Microbial production of rare unsaturated fatty acids and alcohols

Aeromonas hydrophila N‐6, isolated from a soil sample, converted vegetable oils to several rare unsaturated fatty acids and alcohols accumulated inside the cells as a wax ester form. A. hydrophila N‐6 effectively decreased fatty acid chain lengths, and converted rapeseed, safflower and linseed oils into 7‐16:1 and 5‐14:1 fatty acids, 7,10‐16:2 and 5,8‐14:2 fatty acids, and 7,10,13‐16:3 fatty acids, respectively. Furthermore, A. hydrophila N‐6 reduced the resulting fatty acids to rare unsaturated fatty alcohols, such as 7‐16:1, 5‐14:1, 9,12‐18:2, 7,10‐16:2, 9,12,15‐18:3 and 7,10,13‐16:3. Such unsaturated fatty acids and alcohols are rarely found in natural oils. Because decreasing fatty acid carbon chain lengths from the carboxyl end and reducing unsaturated fatty acids to unsaturated fatty alcohols in industrially applicable scale are both difficult reactions to accomplish by chemical means, we suggest that A. hydrophila N‐6 may facilitate the introduction of new bioprocesses for producing rare unsaturated fatty acids and alcohols, especially fatty alcohols with more than two double bonds.     

A Method for Identifying Germplasm that Exhibit Multiple Mutations in the Fatty Acid Synthetic Pathway of Perilla frutescens

A method using gas chromatography–mass spectroscopy (GC–MS) was developed to assess phenotypic differences in the fatty acid composition of 143 accessions of Perilla frutescens varieties that originated from different Chinese provinces, Japan, and Britain. Thirty-six compounds identified in the analysis included a number of saturated fatty acids and positional isomers of unsaturated fatty acids. A wide range in phenotypic variation was noted within each of five major fatty acids [16:0, 6.8–11.5%; 18:0, 1.7–11.9%; 18:1 (n-9), 12.3–28.0%; 18:2 (n-9,12), 9.6–21.6%; 18:3 (n-9,12,15), 41.4–56.6%]. Many accessions exhibited potential gene mutations or differences in gene copy number for multiple steps of fatty acid synthesis. For example, PCA models were developed to distinguish groups of accessions that exhibited low-16:0, low-18:1, high-18:3 traits, and others that exhibited high-16:0, high-18:1, low-18:3 traits. We believe this approach should improve the process for the identification of genetic resources that help reveal the complex nature of biological regulation not only of fatty acid metabolism but other metabolic processes as well.     

Pathways for fatty acid elongation and desaturation in Neurospora crassa

Neurospora crassa incorporated exogenous deuterated palmitate (16∶0) and ¹⁴C-labeled oleate (18∶1^Δ9) into cell lipids. Of the exogenous 18∶1^Δ9 incorporated, 59% was desaturated to 18∶2^Δ9,12 and 18∶3^Δ9,12,15. Of the exogenous 16∶0 incorporated, 20% was elongated to 18∶0, while 37% was elongated and desaturated into 18∶1^Δ9, 18∶2^Δ9,12, and 18∶3^Δ9,12,15. The mass of unsaturated fatty acids in phospholipid and triacylglycerol is 12 times greater than the mass of 18∶0. Deuterium label incorporation in unsaturated fatty acids is only twofold greater than in 18∶0, indicating a sixfold preferential use of 16∶0 for saturated fatty acid synthesis. These results indicate that the release of 16∶0 from fatty acid synthase is a key control point that influences fatty acid composition in Neurospora.     

Conversion of palmitate to unsaturated fatty acids differs in a Neurospora crassa mutant with impaired fatty acid synthase activity

The Neurospora crassa cel (fatty acid chain elongation) mutant has impaired fatty acid synthase activity. The cel mutant requires exogenous 16:0 for growth and converts 16:0 to other fatty acids. In contrast to wild-type N. crassa, which converted only 42% of the exogenous [7,7,8,8-²H₄]16:0 that was incorporated into cell lipids to unsaturated fatty acids, cel converted 72%. In addition, cel contains higher levels of 18:3^δ9,12,15 than wild-type, and synthesizes two fatty acids, 20:2^δ11,14 and 20:3^δ11,14,17, found at only trace levels in wild-type. Thus, the Δ15-desaturase activity and elongation activity on 18-carbon polyunsaturated fatty acids are higher for cel than wild-type. This altered metabolism of exogenous 16:0 may be directly due to impaired flux through the endogenous fatty acid biosynthetic pathway, or may result from altered regulation of the synthesis of unsaturated fatty acids in the mutant.     

