Fatty Acyl Synthetases and Thioesterases in Plant Lipid Metabolism: Diverse Functions and Biotechnological Applications

Plants use fatty acids to synthesize acyl lipids for many different cellular, physiological, and defensive roles. These roles include the synthesis of essential membrane, storage, or surface lipids, as well as the production of various fatty acid-derived metabolites used for signaling or defense. Fatty acids are activated for metabolic processing via a thioester linkage to either coenzyme A or acyl carrier protein. Acyl synthetases metabolically activate fatty acids to their thioester forms, and acyl thioesterases deactivate fatty acyl thioesters to free fatty acids by hydrolysis. These two enzyme classes therefore play critical roles in lipid metabolism. This review highlights the surprisingly complex and varying roles of fatty acyl synthetases in plant lipid metabolism, including roles in the intracellular trafficking of fatty acids. This review also surveys the many specialized fatty acyl thioesterases characterized to date in plants, which produce a great diversity of fatty acid products in a tissue-specific manner. While some acyl thioesterases produce fatty acids that clearly play roles in plant-insect or plant-microbial interactions, most plant acyl thioesterases have yet to be fully characterized both in terms of their substrate specificities and their functions. The biotechnological applications of plant acyl thioesterases and synthetases are also discussed, as there is significant interest in these enzymes as catalysts for the sustainable production of fatty acids and their derivatives for industrial uses.     

Preparation and surfactant properties of diglycerol esters of fatty acids

Fatty acid mono- and diesters of diglycerol constitute the major portion of commercial polyglycerol esters; hence, their composition influences the performance of the latter as emulsifiers. The correlation of structure of the fatty acids in the mono- and diesters with surfactant properties is of interest. Linear diglycerol was isolated from polymerized glycerol by acetonation, fractional distillation and regeneration. Diglycerol mono- and diesters of undecenoic, lauric, stearic, oleic and ricinoleic acids were prepared by reacting diglycerol and fatty acids in a refluxing mixture of acetonitrile-tetrahydrofuran (75:25) in the presence of p-toluenesulfonic acid and molecular sieves. Mono- and diesters were separated by silica gel column chromatography, and their purities were ascertained by thin layer chromatography and determination of saponification value. The structures were confirmed by periodic acid oxidation, chemical-ionization mass spectrometry and 13 C-NMR spectroscopy. Surfactant properties of the esters were determined. Monoesters showed higher ability in surface tension reduction, emulsification and foaming than the diesters. Short-chain fatty acid esters showed better surfactant properties than the long-chain fatty acid esters. The presence of a central double bond in the lipophilic part of the monoesters reduced emulsion stability. The presence of a hydroxy group in acyl chain retarded foaming and surface tension reducing power.     

Synthetic Base Stock Based on Guerbet Alcohols

Guerbet (β branched) alcohols of varying chain length of even carbon numbers were synthesized by using single linear fatty alcohols ranging from 1-octanol to 1-dodecanol. All Guerbet alcohols having fewer than 28 carbon atoms and are liquid at 0 °C due to β branching. Synthetic base oils were prepared by reacting commonly available unsaturated fatty acids and dicarboxylic acids with Guerbet alcohols using p-toluenesulfonic acid as a catalyst. These base oils were characterized by physical and tribological properties like viscosity, viscosity index, pour point, flash point, wear scar, weld load, coefficient of friction etc. and compared with commercially available 150 and 500 N base oils.     

Fatty acid composition of adultSchistosoma mansoni

The fatty acid composition of triglyceride and total phospholipid fractions of adultSchistosoma mansoni has been examined. Both triglyceride and phospholipid contained fatty acids varying in chain length from 12 through 24 carbons; trace amounts of shorter chain components were found in the triglyceride fraction. A docosahexaenoic acid in the triglyceride fraction represented the highest degree of unsaturation encountered. Branched chain fatty acids of 16 and 18 carbons were found in both phospholipid and triglyceride. Examination of fatty acids from fluke total lipid revealed the presence of small amounts of odd numbered carbon fatty acids varying in chain length from 13 through 23 carbons.     

