Showing papers on "Acyl-CoA published in 2013"

AtABCA9 transporter supplies fatty acids for lipid synthesis to the endoplasmic reticulum

Abstract: Fatty acids, the building blocks of biological lipids, are synthesized in plastids and then transported to the endoplasmic reticulum (ER) for assimilation into specific lipid classes. The mechanism of fatty acid transport from plastids to the ER has not been identified. Here we report that AtABCA9, an ABC transporter in Arabidopsis thaliana, mediates this transport. AtABCA9 was localized to the ER, and atabca9 null mutations reduced seed triacylglycerol (TAG) content by 35% compared with WT. Developing atabca9 seeds incorporated 35% less (14)C-oleoyl-CoA into TAG compared with WT seeds. Furthermore, overexpression of AtABCA9 enhanced TAG deposition by up to 40%. These data strongly support a role for AtABCA9 as a supplier of fatty acid substrates for TAG biosynthesis at the ER during the seed-filling stage. AtABCA9 may be a powerful tool for increasing lipid production in oilseeds.

Phospholipid:diacylglycerol acyltransferase-mediated triacylglycerol biosynthesis is crucial for protection against fatty acid-induced cell death in growing tissues of Arabidopsis.

Abstract: Summary Phospholipid:diacylglycerol acyltransferase (PDAT) and diacylglycerol:acyl CoA acyltransferase play overlapping roles in triacylglycerol (TAG) assembly in Arabidopsis, and are essential for seed and pollen development, but the functional importance of PDAT in vegetative tissues remains largely unknown. Taking advantage of the Arabidopsis tgd1–1 mutant that accumulates oil in vegetative tissues, we demonstrate here that PDAT1 is crucial for TAG biosynthesis in growing tissues. We show that disruption of PDAT1 in the tgd1–1 mutant background causes serious growth retardation, gametophytic defects and premature cell death in developing leaves. Lipid analysis data indicated that knockout of PDAT1 results in increases in the levels of free fatty acids (FFAs) and diacylglycerol. In vivo 14C-acetate labeling experiments showed that, compared with wild-type, tgd1–1 exhibits a 3.8-fold higher rate of fatty acid synthesis (FAS), which is unaffected by disruption or over-expression of PDAT1, indicating a lack of feedback regulation of FAS in tgd1–1. We also show that detached leaves of both pdat1–2 and tgd1–1 pdat1–2 display increased sensitivity to FFA but not to diacylglycerol. Taken together, our results reveal a critical role for PDAT1 in mediating TAG synthesis and thereby protecting against FFA-induced cell death in fast-growing tissues of plants.

Fatty alcohol production in engineered E. coli expressing Marinobacter fatty acyl-CoA reductases

Abstract: Although successful production of fatty alcohols in metabolically engineered Escherichia coli with heterologous expression of fatty acyl-CoA reductase has been reported, low biosynthetic efficiency is still a hurdle to be overcome. In this study, we examined the characteristics of two fatty acyl-CoA reductases encoded by Maqu_2220 and Maqu_2507 genes from Marinobacter aquaeolei VT8 on fatty alcohol production in E. coli. Fatty alcohols with diversified carbon chain length were obtained by co-expressing Maqu_2220 with different carbon chain length-specific acyl-ACP thioesterases. Both fatty acyl-CoA reductases displayed broad substrate specificities for C12-C18 fatty acyl chains in vivo. The optimized mutant strain of E. coli carrying the modified tesA gene and fadD gene from E. coli and Maqu_2220 gene from Marinobacter aquaeolei VT8 produced fatty alcohols at a remarkable level of 1.725 g/L under the fermentation condition.