GC-FID/MS Analysis of Fatty Acids in Indian Cultivars of Moringa oleifera: Potential Sources of PUFA

In the present investigation, the fatty acid profile was analysed in vegetative and reproductive parts of eight commercially cultivated Indian cultivars of Moringa oleifera and verified by gas chromatography mass spectra. In leaves, α-linolenic acid (C_18:3, cis-9,12,15) was found in the highest quantity (49–59 %) followed by palmitic acid (C_16:0) (16–18 %), and linoleic acid (C_18:2, cis-9,12) (6–13 %). The total content of saturated fatty acids and unsaturated fatty acids showed a ratio of 0.33 (cv. DHANRAJ) to 0.39 (cv. PKM-2) in leaves, 0.53 in flowers and 0.56 in tender pods. Similarly, polyunsaturated fatty acids and total monounsaturated fatty acids were found in a ratio of 5.68 (cv. DHANRAJ) to 9.71 (cv. CO-1) in leaves, 1.11 in flowers and 2.79 in tender pods. The total lipid content was recorded in the range of 1.92 % (flowers) to 4.82 % (leaves, cv. CO-1). When considering health benefits, M. oleifera leaves contain low amounts of saturated fatty acids, a high mono- and polyunsaturated fatty acid content, which can enhance the health benefits of Moringa-based products.     

Synthesis and Surface Active Properties of Sulfonated Acrylate Esters Based on Al‐Cedre Oil

Sulfonated acrylate esters have been synthesized by using renewable raw materials such as fatty alcohols of Al‐Ceder oil. Mixed fatty acids were isolated from Al‐Ceder oil by hydrolysis; both saturated and unsaturated fatty acids were isolated from the mixed fatty acids. The methyl esters of mixed fatty acid, saturated and unsaturated acids of Al‐Cedre oil were subjected to reduction with (LiAlH4) to give the corresponding fatty alcohols. The products of the reduction process were saponified and the hydroxyl values were estimated to further confirm the reduction occurrence. The acrylate esters were synthesized by esterification of acrylic acid with fatty alcohols of C_16:0, C_18:0, C_18:1, and C_18:2 mixed saturated, mixed unsaturated and mixed fatty acids of Al‐Cedre oil, respectively. This esterification was followed by addition of NaHSO₃ to form bisulfite adducts. The structures of the prepared surfactants were characterized by IR and ¹HNMR spectroscopy. A series of useful surface parameters, stability towards acids and base hydrolysis and calcium stability have been determined.     

中国南海总状蕨藻中脂肪酸和甾醇GC-MS分析

采用GC—MS分析中国南海总状蕨藻中的脂肪酸及甾醇化合物,鉴定出二十三种脂肪酸及四种甾醇。其中脂肪酸主要为十六碳酸(34．42％)、9,12,15-十八碳三烯酸(10．39％)、9,12-十八碳二烯酸(7．63％)、7,10．13-十六三烯酸(5．60％);甾醇主要为穿贝海绵甾醇。     

Overexpression of a FAD3 Desaturase Increases Synthesis of a Polymethylene-Interrupted Dienoic Fatty Acid in Seeds of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> L.

A cDNA encoding the Arabidopsis extraplastidic linoleate desaturase (FAD3) was overexpressed in the seeds of wild-type Arabidopsis and in a mutant line that accumulates high levels of oleic acid. In the transformed wild-type plants, linolenic acid (18:3Δ9,12,15) increased from 19% to nearly 40% of total seed fatty acids, with a corresponding decrease in linoleate content (18:2Δ9,12). In the high oleate mutant, a large increase in the level of a fatty acid identified by gas-chromatography/mass-spectrometry as mangiferic acid (18:2Δ9,15) was observed. The results demonstrate that the polymethylene-interrupted dienoic fatty acid, mangiferic acid, can be produced in seed oil through the overexpression of a fatty acid n-3 desaturase.

