Biochemical characterization of lipid metabolic genes of Aurantiochytrium limacinum.

Docosahexaenoic acid (DHA) is known to have numerous health benefits and immense dietary value. There is a pressing need to have a deeper understanding of DHA metabolism. Acyl CoA: Diacylglycerol Acyltransferase (DGAT) is an important enzyme of lipid anabolism and an essential piece of the puzzle. Aurantiochytrium limacinum, a primary producer of DHA, is a good model for studying DHA metabolism. Thus, we aimed to investigate important lipid metabolic genes from A. limacinum. We cloned four putative DGATs (DGAT2a, DGAT2b, DGAT2c, and DGAT2d) from A. limacinum and performed detailed in vivo and in vitro characterization. Functional characterization showed that not all the studied genes exhibited DGAT activity. DGAT2a and DGAT2d conferred DGAT activity whereas DGAT2b showed wax synthase (WS) activity and DGAT2c showed dual function of both WS and DGAT. Based on their identified function, DGAT2b and DGAT2c were renamed as AlWS and AlWS/DGAT respectively. DGAT2a was found to exhibit a preference for DHA as a substrate. DGAT2d was found to have robust activity and emerged as a promising candidate for genetic engineering aimed at increasing oil yield. The study enriches our knowledge of lipid biosynthetic enzymes in A. limacinum, which can be utilized to design suitable application strategies.

Extraction, fatty acid profile, phytochemical composition and antioxidant activities of fixed oils from spices belonging to Apiaceae and Lamiaceae family.

Fixed oil, the non-volatile fraction of oil from spices remains an unexplored entity. The study aimed to extract fixed oils from 13 Indian spices belonging to the Apiaceae and Lamiaceae family. Further fatty acid composition, antioxidant activities and phytochemical profile of the fixed oils was estimated. Among the studied spices, Ferula assa-foetida had the highest amount of fixed oil (19.93%). GC-MS profiling of the fixed oils showed palmitic, stearic, oleic, linoleic and alpha-linolenic to be the major fatty acids. The study further identified fixed oils from Trachyspermum ammi L., Petroselinum crispum L., Ocimum basilicum L. and Rosmarinus officinalis L. to be major source of protocatecheuic, 4,hydroxybenzoic, trans-cinnamic, myristein and trans-ferulic acid. R. officinalis, O. basilicum and Thymus vulgaris L. fixed oils also showed highest radical scavenging property. T. vulgaris, Majorana hortensis Moench. and O. Basilicum fixed oils confirmed high amounts of α-, ß + γ- and δ-tocopherol respectively. ß-sitosterol was found to be the dominating phytosterol in all fixed oils. Principal component analysis revealed existence of variation among spice fixed oils concerning to their fixed oil composition. The study thus identifies spice fixed oils as a rich source of lipid soluble bioactive compounds that are of tremendous industrial and pharmaceutical importance. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13197-021-05036-1.

Profiling of Lipids, Nutraceuticals, and Bioactive Compounds Extracted from an Oilseed Rich in PUFA.

Polyunsaturated fatty acid (PUFA) rich vegetable oils are nutritionally and economically important agriculture based commodities. The lipid profile, nutraceutical content, and antioxidant activity of Indian chia seed oil (CSO) were analysed and compared with PUFA rich vegetable oils. The total oil content was 28% (w/w) with α-linolenic acid (ALA; 65%) as the predominant fatty acid and a n-3/n-6 ratio of 3.5. The tocopherol content was 144 mg/kg of oil, with γ + ß being the most abundant. The squalene content was 178.47 mg/100 g of oil, and the total phenolic content was 0.014 mg GAE/g of oil. The identity of major polyphenols in the methanolic extract of CSO were established by LC-HRMS. FTIR spectra of CSO exhibited characteristic features that were identical to other PUFA rich oils. Results demonstrate that the Indian CSO is an excellent source of essential fatty acids and key nutraceuticals.

Physicochemical characterization and nutraceutical compounds of the selected spice fixed oils.