A Requirement for Fatty Acid Oxidation in the Hormone-Induced Meiotic Maturation of Mouse Oocytes

Abstract: We have previously shown that fatty acid oxidation (FAO) is required for AMP-activated protein kinase (PRKA)-induced maturation in vitro. In the present study, we have further investigated the role of this metabolic pathway in hormone-induced meiotic maturation. Incorporating an assay with 3H-palmitic acid as the substrate, we first examined the effect of PRKA activators on FAO levels. There was a significant stimulation of FAO in cumulus cell-enclosed oocytes (CEO) treated with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and RSVA405. In denuded oocytes (DO), AICAR stimulated FAO only in the presence of carnitine, the molecule that facilitates fatty acyl CoA entry into the mitochondria. The carnitine palmitoyltransferase 1 activator C75 successfully stimulated FAO in CEO. All three of these activators trigger germinal vesicle breakdown. Meiotic resumption induced by follicle-stimulating hormone (FSH) or amphiregulin was completely inhibited by the FAO inhibitors etomoxir, mercaptoacetate, and malonyl CoA. Importantly, FAO was increased in CEO stimulated by FSH and epidermal growth factor, and this increase was blocked by FAO inhibitors. Moreover, compound C, a PRKA inhibitor, prevented the FSH-induced increase in FAO. Both carnitine and palmitic acid augmented hormonal induction of maturation. In a more physiological setting, etomoxir eliminated human chorionic gonadotropin (hCG)-induced maturation in follicle-enclosed oocytes. In addition, CEO and DO from hCG-treated mice displayed an etomoxir-sensitive increase in FAO, indicating that this pathway was stimulated during in vivo meiotic resumption. Taken together, our data indicate that hormone-induced maturation in mice requires a PRKA-dependent increase in FAO.

Targeting of Acyl-CoA synthetase 5 decreases jejunal fatty acid activation with no effect on dietary long-chain fatty acid absorption

Abstract: The absorption of dietary long chain fatty acids (LCFA) largely occurs in the jejunum. LCFA are activated via conjugation with Coenzyme A (CoA), a reaction catalyzed by Acyl-CoA synthetases (ACS). Acyl-CoA sythesis is critical for dietary LCFA absorption; yet, the jejunal ACS enzymes that catalyze the reaction are largely unknown. High throughput mRNA sequencing of the mouse jejunum revealed that the expression of acyl-CoA synthetase 5 (Acsl5) and fatty-acid transport protein 4 (Fatp4) largely exceeded all other annotated ACS genes that activate LCFA. Interestingly, Acsl5 knockout (KO) mice displayed a decrease of 60% in jejunal total long chain acyl-CoA synthesis rate. Nevertheless, and despite of this decrease, dietary LCFA absorption and body-weight gain in response to high fat diet remained unaffected. Acsl5 is a major activator of dietary LCFA, yet in Acsl5 KO mice residual ACS activity is sufficient for maintaining a normal LCFA absorption. Our findings provide further evidence for a robust small intestine LCFA absorption capacity.

Expression of yeast acyl-CoA-∆9 desaturase leads to accumulation of unusual monounsaturated fatty acids in soybean seeds.

Abstract: An acyl-CoA-Δ9 desaturase from Saccharomyces cerevisiae was expressed by subcellular-targeting in soybean (Glycine max) seeds with the goal of increasing palmitoleic acid (16:1Δ9), a high-valued fatty acid (FA), and simultaneously decreasing saturated FA in oil. The expression resulted in the conversion of palmitic acid (16:0) to 16:1Δ9 in soybean seeds. 16:1Δ9 and its elongation product cis-vaccenic acid (18:1Δ11) were increased to 17 % of the total fatty acids by plastid-targeted expression of the enzyme. Other lipid changes include the decrease of polyunsaturated FA and saturated FA, suggesting that a mechanism exists downstream in oil biosynthesis to compensate the FA alternation. This is the first time a cytosolic acyl-CoA-∆9 desaturase is functionally expressed in plastid and stronger activity was achieved than its cytosolic expression. The present study provides a new strategy for converting 16:0 to 16:1Δ9 by engineering acyl-CoA-Δ9 desaturase in commercialized oilseeds.