Omega-Functionalized Fatty Acids,Alcohols, and Ethers via Olefin Metathesis

Methyl 17-hydroxy stearate was converted to methyl octadec-16-enoate using copper sulfate adsorbed on silica gel. This compound served as a useful substrate for the olefin metathesis reaction. As a result, several fatty acids with novel functional groups at the ω-end were prepared: a glyceryl ether attached at the 18-carbon, an aromatic fatty acid from eugenol, and a ferrocenyl fatty acid. By employing the unsaturated fatty alcohol, other groups were introduced, namely the terminal fluoride, bromide, and iodide were prepared, as was a thiol derivative. The penultimate and omega olefins reported here should serve as building blocks that allow fatty acids to make a greater contribution to a range of emerging technological areas.     

TheTrans-6 fatty acids ofPicramnia sellowii seed oil

The C₁₈ monoenoic acids inPicramnia sellowii Planch. seed oil include bothcis-andtrans-6-octadecenoic acids, as well as oleic acid. The hexadecenoic acids are also thecis- andtrans-Δ6-isomers, and the eicosenoic acids have Δ6-unsaturation of undetermined geometric configuration. The C₁₈ polyenoic acids detected are 9,12- and 6,9-octadecadienoic and 9,12,15- and 6,9,12-octadecatrienoic acids. Partial investigation of another species,P. pentandra Sw., revealed its oil to have a similar fatty acid composition. Presented in part at AOCS Meeting, New York, October 1968. No. Utiliz. Res. Dev. Div., ARS, USDA.     

X-Ray diffraction study of sodium soaps of monounsaturated and polyunsaturated fatty acids

X-Ray powder diffraction patterns of the sodium soaps of 14 monounsaturated and polyunsaturated fatty acids were obtained at room temperature. The patterns of the soaps of 9,12-trans,trans-octadecadienoic acid, 11,14-cis,cis-eicosadienoic acid 11,14,17-allcis-eicosatrienoic acid and 5 monounsaturated fatty acids were typical of the crystalline lamellar phase. The patterns of the soaps of 9,12-cis,cis-octadecadienoic, allcis-9,12,15-octadecatrienoic, allcis-8,11,14-eicostrienoic, allcis-5,8,11,14-eicosatetraenoic, allcis-5,8,11,14,17-eicosapentaenoic and allcis-4,7,10,13,16,19-docosahexaenoic acids were indicative of the less ordered forms reported for sodium oleate at elevated temperature. The diffraction data from the less ordered soaps are consistent with the melted form of the hydrocarbon chains of the unsaturated acids at room temperature.     

Characterization of wax esters in the roe oil of amber fish,Seriola aureovittata

The wax esters of the roe oil of the amber fish,Seriola aureovittata, have been resolved by high-performance liquid chromatography (HPLC) in the silver-ion mode. Each of the fractions collected was transmethylated, and the fatty acids and alcohols were identified by gas chromatography/mass spectrometry (GC/MS) as the picolinyl esters and nicotinates, respectively. Their compositions were determined by GC. The fatty acid composition is complex, and the main components are C18:1n-9 (35.5 mol%), C22:6n-3 (20.3 mol%), and C16:1n-7 (10.7 mol%), while fatty alcohols are limited to saturated (C16:0, 60.3 mol%; C18:0, 15.3 mol%; C14:0, 5.1 mol%) and monoenoic alcohols (C18:1n-9, 6.5 mol%; C16:1n-7, 4.5 mol%) with traces (<0.1 mol%) of polyunsaturated fatty alcohols such as C20:3n-3, C20:4n-6, C20:5n-3, and C22:5n-3. Silver-ion HPLC exhibited excellent resolution in which fractions were resolved on the basis of the number and configuration of double bonds as well as the distribution pattern between the acid and alcohol moieties of the molecules with a given number of double bonds. The main wax ester fraction are those of monoenoic acid-saturated alcohol species, hexaenoic acid/saturated alcohol species, and pentaenoic acid/saturated alcohol species. Appreciable specificity was observed in the esterification of fatty acids with alcohols, and surprisingly, no saturated acid-monoenoic alcohol species were detected.     