Spices and herbs are well appreciated for their medicinal properties since ancient times. Till date, spices are being explored for volatile oils (essential), flavour and for addressing many chronic diseases. In the present study, we investigated the physicochemical properties, fatty acid composition, differential scanning calorimetry (DSC), elemental composition and nutraceutical compounds of fixed oils (non-volatile) from five selected spices viz., Alpinia galanga, Cinnamomum zeylanicum, Trigonella foenum-graecum, Foeniculum vulgare, and Myristica fragrans. The fixed oil (FO) content of volatiles-free powders of the five selected spices ranged from 1.58% (C. zeylanicum) to 26.43% (M. fragrans). The studied FO showed a good quality index which was analysed by estimation of free fatty acids, iodine value and unsaponifiable matter. The fatty acid analysis showed high palmitic acid in the FO of A. galanga and C. zeylanicum. High linoleic, oleic, and myristic acid levels were observed in T. foenum-graecum, F. vulgare and M. fragrans FOs, respectively. The nutraceutical compounds such as total phenolics were high in C. zeylanicum FO (0.53%). Hence the studied FO could be an excellent alternative to oil nutraceutical compounds. It may be used as a functional ingredient in foods which needs further validation for value addition.

Biochemical Characterization of Acyl-CoA: Lysophosphatidylcholine Acyltransferase (LPCAT) Enzyme from the Seeds of Salvia hispanica.

Salvia hispanica (chia) is the highest reported terrestrial plant source of alpha-linolenic acid (ALA, ~ 65%), an ω-3 polyunsaturated fatty acid with numerous health benefits. The molecular basis of high ALA accumulation in chia is yet to be understood. We have identified lysophosphatidylcholine acyltransferase (LPCAT) gene from the developing seed transcriptome data of chia and carried out its biochemical characterization through heterologous expression in Saccharomyces cerevisiae. Expression profiling showed that the enzyme was active throughout the seed development, indicating a pivotal role in oil biosynthesis. The enzyme could utilize both saturated and unsaturated lysophosphatidylcholine substrates at the same rate, to synthesize phosphatidylcholine (PC). The enzyme also exhibited lysophosphatidic acid acyltransferase (LPAAT) activity, by preferring lysophosphatidic acid substrate. Substrate specificity studies showed that the enzyme preferred both monounsaturated and polyunsaturated fatty acyl CoAs over saturated CoAs. This activity may play a key role in enriching the PC fraction with polyunsaturated fatty acids (PUFAs). PUFAs present on PC can be transferred to oil through the action of other acyltransferases. Our results describe a new LPCAT enzyme that can be used to biotechnologically alter oilseed crops to incorporate more PUFA in its seed oil.

Functional characterization of acyltransferases from Salvia hispanica that can selectively catalyze the formation of trilinolenin.

Salvia hispanica (chia) is an important oilseed crop cultivated commercially in South America, Australia, and India. It is the richest terrestrial natural source of α-linolenic acid (ALA), an ω-3 polyunsaturated fatty acid with varied health benefits. In this study, we have measured the total lipid content, fatty acid composition in four phases of seed development and analyzed the major triacylglycerol (TAG) molecular species present in Indian chia seed oil. We found that the mature seeds produced 28% oil, 65% of ALA, and trilinolenin as the major TAG species. To make TAG rich in ALA, there should be specialized enzymes that can efficiently transfer ALA to TAG. To study this hypothesis, we performed a characterization of TAG synthesizing enzymes present in chia. We have identified two acyl CoA:diacylglycerol acyltransferases (ShDGAT1 and ShDGAT2) and one phospholipid:diacylglycerol acyltransferase (ShPDAT1) from the chia transcriptome data. Functional characterization of these enzymes was conducted by heterologous expression in a TAG deficient mutant of Saccharomyces cerevisiae. Substrate specificity studies showed that ShDGAT2-1 and ShPDAT1 exhibited a strong preference towards substrates containing ALA and could incorporate 45% and 80% ALA into TAG, respectively. Both enzymes incorporated ALA in a concentration-dependent manner into TAG and were able to form trilinolenin in yeast. Our results provide a first insight into the high ALA accumulation in chia and the first demonstration of trilinolenin formation by DGAT2. The two identified enzymes (ShDGAT2-1 and ShPDAT1) can be used to metabolically engineer other oilseed crops to produce high levels of ALA.

