Triacylglycerol Formation Research Articles

Mechanism Controlling High‐Temperature Degradation of Sunflower Oil Triacylglycerols in the Absence of Oxygen

AbstractThe aim of the present study is to describe the mechanism controlling heat‐induced formation of sunflower oil triacylglycerol and fatty acid methyl ester oligomers. The unique combination of high‐performance size‐exclusion chromatography with hyphenated electrospray ionization mass spectrometry (MS), atmospheric pressure chemical ionization‐MS, and high‐temperature gas chromatography‐MS techniques allows differentiating between radical coupling species and Diels–Alder cycloadducts. Targeted analysis of thermally degraded sunflower oils confirms the exact structures of various acyclic oligomers accompanied by less‐abundant products of pericyclic transformations. A series of model experiments simulate the impact of dienophile nature on the course of Diels–Alder reactions. Thus, α‐tocopherylquinone, δ‐tocopherylquinone, and methyl‐(E)‐11‐oxoundec‐9‐enoate are synthesized as naturally occurring dienophiles bearing electron‐withdrawing groups. The geometry of poor dienophiles does not affect concerted cyclization, while the structure of electron deficient dienophiles can overcome low reactivity.Practical Application: In the absence of oxygen, heat‐induced degradation of polyunsaturated triacylglycerols proceed predominantly via a radical pathway, whereas concerted reactions represent minor mechanisms. Sunflower oil triacylglycerol molecules in the system without propagation stage can be effectively protected by natural and/or synthetic antioxidants. Application of chelates is also recommended. However, antioxidant‐derived quinones, such as α‐tocopherylquinone, can enter the Diels–Alder reaction even more easily than dienophiles without electron‐withdrawing groups. Unsaturated core aldehydes possess the same reactivity. Examination of the mechanism controlling high‐temperature degradation of triacylglycerols is especially important for processing engineers in edible oil refineries and food technologists. New perspective may help them to minimize undesirable changes in polyunsaturated species.

Degradation of Lipid Droplets in Plants and Algae—Right Time, Many Paths, One Goal

In eukaryotic cells, lipids in the form of triacylglycerols (TAGs) are the major reservoir of cellular carbon and energy. These TAGs are packed into specialized organelles called lipid droplets (LDs). They can be found in most, if not all, types of cells, from bacteria to human. Recent data suggest that rather than being simple storage organelles, LDs are very dynamic structures at the center of cellular metabolism. This is also true in plants and algae, where LDs have been implicated in many processes including energy supply; membrane structure, function, trafficking; and signal transduction. Plant and algal LDs also play a vital role in human life, providing multiple sources of food and fuel. Thus, a lot of attention has been paid to metabolism and function of these organelles in recent years. This review summarizes the most recent advances on LDs degradation as a key process for TAGs release. While the initial knowledge on this process came from studies in oilseeds, the findings of the last decade revealed high complexity and specific mechanisms of LDs degradation in plants and algae. This includes identification of numerous novel proteins associated with LDs as well as a prominent role for autophagy in this process. This review outlines, systemizes, and discusses the most current data on LDs catabolism in plants and algae.

The Phospholipid:Diacylglycerol Acyltransferase-Mediated Acyl-Coenzyme A-Independent Pathway Efficiently Diverts Fatty Acid Flux from Phospholipid into Triacylglycerol in Escherichia coli.