Gas-liquid chromatographic analysis of cyclopropene fatty acids

A gas-liquid chromatographic method is described for the quantitative estimation of cyclopropene fatty acids as their methyl mercaptan derivatives. This method estimates individual cyclopropene acids as well as normal and cyclopropane acids. Nine seed oils were analyzed for their cyclopropene fatty acid content. Evidence was obtained for the presence of a cyclopropene fatty acid of shorter chain length than malvalic inAlthaea rosea cav and one with a higher chain length than sterculic inBombacopsis glabra seed oil. This method is less accurate for cottonseed oil than for the other oils tested because of the appearance of some unsymmetrical peaks of unknown origin. The mercaptan derivatives of the cyclopropene acids may be isolated by silver ion thin-layer chromatography. Small amounts of cyclopropane fatty acids were found in a number of the oils analyzed for cyclopropene fatty acids. Presented at the AOCS meeting, Chicago, October 1964.     

Polyhydroxy Fatty Acids Derived from Sophorolipids

Starting from 17-hydroxyoleic acid, which is readily available from acid alcoholysis of sophorolipids, several new polyhydroxy fatty acids have been synthesized. These compounds contain from 2 to 4 hydroxy groups, in some instances combined with other functional groups. The added hydroxy groups can be incorporated in the C₁₈ chain in a variety of geometries, for example spaced widely throughout the chain at C1, C8, and C17. This regiochemical control will be of use in structure/function studies involving materials constructed from these hydroxy fatty acids. A further benefit is that the hydroxy groups can be present in protected or free states. The principal reactions used to introduce extra hydroxy groups are selenium oxide-mediated allylic hydroxylation, osmium-catalyzed dihydroxylation, and borohydride reduction of a carboxylic ester. These new compounds are expected to be of use in a number of areas, but particularly as building blocks for polymers or components of lubricant formulations. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.     

DC-Trennung von Ricinusölfettsäure-Estoliden

TLC Separation of Estolides of Castor Oil Fatty Acids Estolides of castor oil fatty acids (polyricinoleic acids) with varying degrees of condensation were synthesized by heating the castor oil fatty acids at 120° C–240° C under vacuum and CO₂ circulation. These products were analyzed by chemical constants and fractionated by TLC on silicagel 60 precoated plates into ricinoleic acid and di-, tri- as well as tetra-ricinoleic acids. Furthermore, the estolides were separated by two-dimensional TLC into two series of estolides, i.e. estolides containing only ricinoleic acid and those which contain fatty acids other than ricinoleic acid at the chain terminal. Hydrogenated castor oil fatty acids (technical 12-hydroxystearic acid) also form estolides which can be fractionated in a similar manner. Thus, TLC provides information on the oligomeric and polymeric character of the estolides of castor oil fatty acids and permits separation even of the decamers.     

Sophorolipid-Derived Unsaturated and Epoxy Fatty Acid Estolides as Plasticizers for Poly(3-Hydroxybutyrate)

Unsaturated and epoxy fatty acid estolides were synthesized from the ω and ω‐1 hydroxy fatty acids derived from sophorolipids (SL). These estolides were utilized as additives in solution‐cast poly(3‐hydroxybutyrate) (P3HB) films and their plasticizing effects reported. Estolides in the P3HB film matrix resulted in slight reductions of melting and glass transition temperatures while the crystallinity remained relatively constant (±2.0 %). Scanning electron microscopy revealed irregular film surfaces in the presence of both estolides and the formation of pores within the P3HB film matrix. These irregularities influenced the tensile properties of the films by incrementally decreasing the tensile strength and moduli of the P3HB films and increasing their elongation. Curing the films for 3 months in the presence of estolide triggered an enhanced tensile strength and modulus when compared to the initial films. Irradiation was implemented in an attempt to crosslink the estolides. Results showed that radiation did not result in crosslinking but rather instigated chain scission and reduced the molecular weights by up to 81 % which further reduced the tensile strength, elongation and modulus of the P3HB films. These tensile property variations showed that while the tensile strength of the P3HB films declined in the presence of both estolides, the plasticity and elongation improved validating that these unique SL‐derived estolides can be effectively used as plasticizers in P3HB films.     