Role of Plant Fatty acid Elongase (3 keto acyl-CoA Synthase) gene in Cuticular Wax Biosynthesis

Abstract: Plant surfaces are ensheathed by cuticular wax, amorphous intra-cuticular embedded in cutin polymer and crystalloid epi-cuticular that imparts a whitish appearance, confers drought resistance by reducing stomatal transpiration and also protects from U.V Radiation, phytophagous insects etc. Very long chain fatty acids acts as precursors for cuticular wax bio-synthesis. Wax bio-synthesis begins with fatty acid synthesis in the plastid (de novo synthesis of C16 and C18) and elongation of fatty acids in endoplasmic reticulum (C20 – C34) by four distinct enzymes 3-ketoacyl-CoA synthase, 3-ketoacyl-CoA reductase, 3-hydroxacyl-CoA dehydratase, trans-2,3-enoyl-CoA reductase (KCS, KCR, HCD, ECR). The KCS, a fatty acid elongase, determines the chain length and substrate specificity of the condensation reaction, a rate limiting step and the subsequent elongated products alkanes, aldehydes, primary alcohols, secondary alcohols, ketones and wax esters. 21 KCS genes were annotated in Arabidopsis thaliana Genome of which some KCSs were identified involved in cuticle formation (CER6) (CUT1), KCS1, KCS2, (DAISY), KCS20 and FDH).The current review will focus on the bio-chemical, genetic and molecular approaches on KCSs genes, predominantly KCS1 in plants particularly useful in identifying and characterizing gene products involved in wax bio-synthesis, secretion and function for developing transgenic crops that combat various stresses. INTRODUCTION

Conjugated linoleic acid isomers and their precursor fatty acids regulate peroxisome proliferator-activated receptor subtypes and major peroxisome proliferator responsive element-bearing target genes in HepG2 cell model

Abstract: The purpose of this study was to examine the induction profiles (as judged by quantitative reverse transcription polymerase chain reaction (qRT-PCR)) of peroxisome proliferator-activated receptor (PPAR) α, β, Γ subtypes and major PPAR-target genes bearing a functional peroxisome proliferator responsive element (PPRE) in HepG2 cell model upon feeding with cis-9,trans-11-octadecadienoic acid (9-CLA) or trans-10,cis-12-octadecadienoic acid (10-CLA) or their precursor fatty acids (FAs). HepG2 cells were treated with 100 μmol/L 9-CLA or 10-CLA or their precursor FAs, viz., oleic, linoleic, and trans-11-vaccenic acids against bezafibrate control to evaluate the induction/expression profiles of PPAR α, β, Γ subtypes and major PPAR-target genes bearing a functional PPRE, i.e., fatty acid transporter (FAT), glucose transporter-2 (GLUT-2), liver-type FA binding protein (L-FABP), acyl CoA oxidase-1 (ACOX-1), and peroxisomal bifunctional enzyme (PBE) with reference to β-actin as house keeping gene. Of the three housekeeping genes (glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin, and ubiquitin), β-actin was found to be stable. Dimethyl sulfoxide (DMSO), the common solubilizer of agonists, showed a significantly higher induction of genes analyzed. qRT-PCR profiles of CLAs and their precursor FAs clearly showed upregulation of FAT, GLUT-2, and L-FABP (∼0.5–2.0-fold). Compared to 10-CLA, 9-CLA decreased the induction of the FA metabolizing gene ACOX-1 less than did PBE, while 10-CLA decreased the induction of PBE less than did ACOX-1. Both CLAs and precursor FAs upregulated PPRE-bearing genes, but with comparatively less or marginal activation of PPAR subtypes. This indicates that the binding of CLAs and their precursor FAs to PPAR subtypes results in PPAR activation, thereby induction of the target transporter genes coupled with downstream lipid metabolising genes such as ACOX-1 and PBE. To sum up, the expression profiles of these candidate genes showed that CLAs and their precursor FAs are involved in lipid signalling by modulating the PPAR α, β, or Γ subtype for the indirect activation of the PPAR-target genes, which may in turn be responsible for the supposed health effects of CLA, and that care should be taken while calculating the actual fold induction values of candidate genes with reference to housekeeping gene and DMSO as they may impart false positive results.

