caenorhabditis briggsae</Emphasis> produced high proportion of DHA and DPA in transgenic mice

The functions of polyunsaturated fatty acids (PUFAs) have been widely investigated. In mammals, levels of n-3 PUFAs are relatively low compared to those of n-6 PUFAs. Either a lack of n-3 PUFAs or an excess of n-6 PUFAs could potentially cause health problems in humans. Hence, methods to increase the amount of n-3 PUFAs in diet have been intensely sought. In this study, we demonstrated that the n-3 fatty acid desaturase gene (sFat-1) synthesized from revised and optimized codons based on roundworm Caenorhabditis briggsae genomic gene for enhanced expression in mammals was successfully expressed in Chinese hamster ovary (CHO) cells and significantly elevated cellular n-3 PUFA contents. We generated sFat-1 transgenic mice by introducing mammal expression vector DNAs containing the sFat-1 gene into regular mice through the method of microinjection. Fatty acid compositions were then altered and the levels of docosahexaenoic acid (DHA, 22:6n-3) and docosapentaenoic acid (DPA, 22:5n-3) were greatly increased in these transgenic mice. Various types of tissues in the transgenic mice produced many types of n-3 PUFAs, such as alpha-linolenic acid (ALA; 18:3n-3), eicosapentaenoic acid (EPA, 20:5n-3), DPA, and DHA, for example, muscle tissues of the transgenic mice contained 12.2% DHA, 2.0% DPA, and 23.1% total n-3 PUFAs. These research results demonstrated that the synthesized sFat-1 gene with modified and optimized codons from C. briggsae possess functional activity and greater capability of producing n-3 PUFAs, especially DHA and DPA, in transgenic mice.     

Molecular cloning of a Pinguiochrysis pyriformis oleate-specific microsomal Δ12-fatty acid desaturase and functional analysis in yeasts and thraustochytrids

We isolated a putative desaturase gene from a marine alga, Pinguiochrysis pyriformis MBIC 10872, which is capable of accumulating eicosapentaenoic acid (C20:5(Δ5,8,11,14,17)). The gene possessed an open reading frame of 1,314 bp encoding a putative 437 amino acid residues showing high sequence identity (37-48%) with fungal and nematode Δ12-fatty acid desaturases. Yeast cells transformed with the gene converted endogenous oleic acid (C18:1(Δ9)) to linoleic acid (C18:2(Δ9,12)). However, no double bonds were introduced into other endogenous fatty acids or exogenously added fatty acids. Flag-tagged enzyme was recovered in the micosome fraction when expressed in yeast cells. To express the gene in thraustochytrids, a construct driven by the thraustochytrid-derived ubiquitin promoter was used. Interestingly, exogenously added oleic acid was converted to linoleic acid in the gene transformants but not mock transformants of Aurantiochytrium limacinum mh0186. These results clearly indicate that the gene encodes a microsomal Δ12-fatty acid desaturase and was expressed functionally in not only yeasts but also thraustochytrids. This is the first report describing the heterozygous expression of a fatty acid desaturase in thraustochytrids, and could facilitate a genetic approach towards fatty acid synthesis in thraustochytrids which are expected to be an alternative source of polyunsaturated fatty acids.