Researchers have long endeavored to accumulate triacylglycerols (TAGs) or their derivatives in easily managed microbes. The attempted production of TAGs in Escherichia coli has revealed barriers to the broad applications of this technology, including low TAG productivity and slow cell growth. We have demonstrated that an acyl-CoA-independent pathway can divert phospholipid flux into TAG formation in E. coli mediated by Chlamydomonas reinhardtii phospholipid:diacylglycerol acyltransferase (CrPDAT) without interfering with membrane functions. We then showed the synergistic effect on TAG accumulation via the acyl-CoA-independent pathway mediated by PDAT and the acyl-CoA-dependent pathway mediated by wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT). Furthermore, CrPDAT led to synchronous TAG accumulation during cell growth, and this could be enhanced by supplementation of arbutin. We also showed that rationally mutated CrPDAT was capable of decreasing TAG lipase activity without impairing PDAT activity. Finally, ScPDAT from Saccharomyces cerevisiae exhibited similar activities as CrPDAT in E. coli Our results suggest that the improvement in accumulation of TAGs and their derivatives can be achieved by fine-tuning of phospholipid metabolism in E. coli Understanding the roles of PDAT in the conversion of phospholipids into TAGs during the logarithmic growth phase may enable a novel strategy for the production of microbial oils.IMPORTANCE Although phospholipid:diacylglycerol acyltransferase (PDAT) activity is presumed to exist in prokaryotic oleaginous bacteria, the corresponding gene has not been identified yet. In this article, we have demonstrated that an acyl-CoA-independent pathway can divert phospholipid flux into TAG formation in Escherichia coli mediated by exogenous CrPDAT from Chlamydomonas reinhardtii without interfering with membrane functions. In addition, the acyl-CoA-independent pathway and the acyl-CoA-dependent pathway had the synergistic effect on TAG accumulation. Overexpression of CrPDAT led to synchronous TAG accumulation during cell growth. In particular, CrPDAT possessed multiple catalytic activities, and the rational mutation of CrPDAT led to the decrease of TAG lipase activity without impairing acyltransferase activity. The present findings suggested that applying PDAT in E. coli or other prokaryotic microbes may be a promising strategy for accumulation of TAGs and their derivatives.

Lipid droplets formation and their effects on phosphatidylinositol level in yeasts

Lipid droplets (LDs) are conserved specialized organelles that are surrounded by a single phospholipid layer, enclosing neutral lipids in form of triacylglycerol (TAG) and/or sterol ester. They are formed at the endoplasmic reticulum (ER), and stay connected to it in the yeast cell. These LDs mainly play major roles in lipid storage and homeostasis. Moreover, they have several functions in the yeast cell including; protein degradation, protein glycosylation, response to ER stress, in addition to providing substrate for energy metabolism and for membrane synthesis. In general, TAG and phosphatidylinositol (PI) synthesis share the same precursor; phosphatidic acid (PA), which controls their level. By the actions of lipases, phosphatases and/or kinases; one can act as a precursor for another, thus explaining the reason of their inverse correlation. TAG lipolysis is catalyzed by TAG lipases producing fatty acids and diacylglycerol (DAG). The DAG produced from lipolysis is phosphorylated by diacylglycerol kinase (Dgk1) to form PA. On the other hand, PA is a cell signaling molecule and once increased, it upregulates cytidine diphosphate diacylglycerol (CDP-DAG) pathway for the PI synthesis. Major TAG lipases are mainly localized in LD, whereas minor amounts of them are localized in ER. Once DAG is produced inside the LD, it is transferred to the ER by inheritance cortical ER protein, Ice2, for further reactions. The TAG and PI levels are also controlled by transcriptional regulation in response to inositol and zinc. The aim of this study was to clarify how LDs regulate lipid homeostasis in the yeast cell, and particularly focuses on the balance between TAG and PI.

Effects of exogenous C18 unsaturated fatty acids on milk lipid synthesis in bovine mammary epithelial cells.

We determined the effects of a combination of C18 unsaturated fatty acids (C18-UFAs) consisting of oleic, linoleic, and linolenic acids on milk lipogenesis in bovine mammary epithelial cells (BMECs). By orthogonal experiments to determine cellular triacylglycerol (TAG) accumulation, a combination of 200 μmol/l C18 : 1, 50 μmol/l C18 : 2, and 2 μmol/l C18 : 3 was selected as C18-UFAs combination treatment, and culture in medium containing fatty acid-free bovine serum albumin was used as the control. The expression of genes related to milk lipid synthesis and intracellular FA composition was measured. The results showed that cytosolic TAG formation was higher under C18-UFAs treatment than under control treatment. The mRNA expression of acetyl-CoA carboxylase-α (ACACA), fatty acid synthase (FASN), and peroxisome proliferator-activated receptor gamma (PPARG) did not differ between treatments. The abundance of stearoyl-CoA desaturase (SCD) and acyl-CoA synthetase long-chain family member 1 (ACSL1) was higher, whereas that of sterol regulatory element binding transcription factor 1 (SREBF-1) was lower after C18-UFAs treatment compared to control treatment. The C16 : 0 and SFA content was decreased following C18-UFAs treatment compared to control treatment, while the cis-9 C18 : 1 and UFA content was increased. In conclusion, C18-UFAs could stimulate triglyceride accumulation, increase the cellular UFA concentration, and regulate lipogenic genes in BMECs.