Comparison of catabolism rate of fatty acids to carbon dioxide in mice

The catabolism rates of a medium chain fatty acid (octanoic acid), an even‐numbered fatty acid (palmitic acid), and odd‐numbered fatty acids (pentadecanoic acid and heptadecanoic acid) in mice were compared using stable isotope (¹³C) labeled fatty acids and isotope‐ratio MS (IRMS). The catabolism rates of respective fatty acids were evaluated by the ratio of ¹³C and ¹²C in carbon dioxide expired from mice. The results show that the catabolism rate of octanoic acid is three times faster than that of palmitic acid. This result is in agreement with previous knowledge that medium chain fatty acids are easily beta‐oxidized as compared to long chain fatty acids. The catabolism rates of odd‐numbered fatty acids such as pentadecanoic acid and heptadecanoic acid were significantly lower as compared to those of even‐numbered fatty acids such as palmitic acid. This finding supports our previous report that odd‐numbered fatty acids are easily accumulated into body fat. The high accumulation of odd‐numbered fatty acids in body fat would be a direct result of their low beta‐oxidizability. Practical applications: ¹³C‐labeled fatty acids were administered to mice and the rates of ¹³CO₂ formation were compared among medium chain, even‐numbered, and odd‐numbered fatty acids using IRMS. We found that the catabolism rates of odd‐numbered fatty acids such as pentadecanoic acid and heptadecanoic acid were significantly lower in comparison to those of even‐numbered fatty acids such as palmitic acid. These findings could be valuable for the development of the lipid metabolism field.     

Different metabolism of saturated and unsaturated long chain plasma free fatty acids by intestinal mucosa of rats

During fat absorption, unsaturated long chain fatty acids are esterified at a higher rate than saturated fatty acids of similar chain length. This phenomenon has been attributed to differences in the binding affinity of fatty acids to a cytosolic fatty acid-binding protein. As intestinal mucosa utilizes plasma free fatty acids as well, we investigated whether long chainplasma free fatty acids of different degree of saturation are metabolized also at different rates.³H-Palmitic and¹⁴C-linoleic acid complexed to rat serum were injected rapidly into a tail vein of fasting rats. One, 2 and 4 min later there was no difference between³H and¹⁴C-radioactivity in intestinal mucosa, suggesting equal initial uptake of the two labeled fatty acids from plasma. Despite their equal uptake, the incorporation of the isotopes into ester lipids was significantly different, however: at 2 min, 53.1±3.9% of³H and 73.8±4.6% of¹⁴C were recovered in ester lipids. Phospholipids and triglycerides accounted for most of the mucosal³H and¹⁴C. At 4 min, a similar distribution of isotopes in intestinal mucosal metabolites was found. These data show that despite equal initial uptake by intestinal mucosa unsaturated long chain fatty acids taken up from plasma are esterified to a higher and oxidized to a lower extent than saturated plasma free fatty acids. Unsaturated plasma free fatty acids, therefore, may provide a more important source of fatty acids for endogenous intestinal lipoprotein lipids than saturated plasma free fatty acids. It is speculated that the fatty acid binding protein might be operative not only in the intracellular transport and metabolism of luminal fatty acids but of plasma free fatty acids as well.     

The question of differences between iodine numbers of coconut oil and of the corresponding soapstock fatty acids

Summary Experiments have been made on coconut oil from pure endosperm, pure testa, and normal mixtures of the two. These experiments have shown that the spread in iodine value between refined coconut oil and the fatty acids found on the corresponding soapstock are greater than can be accounted for by the proportion of testa oil present in extracted whole crude oils. Furthermore the iodine value of the free fatty acid fraction of pure endosperm oils was found to be higher than that of the combined fatty acids in the same oils by an amount which varied inversely as the degree of hydrolysis which had occurred in the oil. From this it appears that preferential hydrolysis plays an important part in the production of coconut oil soapstock having higher iodine values than those of the corresponding refined oils. Attention is also called to some European publications which deal with this question and to the possibility that molds may be involved through their ability to decompose short chain acids to ketones.     

Synthesis and physical properties of isostearic acids and their esters

Saturated branched‐chain fatty acids (sbc‐FAs) are found as minor constituents in several natural fats and oils. Sbc‐FAs are of interest since they have lower melting points than their linear counterparts and exhibit good oxidative stability; properties that make them ideally suited in a number of applications. We (and others) have previously synthesized sbc‐FAs by clay‐ or zeolite‐catalyzed isomerization of unsaturated linear‐chain fatty acids (ulc‐FAs) to unsaturated branched‐chain fatty acids (ubc‐FAs) that were subsequently hydrogenated to the desired sbc‐FAs. These acid‐catalyzed isomerization reactions, however, proceed in moderate conversion and selectivity. Recently, our group found that H‐Ferrierite zeolite catalyst isomerized ulc‐FAs to their branched‐chain counterparts in high conversion (>95%) and selectivity (85%). This paper reports the use of this type of catalyst for the preparation of a series of sbc‐FAs and their ester derivatives. Selected physical properties of these branched acids and esters such as cloud point (CP) and pour point (PP), cold filter plugging point (CFPP), viscosity index (VI), thermo‐oxidation stability, iodine value, and lubricity are also reported.     