Fatty Acyl-CoA Reductase and Wax Synthase from <Emphasis Type="Italic">Euglena gracilis</Emphasis> in the Biosynthesis of Medium-Chain Wax Esters

Euglena gracilis, a unicellular phytoflagellate, can accumulate a large amount of medium-chain wax esters under anaerobic growth conditions. Here we report the identification and characterization of two genes involved in the biosynthesis of wax esters in E. gracilis. The first gene encodes a fatty acyl-CoA reductase (EgFAR) involved in the conversion of fatty acyl-CoAs to fatty alcohols and the second gene codes for a wax synthase (EgWS) catalyzing esterification of fatty acyl-CoAs and fatty alcohols, yielding wax esters. When expressed in yeast (Saccharomyces cerevisiae), EgFAR converted myristic acid (14:0) and palmitic acid (16:0) to their corresponding alcohols (14:0Alc and 16:0Alc) with myristic acid as the preferred substrate. EgWS utilized a broad range of fatty acyl-CoAs and fatty alcohols as substrates with the preference towards myristic acid and palmitoleyl alcohol. The wax biosynthetic pathway was reconstituted by co-expressing EgFAR and EgWS in yeast. When myristic acid was fed to the yeast, myristyl myristate (14:0–14:0), myristyl palmitoleate (14:0–16:1), myristyl palmitate (14:0–16:0) and palmityl myristate (16:0–14:0) were produced. These results indicate EgFAR and EgWS are likely the two enzymes involved in the biosynthesis of medium-chain wax esters in E. gracilis.     

Chemical Composition and Fatty Acid Profile of Khat (Catha edulis) Seed Oil

The proximate, physicochemical, and fatty acid compositions of seed oil extracted from khat (Catha edulis) were determined. The oil, moisture, crude protein, crude fiber, crude carbohydrate, and ash content in seeds were 35.54, 6.63, 24, 1.01, 30.4 %, and 1.32 g/100 g DW respectively. The free fatty acids, peroxide value, saponification value, and iodine value were 2.98 %, 12.65 meq O₂/kg, 190.60 mg KOH/g, and 145 g/100 g oil, respectively. Linolenic acid (C_18:3, 50.80 %) and oleic (C_18:1, 16.96 %) along with palmitic acid (C_16:0, 14.60 %) were the dominant fatty acids. The seed oil of khat can be used in industry for the preparation of liquid soaps and shampoos. Furthermore, high levels of unsaturated fatty acids make it an important source of nutrition especially as an animal product substitute for omega‐3 fatty acids owing to the high content of linolenic acid.     

Neutral lipids of chickpea flour and protein isolates

The neutral lipids composition of defatted chickpea flour and two types of protein isolates has been studied. The main compounds in neutral lipids are triacylglycerols, free fatty acids, and diacylglycerols. Other compounds present are wax esters, free fatty alcohols, and free sterols. The main fatty acids in neutral lipids are C_18:2 and C_18:1 among the unsaturated, and C_16:0 and C_18:0 among the saturated acids. Free and esterified alcohols range from C_16:0 to C_28:0, the majority being those with an even number of carbon atoms. Sterols observed are β-sito-sterol, campesterol, stigmasterol, and δ-5-avenasterol. Triacyl-glycerols are partially hydrolyzed, and the amounts of unsaturated sterols and unsaturated fatty acids are reduced as a result of the chemical treatment during production of the protein isolates.     

Specificity of papaya lipase in esterification of aliphatic alcohols a comparison with microbial lipases

Straight-chain saturated C4 to C18 alcohols and unsaturated C18 alcohols such as cis-9-octadecenyl (oleyl) cis-6-octadecenyl (petroselinyl), cis-9, cis-12-octadecadienyl (linoleyl), all-cis-9,12,15-octadecatrienyl (α-linolenyl), and all-cis-6,9,12-octadecatrienyl (γ-linolenyl) alcohols, were esterified with caprylic acid using papaya (Carica papaya) latex lipase (CPL) and immobilized lipase from Candida antarctica (Lipase B, Novozym, NOV) and Rhizomucor miehei (Lipozyme, LIP) as biocatalysts. With CPL, highest activity was found for octyl and decyl caprylate syntheses, whereas both NOV and LIP showed a broad chain-length specificity toward the alcohol substrates. CPL strongly discriminated against all C18 alcohols studied, relative to n-hexanol, whereas the microbial lipases accepted the C18 alcohols as substrates nearly as well as n-hexanol. Both petroselinyl and γ-linolenyl alcohol were very well accepted as substrates by NOV as well as LIP, although the corresponding fatty acids, i.e., petroselinic and γ-linolenic acid, are strongly discriminated against by several microbial and plant lipases, including LIP and CPL.     