Modeling the structure and infrared spectra of omega-3 fatty acid esters.

Omega-3 dietary supplements provide a rich source of the active moieties eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which exist in the form of triacylglycerols or ethyl esters. Infrared (IR) spectroscopy provides a rapid and quantitative tool to assess the quality of these products as specific normal modes, in particular the ester carbonyl stretch modes, exhibit characteristic spectral features for the two ester forms of omega-3 fatty acids. To uncover the origin of the observed spectra, in this work, we perform molecular dynamics simulations of EPA and DHA ethyl esters and triacylglycerols to characterize their conformation, packing, and dynamics in the liquid phase and use a mixed quantum/classical approach to calculate their IR absorption spectra in the ester carbonyl stretch region. We show that the ester liquids exhibit slow dynamics in spectral diffusion and translational and rotational motion, consistent with the diffusion ordered NMR spectroscopy measurements. We further demonstrate that the predicted IR spectra are in good agreement with experiments and reveal how a competition between intermolecular and intramolecular interactions gives rise to distinct absorption peaks for the fatty acid esters.

Alteration of the fatty acid composition of Brassica napus L. via overexpression of phospholipid: Diacylglycerol acyltransferase 1 from Sapium sebiferum (L.) Roxb.

Sapium sebiferum (L.) Roxb. plays an important role in traditional Chinese medicine and is one of major woody oil tree in China. Phospholipid: diacylglycerol acyltransferase 1 (PDAT1), as an important catalytic enzyme for the formation of triacylglycerol (TAG), is mainly responsible for the transfer of an acyl group from the sn-2 position of phospholipids to the sn-3 position of sn-1, 2-diacylglycerol (DAG) to produce TAG and sn-1 lysophospholipids. The importance of PDAT1 in triacylglycerol biosynthesis has been illustrated in previous research, and at least 67 PDAT1 sequences have been identified from 31 organisms. However, little is known about the gene encoding PDAT1 in S. sebiferum (SsPDAT1), which is involved in seed oil biosynthesis. To explore the functional characteristics of SsPDAT1, we cloned and analyzed the full-length cDNA in the coding region of SsPDAT1, which consists of 2040 bp and encodes a putative protein of 680 amino acid (aa) residues. Thin-layer chromatography (TLC) analysis showed that recombinant SsPDAT1 could restore TAG accumulation in TAG-deficient mutant yeast (Saccharomyces cerevisiae) H1246, which revealed the enzyme activity of SsPDAT1. Moreover, transgenic Brassica napus L. W10 plants overexpressing SsPDAT1 showed significant increases of 19.6–28.9 % in linoleic acid levels but decreases of 27.3–37.1 % in linolenic acid. Furthermore, the total oil content increased by 8.1 %–10.8 % in SsPDAT1 transgenic seeds. These results confirmed the role of SsPDAT1 in stabilizing oil biosynthesis and suggested that SsPDAT1 could be exploitable to specifically regulate the oil composition of plants. These experimental results provide a new concept that may enable the industrial development of plants with high-linoleic-acid oil through overexpression of SsPDAT1 in S. sebiferum L.

A Lipidomic Perspective of the Action of Group IIA Secreted Phospholipase A2 on Human Monocytes: Lipid Droplet Biogenesis and Activation of Cytosolic Phospholipase A2α.

Phospholipase A2s constitute a wide group of lipid-modifying enzymes which display a variety of functions in innate immune responses. In this work, we utilized mass spectrometry-based lipidomic approaches to investigate the action of Asp-49 Ca2+-dependent secreted phospholipase A2 (sPLA2) (MT-III) and Lys-49 sPLA2 (MT-II), two group IIA phospholipase A2s isolated from the venom of the snake Bothrops asper, on human peripheral blood monocytes. MT-III is catalytically active, whereas MT-II lacks enzyme activity. A large decrease in the fatty acid content of membrane phospholipids was detected in MT III-treated monocytes. The significant diminution of the cellular content of phospholipid-bound arachidonic acid seemed to be mediated, in part, by the activation of the endogenous group IVA cytosolic phospholipase A2α. MT-III triggered the formation of triacylglycerol and cholesterol enriched in palmitic, stearic, and oleic acids, but not arachidonic acid, along with an increase in lipid droplet synthesis. Additionally, it was shown that the increased availability of arachidonic acid arising from phospholipid hydrolysis promoted abundant eicosanoid synthesis. The inactive form, MT-II, failed to produce any of the effects described above. These studies provide a complete lipidomic characterization of the monocyte response to snake venom group IIA phospholipase A2, and reveal significant connections among lipid droplet biogenesis, cell signaling and biochemical pathways that contribute to initiating the inflammatory response.