Predicting the surface tension of biodiesel fuels from their fatty acid composition

The emergence of biodiesel fuels as diesel fuel substitutes has led to several studies on their properties. Surface tension, which plays a role in atomization, has lacked attention compared to other properties. This paper presents a method to predict the surface tension of biodiesel fuels based on the fatty acid composition. Several binary, ternary, and quaternary mixtures of fatty acid ethyl ester gas chromatographic (GC) standards were prepared, and we found that a mass-average equation predicted the surface tension of these mixtures within ±3.5% of their measured values. Six complex mixtures of fatty acid methyl ester GC standards that simulated typical oils used as biodiesel fuels were also prepared. For these complex mixtures the predicted surface tensions of the mixtures, calculated from a mass-average equation, were 2–6% higher than the measured values. A mass-average equation was developed in which we used a weighted surface tension for the individual components, and we found that this method predicted the surface tension of the simulated oils within ±4.5% of their measured values. Five natural vegetable oils were used to produce biodiesel fuels by the transesterification process. The predicted surface tensions of these fuels were all within ±3.5% of their measured values. The surface tensions of 15 biodiesel types were then predicted, based on their fatty acid composition as published in the literature. These results show that the differences in surface tension between biodiesel types are not the main cause of the reported differences in engine tests.     

Production and Emulsifying Effect of Polyglycerol and Fatty Acid Esters with Varying Degrees of Esterification

Esters with acyl groups can be formed by the esterification of polyglycerol. The purpose of the present study was to produce fatty acid esters [hexanoic (caproic), octanoic (caprylic), decanoic (capric), dodecanoic (lauric), tetradecanoic (myristic), hexadecanoic (palmitic), octadecanoic (stearic)] and polyglycerol (average number‐of degrees of polymerization of 5) with varying degrees of esterification and to examine their emulsifying properties. A number of fundamental catalysts of polyglycerol acylation reactions by methyl esters of carboxylic acid were studied, and sodium methoxide was found to be the best choice. The temperature rate of transesterification increased from 180 to 220 °C with the fatty acid chain alkyl residue. Synthesized mono‐, di‐, tri‐, tetra‐, and heptaesters of various fatty acids and polyglycerol provided the highest hydroxyl values from 15 to 815 mg KOH g^?1 and saponification values from 82 to 321 mg KOH g^?1. The emulsifying properties were assessed for all polyglycerol and fatty acid esters, with results showing maximum emulsifying effect for tri‐ and tetraesters of capric, lauric, and caprylic acids. Regardless of the hydrophilic–lipophilic balance value (HLB) of polyglycerol esters and carboxylic acid, a 4:1 ratio of sunflower oil to water formed a water‐in‐oil type emulsion. When mixing oil and water in a 1:1 ratio, mono‐ and diesters of polyglycerol formed an oil‐in‐water type emulsion, heptaesters formed a water‐in‐oil type emulsion, and tri‐ and tetraesters formed both of types of emulsions, depending on the length of the acid hydrocarbon radicals.     

Effect of substrate on sophorolipid properties

This research demonstrates that the formation of a crystalline sophorolipid product, by Candida bombicola can be attributed to two overlapping types of discrimination by the enzymes of this yeast. The first of these is a preference for direct incorporation of hydrocarbons having a chain length between 15 and 18 carbon atoms if these are in the medium. The second is a preference for the formation of the diacetylated lactone structure if hydroxycarboxylic acids that are 16 or 17 carbon atoms long are attached to the disaccharide. The combined effect is to produce a product mixture that contains a large amount of a single structure of sophorolipid that can result, in a crystalline product if either hexadecane or heptadecane is the sole lipophilic carbon source. Surface tension measurements of the various components isolated from the sophorolipid mixtures showed that the mono-and diacetylated lactones were the most effective surfactants. This is consistent with literature reports that the lactone form of the sophorolipids is more useful for a number of applications than the open acidic structure. The surface tension behavior of some of the sophorolipids demonstrated patterns similar to those usually observed for carboxylic acids and other sparingly soluble amphipathic compounds.     