LONG CHAIN OMEGA-3 AND OMEGA-6 POLYUNSATURATED FATTY ACID BIOSYNTHESIS BY EXPRESSION OF ACYL-CoA LYSOPHOSPHOLIPID ACYLTRANSFERASES

Abstract: Methods for increasing C18 to C20 elongation conversion efficiency and/or Δ4 desaturation conversion efficiency in long-chain polyunsaturated fatty acid [“LC-PUFA”]-producing recombinant oleaginous microbial host cells are provided herein, based on over-expression of acyl-CoA:lysophospholipid acyltransferases [“LPLATs”] (e.g., Ale1, LPAAT, LPCAT). Production host cells and oils produced by the methods of the invention are also claimed.

Fatty Acyl-CoA Synthetases

Abstract: Fatty acyl-CoA synthetase (ACS) catalyzes the adenosine triphosphate dependent formation of a thioester bond between a fatty acid and coenzyme A. This fundamental reaction allows the fatty acid to be degraded for energy production, incorporated into complex lipids, or participate in other metabolic pathways. Different ACS proteins exhibit preferences for fatty acid substrates of varying chain lengths, ranging from 2 to more than 30 carbon atoms. ACSs also differ in their tissue and cellular expression patterns, and subcellular locations. Humans and mice have 26 different ACSs. Emerging evidence suggests that each may perform a unique function essential for the maintenance of normal lipid homeostasis.

Fatty Acid Oxidation

Abstract: Fatty acid oxidation is the process by which fatty acids are degraded in living organisms. The initial event is the activation of fatty acids by esterification with coenzyme A (CoA). The degradation of the resultant fatty acyl-CoA thioesters proceeds by a cyclic process named β -oxidation. Passage through one cycle of β -oxidation produces fatty acids that are shortened by two carbon atoms and yields acetate in the form of acetyl-CoA. Repetitive cycling through β -oxidation results in the complete degradation of fatty acids.

A method for producing acyl amino acids

Abstract: The present invention relates to a cell expressing an amino acid-N-acyl-transferase, which is preferably recombinant, and an acyl-CoA synthetase, which is preferably recombinant, wherein the cell has a reduced fatty acid degradation capacity, a method for producing acyl amino acids, comprising the step contacting an amino acid and an acyl CoA in the presence of an amino acid-N-acyl-transferase, which is preferably isolated and/or recombinant, wherein the amino acid-N-acyl-transferase is preferably a human amino acid-N-acyl-transferase, or culturing the cell and a reaction mixture comprising an amino acid-N-acyl-transferase, which is preferably isolated and/or recombinant, an acyl-CoA synthetase, which is preferably isolated and/or recombinant, an amino acid and either a fatty acid-CoA or a fatty acid and an acyl-CoA-synthase.

Expression and distribution of acyl-CoA thioesterases in the white adipose tissue of rats.

Abstract: Acyl-CoA thioesterases (Acots) are enzymes that catalyze the hydrolysis of fatty acyl-CoAs to free fatty acids and coenzyme A, and have the potential to regulate the intracellular levels of these molecules. In this study, we show that a cytosolic isoform, Acot1, is expressed and distributed in immature adipocytes located in the perivascular region of the white adipose tissue (WAT) of rats. Immunoblot analyses detected Acot1 in all of the WATs examined, while immunohistochemistry revealed positively stained layered structures surrounding the adventitia of blood vessels in the subcutaneous WAT. When the subcutaneous WAT was digested with collagenase and centrifuged, Acot1 was recovered in the stromal vascular fraction (SVF), and not in the large mature adipocytes. In the SVF, undigested cells attached to short tubular fragments of blood vessels showed positive immunostaining, as well as a proportion of the dispersed cells. These fibroblast-like cells contained fine particulate lipid droplets, stained by oil-red O dye, in their cytoplasm, or expressed fatty acid-binding protein 4, an adipocyte marker. After induction of adipocyte differentiation following a 15-day preculture without insulin, the dedifferentiated cells showed increased Acot1 expression with a diffuse distribution throughout the cytosol. These findings suggest that Acot1 expression is transiently upregulated at an early stage of adipocyte maturation, possibly to maintain cytosolic acyl-CoAs below a certain level until the cells acquire their full capability for fat storage.