Construction of a new lipase- and xylanase-producing oleaginous yeast consortium capable of reactive azo dye degradation and detoxification

A new oleaginous yeast consortium Y-BC-SH which stands for molecularly identified species Yarrowia sp., Barnettozyma californica and Sterigmatomyces halophilus was successfully constructed in this study. This multipurpose oleaginous yeast consortium was developed based on its higher ability to accumulate large amounts of lipids in the form of triacylglycerol, grow on xylose, produce lipase and xylanase and it could rapidly decolorize and degrade commonly-used textile reactive azo dyes. The specific enzyme activities of lipase, xylanase, xylan esterase, β-xylosidase, CMCase, β-glucosidase and cellobiohydrolase produced by Y-BC-SH were significantly higher than that of individual strains. As chemical oxygen demand reduction had occurred in the dye mixture solutions, it was evidence of their color removal and mineralization by Y-BC-SH. The significant induction of oxidoreductive enzymes by Y-BC-SH was probably due to the coordinated metabolic interactions of the individual strains. Phytotoxicity assay confirmed that metabolites generated after dye degradation by Y-BC-SH are non-toxic.

Towards oilcane: Engineering hyperaccumulation of triacylglycerol into sugarcane stems

AbstractMetabolic engineering to divert carbon flux from sucrose to oil in high biomass crop like sugarcane is an emerging strategy to boost lipid yields per hectare for biodiesel production. Sugarcane stems comprise more than 70% of the crops' biomass and can accumulate sucrose in excess of 20% of their extracted juice. The energy content of oils in the form of triacylglycerol (TAG) is more than twofold that of carbohydrates. Here, we report a step change in TAG accumulation in sugarcane stem tissues achieving an average of 4.3% of their dry weight (DW) in replicated greenhouse experiments by multigene engineering. The metabolic engineering included constitutive co‐expression of wrinkled1; diacylglycerol acyltransferase1‐2; cysteine‐oleosin; and ribonucleic acid interference‐suppression of sugar‐dependent1. The TAG content in leaf tissue was also elevated by more than 400‐fold compared to non‐engineered sugarcane to an average of 8.0% of the DW and the amount of total fatty acids reached about 13% of the DW. With increasing TAG accumulation an increase of 18:1 unsaturated fatty acids was observed at the expense of 16:0 and 18:0 saturated fatty acids. Total biomass accumulation, soluble lignin, Brix and juice content were significantly reduced in the TAG hyperaccumulating sugarcane lines. Overcoming this yield drag by engineering lipid accumulation into late stem development will be critical to exceed lipid yields of current oilseed crops.

Inhibition of TOR in Chlamydomonas reinhardtii Leads to Rapid Cysteine Oxidation Reflecting Sustained Physiological Changes