Tissue distribution of phytanic acid and its analogues in a kinship with Refsum's disease

Three of six kin were identified, by high performance thin layer chromatography, capillary gas chromatography and mass spectrometry, as having phytanic acid storage disease. Phytanic acid was found in triacylglycerol and, to a lesser degree, in phosphatidylcholine and free fatty acids. An unsaturated analogue of phytanic acid was additionally identified in plasma and erythrocyte triacylglycerols. In plasma, branched chain fatty acids were primarily localized in the low density lipoprotein fraction. The concentration of plasma major fatty acids was not affected by the presence of these branched chain fatty acids. In contrast to plasma, only small amounts of phytanic acid were found in cerebrospinal fluid and biopsied sural nerve. The nerve phytanate was mainly associated with triacylglycerol in epineurial and perineurial tissues. Lack of phytanate accumulation in sural endoneurium, even in cases with severe fiber degeneration, suggests that demyelination in Refsum's disease may not be due to myelin instability resulting from the incorporation of branched chain fatty acids into peripheral nerve membrane.     

Differently sized native milk fat globules separated by microfiltration: fatty acid composition of the milk fat globule membrane and triglyceride core

The aim of this study was to characterize the fatty acid composition of the core and membrane of differently sized milk fat globules separated by microfiltration, which can now be used to manufacture dairy products. Native milk fat globules of various mean diameters, ranging from d₄₃ = 2.3 µm to 8.0 µm, were obtained using microfiltration of raw whole milk. After milk fat globule washing, the milk fat globule membrane (MFGM) and the triglyceride core (TC) were separated by manual churning. After total lipid extraction from each fraction, their fatty acid composition was characterized using methyl ester analysis by gas chromatography. Regardless of season, no significant differences were observed in the fatty acid composition of the MFGM phospholipids. Conversely, significant differences were found in the fatty acid composition of TC; particularly, small fat globule TC contained more medium‐chain fatty acids and less stearic acid than large fat globule TC. These results show that the previously observed differences in total fatty acid composition among differently sized milk fat globules are due to their triglyceride composition and MFGM amount rather than to the composition of the MFGM.     

Engineering Oilseed Plants for a Sustainable,Land-Based Source of Long Chain Polyunsaturated Fatty Acids

Numerous clinical studies have demonstrated the cardiovascular and mental health benefits of including very long chain omega-3 polyunsaturated fatty acids, namely eicospentaenoic acid (EPA) and docosohexaenoic acid (DHA) in the human diet. Certain fish oils can be a rich source of omega-3 long chain polyunsaturated fatty acids although processed marine oils are generally undesirable as food ingredients because of the associated objectionable flavors and contaminants that are difficult and cost-prohibitive to remove. Oilseed plants rich in omega-3 fatty acids, such as flax and walnut oils, contain only the 18-carbon omega-3 polyunsaturated fatty acid alpha-linolenic acid, which is poorly converted by the human body to EPA and DHA. It is now possible to engineer common omega-6 rich oilseeds such as soybean and canola to produce EPA and DHA and this has been the focus of a number of academic and industrial research groups. Recent advances and future prospects in the production of EPA and DHA in oilseed crops are discussed here.     

Sophorolipid Production by <Emphasis Type="Italic">Starmerella bombicola</Emphasis> (ATCC 22214) from Virgin and Waste Frying Oils,and the Effects of Activated Earth Treatment of the Waste Oils

Frying is one of the most common processes in households, restaurants and food industries. During frying, oil undergoes degradation and hence has to be replaced time to time. This creates large amounts of waste causing disposal and environmental problems. Sophorolipids are produced by microbial bioconversion of refined vegetable oils along with glucose. The raw material cost accounts for 10–30% of the overall cost of biosurfactant production that can be reduced by using a low cost substrate like waste frying oil. In the present work, waste frying oils were used in the production of sophorolipids at the shake flask level. It gave mainly (70–80%) the acidic form of sophorolipids. It was observed that the linoleic acid was preferentially consumed over other fatty acids by the organism (Starmerella bombicola). The activated earth treatment was found to improve the yield of sophorolipids and hence the treatment can be used to convert waste frying oil as a low cost substrate into a cost effective carbon source.