Determination of the Fatty Acid Composition of Acorn (Quercus), Pistacia lentiscus Seeds Growing in Algeria

The fruits of two plants from Algeria (Quercus and Pistacia lentiscus) were investigated. The paper reports the chemical characteristics and the fatty acid composition of the oil extracts from the fruits. The black fruits of P. lentiscus has the highest crude fat of 32.8%, followed by the red fruits with 11.7%, and the lowest value of 9% in Quercus (acorn). The acid value was highest in red fruits of P. lentiscus oil (24.0 mg KOH/g), followed by the black fruits oil and lowest in acorn oil. The relatively high iodine value in the oils indicates the presence of many unsaturated bonds. Saponification value was highest in the Quercus ilex oil (166.7 mg KOH/g), while the lowest value was in the black fruits of P. lentiscus oil. Gas-liquid chromatography revealed that the three dominant fatty acids found are: palmitic C16:0 (16.3–19.5%), oleic C18:1 (55.3–64.9%), linoleic C18:2 (17.6–28.4%). The oils contain an appreciable amount of unsaturated fatty acids (78.8–83.5%).     

γ-Linolenic acid inAcer seed oils

The octadecatrienoic acids inAcer negundo L. (maple family) seed oil include both 9,12,15- (1%) and 6.9,12-(7%) isomers. The chief monoenoic acids identified were 9-octadecenoic (21%), 11-eicosenoic (7%), 13-docosenoic (15%), and 15-tetracosenoic (7%). Also present is a considerable amount of 9,12-octadecadienoic acid. Investigation of ten other Aceraceae revealed their seed oils to have a similar fatty acid composition.     

Divergent Response of Murine and Porcine Adipocytes to Stimulation of Browning Genes by 18‐Carbon Polyunsaturated Fatty Acids and Beta‐Receptor Agonists

Long‐chain fatty acids (LCFA) are known to activate brown and beige adipocytes. However, very little is known about the effects of the number and the position of double bonds in LCFA with the same length on brown fat‐specific gene expression. To determine the specificity of LCFA in the regulation of these genes in different adipocyte models, fully differentiated 10T1/2, 3T3‐L1, murine, or porcine primary adipocytes (obtained from the subcutaneous fat pad of C57BL/6 mice or Landrace × Yorkshire × Duroc crossbred piglets) were treated with 50 μM of the following 18‐carbon fatty acids: stearic acid (STA; 18:0), oleic acid (OLA; 18:1, Δ9), linoleic acid (LNA; 18:2, Δ9,12), α‐linolenic acid (ALA; 18:3, Δ9,12,15), γ‐linolenic acid (GLA; 18:3, Δ6,9,12), or pinolenic acid (PLA; 18:3, Δ5,9,12) for 24 h with or without 4‐h norepinephrine (NE) treatment. Expression levels of thermoregulatory markers were measured by quantitative real‐time PCR. LNA, ALA, GLA, and PLA upregulated Ucp1 expression and tended to upregulate Pgc1a expression in murine primary adipocytes, but not in 10T1/2, 3T3‐L1, and porcine primary adipocytes. In murine primary adipocytes, NE induced a higher expression of Ucp1 and Pgc1a than non‐NE‐treated cells, and PLA augmented the NE effect. In 10T1/2 cells, NE upregulated Ucp1 and Pgc1a expression, but there was no fatty acid effect. However, 3T3‐L1 cells were insensitive to both fatty acid and beta‐adrenergic agonist stimulation. These results indicate that different adipocyte cell types have different levels of sensitivity to both LCFA and beta agonists in regard to induction of brown fat‐specific gene expression.