The target of rapamycin (TOR) kinase is a Ser/Thr kinase and master regulator, with roles in the regulation of nutritional sensing, protein translation, and autophagy, among others. In Chlamydomonas reinhardtii, a unicellular green algae and model organism for biofuel production and optimization, TOR has been linked to increased triacylglycerol (TAG) accumulation, suggesting that TOR or a downstream target is responsible for the elusive “lipid switch,” the protein or proteins responsible for increasing TAG formation under nutrient limitation and environmental stressors. However, when this lipid switch is activated, overall culture growth is arrested, truncating the alga’s ability to continuously produce TAGs at a level needed for widespread biofuel use.In order to engineer an effective strain for algal biofuels, the targets and signaling pathways of lipid regulatory networks must be determined. However, while TOR has been well characterized in mammalian systems, it is still poorly understood in photosynthetic systems. Previous research has used TOR inhibitors to follow phosphorylation pathways and identify potential downstream targets of TOR but little work has uncovered the role of oxidative signaling in regulation. Since TOR inhibition results in an increase in reactive oxygen species analogous to that of nitrogen deprivation, the opportunity for redox control mechanisms is significant.This study inhibited TOR using AZD8055 and monitored reversible thiol oxidation while simultaneously tracking physiological changes. By pairing these data together, reversible thiol oxidation was related to nearly every major metabolic pathway in the cell, including TAG accumulation, the tricarboxylic acid cycle, and protein translation. Additionally, reversible thiol oxidation corresponded to changes in photosynthetic activity measured through chlorophyll a fluorescence, showing a novel role for TOR regulation of photosynthesis. The delineation of redox‐controlled proteins under TOR inhibition provides a framework for further characterization of the TOR pathway in photosynthetic eukaryotes.Support or Funding InformationThis research was supported by a National Science Foundation CAREER award (MCB‐1552522) awarded to L.M.H and a National Science Foundation award (Cooperative Agreement OIA‐1458,952) to D.R.J.K. A.L.S. was funded by the NASA West Virginia Space Grant Consortium (NNX15AK74A and NNX15AI01H).

Adipocyte lipolysis: from molecular mechanisms of regulation to disease and therapeutics.

Fatty acids (FAs) are stored safely in the form of triacylglycerol (TAG) in lipid droplet (LD) organelles by professional storage cells called adipocytes. These lipids are mobilized during adipocyte lipolysis, the fundamental process of hydrolyzing TAG to FAs for internal or systemic energy use. Our understanding of adipocyte lipolysis has greatly increased over the past 50 years from a basic enzymatic process to a dynamic regulatory one, involving the assembly and disassembly of protein complexes on the surface of LDs. These dynamic interactions are regulated by hormonal signals such as catecholamines and insulin which have opposing effects on lipolysis. Upon stimulation, patatin-like phospholipase domain containing 2 (PNPLA2)/adipocyte triglyceride lipase (ATGL), the rate limiting enzyme for TAG hydrolysis, is activated by the interaction with its co-activator, alpha/beta hydrolase domain-containing protein 5 (ABHD5), which is normally bound to perilipin 1 (PLIN1). Recently identified negative regulators of lipolysis include G0/G1 switch gene 2 (G0S2) and PNPLA3 which interact with PNPLA2 and ABHD5, respectively. This review focuses on the dynamic protein-protein interactions involved in lipolysis and discusses some of the emerging concepts in the control of lipolysis that include allosteric regulation and protein turnover. Furthermore, recent research demonstrates that many of the proteins involved in adipocyte lipolysis are multifunctional enzymes and that lipolysis can mediate homeostatic metabolic signals at both the cellular and whole-body level to promote inter-organ communication. Finally, adipocyte lipolysis is involved in various diseases such as cancer, type 2 diabetes and fatty liver disease, and targeting adipocyte lipolysis is of therapeutic interest.

Novel insights into type 2 diacylglycerol acyltransferases in microalga Myrmecia incisa

The green alga Myrmecia incisa is rich in arachidonic acid (ArA), which mainly stores in lipid droplets in the form of triacylglycerol (TAG). In the de novo biosynthesis of TAG, diacylglycerol acyltransferase (DGAT) catalyzes the final and committed step as a rate-limiting enzyme. Previously, our lab has reported the first DGAT in M. incisa, and in this study, a new DGAT was further identified. Through homology search against our transcriptome database and annotation, a new contig corresponding to the putative DGAT2 was targeted, whose full-length cDNA was cloned by RACE and designated as MiDGAT2C. For this new MiDGAT2C, bioinformatics analyses revealed the presence of multiple conserved amino acid residues and sequences. Its function to synthesize TAG was determined by complementation assay using the TAG-deficient yeast system. Besides, MiDGAT2C was compared with other MiDGATs with respect to their substrate preference. Results of fatty acid feeding and in vitro enzymatic assays indicated diverse substrate specificities among MiDGAT members. While MiDGAT2C showed no activity on ArA, MiDGAT2A made the most contribution to incorporate ArA into TAG. Findings of this study expand our knowledge on the molecular basis of DGAT, shedding more light on M. incisa as a promising feedstock for ArA-rich TAG.