Improved production of polyunsaturated fatty acids by coexpression of acyl-coa:lysophosphatidylcholine acyltransferases and phospholipid:diacylglycerol acyltransferases

Abstract: Acyl-CoA:lysophosphatidylcholine acyltransferase ["LPCAT"] having the ability to convert acyl-CoA + 1-acyl-sn-glycero-3-phosphocholine to CoA + 1,2-diacyl-sn-glycero-3-phosphocholine (EC 2.3.1.23) is disclosed herein to be over-expressed along with the over-expression of phospholipid:diacylglycerol acyltransferase ["PDAT"] having the ability to transfer a fatty acyl group from the sn-2 position of a phospholipid (e.g., phosphatidylcholine) to the sn-3 position of 1,2-diacylglycerol [E.C.2.3.1.158], thus resulting in a lysophospholipid and TAG. Co-expression of these enzymes in a recombinant microbial host cell resulted in increased production of long chain polyunsaturated fatty acids ["PUFAs"].

Molecular Modeling and Simulation Studies of Acyl CoA Synthetaseof Mycobacteriumleprae

Abstract: Leprosy or Hansen’s disease is caused by an obligate intracellular pathogen i.e. Mycobacterium leprae. Leprosy is a granulomatous disease of peripheral nerves and mucosa of the upper respiratory tract. This infectious disease results in Leprosy reactions that cause irreversible nerve damage and disabilities. The organism requires minimal set of functional genes for its survival. Most of the genes are involved in biosynthetic and metabolic pathways, so the product of these genes can be aimed for the novel drug target. Acyl CoA Synthetase is an enzyme that participates in fatty acid biosynthesis. The activation of fatty acids by Acyl-CoA Synthetase is the need of de novo lipid biosynthesis, fatty acid catabolism and remodeling of biological membranes. Therefore by emphasizing this protein as a drug target can help in the identification of novel drugs to cure leprosy. A well organized research comprising of analogue based drug design and molecular dynamics plays a major role in obtaining the lead molecules. The bacteria have developed resistance against many of the drugs available in the market. Therefore identification of the novel drug target and potent drug can be helpful in better prevention of the disease.

PRODUCTION OF POLYUNSATURATED FATTY ACIDS BY COEXPRESSION OF ACYL-CoA:LYSOPHOSPHATIDYLCHOLINE ACYLTRANSFERASES AND PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASES

Abstract: Acyl-CoA:lysophosphatidylcholine acyltransferase [“LPCAT”] having the ability to convert acyl-CoA+1-acyl-sn-glycero-3-phosphocholine to CoA+1,2-diacyl-sn-glycero-3-phosphocholine (EC 2.3.1.23) is disclosed herein to be over-expressed along with the over-expression of phospholipid:diacylglycerol acyltransferase [“PDAT”] having the ability to transfer a fatty acyl group from the sn-2 position of a phospholipid (e.g., phosphatidylcholine) to the sn-3 position of 1,2-diacylglycerol [E.C.2.3.1.158], thus resulting in a lysophospholipid and TAG. Co-expression of these enzymes in a recombinant microbial host cell resulted in increased production of long chain polyunsaturated fatty acids [“PUFAs”].

Nucleic acids encoding mutant acyl-CoA:lysophosphatidylcholine acyltransferases

Abstract: Mutant acyl-CoA:lysophosphatidylcholine acyltransferases [“LPCATs”] having the ability to convert acyl-CoA+1-acyl-sn-glycero-3-phosphocholine to CoA+1,2-diacyl-sn-glycero-3-phosphocholine (EC 2.3.1.23) are disclosed herein. Isolated nucleic acid fragments and recombinant constructs comprising such fragments encoding mutant LPCATs, along with a method of making long chain polyunsaturated fatty acids [“PUFAs”] using these mutant LPCATs in oleaginous yeast, are also disclosed.