Kinetic improvement of an algal diacylglycerol acyltransferase 1 via fusion with an acyl-CoA binding protein.

Microalgal oils in the form of triacylglycerols (TAGs) are broadly used as nutritional supplements and biofuels. Diacylglycerol acyltransferase (DGAT) catalyzes the final step of acyl-CoA-dependent biosynthesis of TAG, and is considered a key target for manipulating oil production. Although a growing number of DGAT1s have been identified and over-expressed in some algal species, the detailed structure-function relationship, as well as the improvement of DGAT1 performance via protein engineering, remain largely untapped. Here, we explored the structure-function features of the hydrophilic N-terminal domain of DGAT1 from the green microalga Chromochloris zofingiensis (CzDGAT1). The results indicated that the N-terminal domain of CzDGAT1 was less disordered than those of the higher eukaryotic enzymes and its partial truncation or complete removal could substantially decrease enzyme activity, suggesting its possible role in maintaining enzyme performance. Although the N-terminal domains of animal and plant DGAT1s were previously found to bind acyl-CoAs, replacement of CzDGAT1 N-terminus by an acyl-CoA binding protein (ACBP) could not restore enzyme activity. Interestingly, the fusion of ACBP to the N-terminus of the full-length CzDGAT1 could enhance the enzyme affinity for acyl-CoAs and augment protein accumulation levels, which ultimately drove oil accumulation in yeast cells and tobacco leaves to higher levels than the full-length CzDGAT1. Overall, our findings unravel the distinct features of the N-terminus of algal DGAT1 and provide a strategy to engineer enhanced performance in DGAT1 via protein fusion, which may open a vista in generating improved membrane-bound acyl-CoA-dependent enzymes and boosting oil biosynthesis in plants and oleaginous microorganisms.

Galactolipid DGDG and Betaine Lipid DGTS Direct De Novo Synthesized Linolenate into Triacylglycerol in a Stress-Induced Starchless Mutant of Chlamydomonas reinhardtii.

The increasing demand for triacylglycerol (TAG) enriching polyunsaturated fatty acids (PUFAs) has led to a surge of interest in microalgal TAG metabolism. Polar membrane lipids serve as the desaturation carrier for PUFA, and the functional group of PUFA can be incorporated into TAG. Monogalactoglycerolipid has been found to provide the de novo synthesized oleate acyl group or the nascent polyunsaturated diacylglycerol backbone for TAG biosynthesis in the model green alga, Chlamydomonas reinhardtii. However, whether other membrane lipids take part in the formation of PUFA-attached TAG has not been clearly discovered. A time course study of glycerolipidomics in the starchless mutant of C. reinhardtii, BAFJ5, which hyper-accumulates TAG, revealed that digalactosyldiacylglycerol (DGDG) and diacylglycerol-N,N,N-trimethylhomoserine (DGTS) turned into the main components of membrane lipids, accounting for 62% of the total polar lipids, under nitrogen deprivation combined with high light conditions. In addition, the membrane lipid molecules DGDG 18:3n3/16:0 and DGTS 16:0/18:3n6 were presumed to be involved in the consecutive integration of the de novo synthesized linolenates into TAG. Based on the stoichiometry calculation, DGDG and DGTS were demonstrated to provide a major contribution to the accumulation of linolenate-attached TAG. Our study gives insights into the potential PUFA-attached TAG formation pathway mediated by the turnover of de novo synthesized DGDG and DGTS in the starchless mutant of Chlamydomonas.

Heart‐Cut Two‐Dimensional Countercurrent Chromatography for the Isolation of a Furan Fatty Acid Triacylglycerol from Latex Gloves and Identification of Further Lipid Compounds

AbstractHevea brasiliensis latex from specific genotypes such as RRIM501 or PB235 is one of the richest natural sources of furan fatty acid 9‐(3‐methyl‐5‐pentylfuran‐2‐yl)‐nonanoic acid (9M5), which is mainly present in the form of triacylglycerols. In this study, we successfully isolated a triacylglycerol esterified with three 9M5 molecules (tri‐9M5) with a purity of 97% from the lipid extract of latex gloves by means of countercurrent chromatography (CCC). The gas chromatography with a mass spectrometry (GC/MS) spectrum of tri‐9M5 not only featured the diagnostic fragment ion [M‐RCOO]+ of triacylglycerols but also a fragment ion shifted by 16 Da to higher mass which corresponded with [M‐RCO]+. [M‐RCO]+ was only detected in triacylglycerols substituted with at least one furan fatty acid. Additionally, five γ‐tocotrienyl fatty acid esters (γ‐T3‐FA esters), namely, γ‐T3‐16:0, γ‐T3‐18:0, γ‐T3‐18:1n‐9, γ‐T3‐18:2n‐6 and γ‐T3‐20:0, were detected in the sample. Contributions of γ‐T3‐FA esters with 18:1n‐9 and 18:2n‐6 which co‐eluted in GC/MS could be calculated after mathematical correction for contributions of the 13C2 isotope peak of γ‐T3‐18:2n‐6 to γ‐T3‐18:1n‐9. This was necessary for quantitation of these two γ‐T3‐FA esters. Improved separation of the γ‐T3‐FA esters could be achieved by the novel heart‐cut recycling CCC mode (four cycles). Sterols detected in disposable latex gloves were β‐sitosterol, Δ5‐avenasterol, and stigmasterol along with small quantities of 24‐methylene‐cycloartenol and Δ5‐citrostadienol.

Stress Induced Production of High-Value Organic Compounds from Freshwater Microalgae

Because of high lipid productivity and potential cultivation in non-fertile areas, microalgae has been renowned as a reliable resource for biodiesel production and various high-value bioactive compounds. They can grow rapidly, synthesize, and accumulate large amounts (20-50% of dry weight) of neutral lipids (mainly in the form of triacylglycerol, TAG). The production of lipids, carotenoids, and algal biomass can be enhanced by inducing various environmental stress factors. The present study investigates on effect of nutrient limitation on biomass and lipid production of freshwater microalgae Grasiella sp. and Chlorella sp. under two different conditions of cultivation viz. normal and nutrient limited conditions. Stress was induced in the form of nitrogen and phosphorous deficiency in the cultivation media. Results found that biomass obtained from nutrient-limited conditions was comparatively higher than normal. The dry weight of biomass for Chlorella sp. under normal and nutrient-limited conditions was found to be 0.78 g/L and 0.96 g/L, respectively, and Grasiella sp. 0.48 g/L and 0.8 g/L respectively. FT-IR analysis revealed nutrient limitation influence the change in the biochemical composition in the biomass and varied between the species. Total lipid content for Grasiella sp. KACC 19 under normal and nutrient limited conditions were found to be 0.0131 g/L and 0.0765 g/L, and Chlorella sp. KACC 21 was found to be 0.1125 g/L and 0.2178 g/L respectively. The fatty acid profiling revealed the presence of saturated and MUFA and accumulation of those fatty acids in nutrient-limited condition thereby considered these microalgae as a potential feedstock for the production of biofuel.

Renewable Biodiesel Production from Oleaginous Yeast Biomass Using Industrial Wastes

The accumulation lipid from oleaginous microorganisms is recognized as a second generation fuel. Biooil is known to as intracellular product of oily yeast utilizing various carbon substrates and converting different quantities of lipids in the form of triacylglycerols. This second generation fuel can be used to make biodiesel via a transesterification process. This study investigated the morphological characteristics of eight strains of Thai oleaginous yeasts via microscopy and analyzed the fatty acid profiling of yeasts cultured in three carbon sources: glucose, sugar cane molasses and crude glycerol in order to estimate biodiesel properties. To approach this goal, batch fermentations were used to culture eight yeast strains,Rhodosporidium toruloidesTISTR 5123, TISTR 5154, TISTR 5149,Yarrowia lipolyticaTISTR 5054, TISTR 5151, TISTR 5621,Rhodotorula glutinisTISTR 5159 andRhodotorula graminisTISTR 5124 for 96 h under 30°C at 250 rpm. Result revealed that eight yeast strains contained significant amounts of fatty acids and lipids and accumulated mainly palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C 18:1) and linoleic acid (C18:2), and they are suitable for the production of biodiesel. Fatty acid productions and profiles indicated that these yeast strains can be potentially used as the triacylglycerols producers for biodiesel production.

Microbiome transfer between IL-1RI-/- and wild-type mice during high or low-fat feeding alters metabolic tissue functionality but not glucose homeostasis.

AbstractReduced inflammatory signaling (IL-1RI-/-) alters metabolic responses to dietary challenges (1). Inflammasome deficiency (e.g. IL-18-/-, Asc-/-) can modify gut microbiota concomitant with hepatosteatosis; an effect that was transferable to wild-type (WT) mice by co-housing (2). Taken together, this evidence suggests that links between diet, microbiota and IL-1RI-signaling can influence metabolic health. Our aim was to determine whether IL-1RI-mediated signaling interacted with the gut microbiome to impact metabolic tissue functionality in a diet-specific fashion. Male WT (C57BL/J6) and IL-1RI-/- mice were fed either high-fat diet (HFD; 45% kcal) or low-fat diet (LFD; 10% kcal) for 24 weeks and were housed i) separately by genotype or ii) with genotypes co-housed together (i.e. isolated vs shared microbial environment; n = 8–10 mice per group). Glucose tolerance and insulin secretion response (1.5 g/kg i.p.), gut microbiota composition and caecal short-chain fatty acids (SCFA) were assessed. Liver and adipose tissue were harvested and examined for triacylglycerol (TAG) formation, cholesterol and metabolic markers (Fasn, Cpt1α, Pparg, Scd1, Dgat1/2), using histology, gas-chromatography and RT-PCR, respectively. Statistical analysis included 1-way or 2-way ANOVA, where appropriate, with Bonferroni post-hoc correction. Co-housing significantly affected gut microbiota composition, illustrated by clustering in PCoA (unweighted UniFrac distance) of co-housed mice but not their single-housed counterparts, on both HFD and LFD. The taxa driving these differences were primarily from Lachnospiraceae and Ruminococcaceae families. Single-housed WT had lower hepatic weight, TAG, cholesterol levels and Fasn despite HFD, an effect lost in their co-housed counterparts, who aligned more to IL-1RI-/- hepatic lipid status. Hepatic Cpt1α was lowest in co-housed WT. Adipose from IL-1RI-/- groups on HFD displayed increased adipocyte size and reduced adipocyte number compared to WT groups, but greater lipogenic potential (Pparg, Scd1, Dgat2) alongside a blunted IL-6 response to pro-inflammatory stimuli (~32%, P = 0.025). Whilst caecal SCFA concentrations were not different between groups, single-housed IL-1RI-/- adipocytes showed greatest sensitivity to SCFA-induced lipogenesis. Interestingly, differences in tissue functionality and gut microbiome occurred despite unaltered glucose tolerance; although there was a trend for phenotypic transfer of body weight via co-housing. For all endpoints examined, similar genotype/co-housing effects were observed for both HFD and LFD with the greatest impacts seen in HFD-fed mice. In conclusion, while the gut microbiome may be an important consideration in dietary interventions, these results question the magnitude of its impact in relation to the IL-1RI-dependent immunometabolism-glucose homeostasis axis.

Characterization of a Type-2 Diacylglycerol Acyltransferase from Haematococcus pluvialis Reveals Possible Allostery of the Recombinant Enzyme.

Haematococcus pluvialis is a green microalga used in the algal biotechnology industry that can accumulate considerable amounts of storage triacylglycerol (TAG) and astaxanthin, which is a high-value carotenoid with strong antioxidant activity, under stress conditions. Diacylglycerol acyltransferase (DGAT) catalyzes the last step of the acyl-CoA-dependent TAG biosynthesis and appears to represent a bottleneck in algal TAG formation. In this study, putative H. pluvialis DGAT2 cDNA (HpDGAT2A, B, D and E) were identified from a transcriptome database and were subjected to sequence-based in silico analyses. The coding sequences of HpDGAT2B, D, and E were then isolated and characterized through heterologous expression in a TAG-deficient Saccharomyces cerevisiae strain H1246. The expression of HpDGAT2D allowed the recovery of TAG biosynthesis in this yeast mutant, and further in vitro enzymatic assays confirmed that the recombinant HpDGAT2D possessed strong DGAT activity. Interestingly, the recombinant HpDGAT2D displayed sigmoidal kinetics in response to increasing acyl-CoA concentrations, which has not been reported in plant or algal DGAT2 in previous studies.