Novel Cell-Based Assay to Investigate Monoacylglycerol Acyltransferase 2 Inhibitory Activity Using HIEC-6 Cell Line.

The dietary triacylglycerol (TAG) gets absorbed and accumulated in the body through the monoacylglycerol (MAG) pathway, which plays a major role in obesity and related disorders. The main enzyme of this pathway, monoacylglycerol acyltransferase 2 (MGAT2), is considered as a potential target for developing antiobesity compounds. Hence, there is a need for in vitro cell-based assays for screening the potential leads for MGAT2 inhibitors. Because of synthetic inhibitor's side effects, there is an increased interest in natural extracts as potential leads. Hence, we have optimized a 2-MAG-induced TAG accumulation inhibitory cell-based assay to screen natural extracts using the HIEC-6 cell line. A concentration-dependent TAG accumulation was observed when the HIEC-6 cells were fed with exogenous 2-MAG. The TAG accumulation was confirmed by in situ BODIPY staining and was quantified. However, no TAG accumulation was seen when the cells were fed with exogenous DAG or TAG, suggesting MGAT2-mediated MAG uptake and its conversion to TAG. We demonstrated the utility of this assay by screening five different plant-based aqueous extracts. These extracts showed various inhibition levels (25% to 30%) of 2-MAG-induced TAG accumulation in the HIEC-6. The MGAT2 inhibitory potential of these extracts was confirmed by an in vitro MGAT2 assay. This cell-based assay adds a new methodology for screening, developing, and evaluating MGAT2 inhibitors for addressing obesity and related disorders.

Spice fixed oils as a new source of γ-oryzanol: nutraceutical characterization of fixed oils from selected spices.

γ-Oryzanol is an important group of nutraceuticals that play a key role in addressing metabolic disorders. This study, for the first time, examined volatile-free spice fixed oils (FOs) as an alternate plant source for γ-oryzanol and other nutraceuticals (phenolics, flavonoids, phytosterols, and tocopherols) using HPLC, HR-MS and NMR. The in vitro antioxidant activities of FOs were also analysed. The selected spices were Alpinia galanga, Cinnamomum zeylanicum, Trigonella foenum-graecum, Foeniculum vulgare and Myristica fragrans. The major polyphenols and flavonoids quantified were gallic, protocatechuic, vanillic, syringic, para-coumaric, ferulic, rutin, trans-cinnamic, and quercetin. T. foenum-graecum FOs recorded high levels of ergosterol (48.56 mg/100 g) and stigmasterol (247.36 mg/100 g). The fucosterol levels were high in A. galanga (268.31 mg/100 g) FOs, whereas C. zeylanicum FOs showed high content of ß-sitosterols (7037.77 mg/100 g). C. zeylanicum and T. foenum-graecum FOs recorded high α-tocopherol content (47.55 and 15.96 mg/100 g respectively). C. zeylanicum FOs showed high levels of three ferulates, namely, cycloartenyl ferulate, 24-methylene cycloartenyl ferulate and ß-sitosteryl ferulate, whose contents were 89.42, 170.23 and 50.23 mg/100 g respectively which was confirmed by HRMS with a molecular mass (m/z) of 601.45, 615.47, and 589.45 respectively. Further, γ-oryzanol ferulates in C. zeylanicum FOs were confirmed by 1H-NMR analysis. The acidified methanolic extractives of FOs showed high free radical scavenging activity and antioxidant potential. These spice FOs have excellent antioxidant activities, and are novel potential functional ingredients against lifestyle disorders.

Chemical composition, nutraceuticals characterization, NMR confirmation of squalene and antioxidant activities of Basella rubra L. seed oil.

Basella rubra (Malabar spinach) is a commonly consumed green leafy vegetable in southern parts of India. The chemical composition, nutraceuticals characterization, squalene Nuclear Magnetic Resonance (NMR), in vitro antioxidant activities and cytotoxicity of B. rubra seed oil (33.08%) was investigated. Gas chromatography-mass spectrometry (GC/MS) analysis revealed the presence of palmitic (27.21 µmol%), oleic (33.83 µmol%) and linoleic acid (26.02 µmol%) with a total of 64.38 µmol% unsaturated fatty acids respectively. HPLC nutraceutical characterization showed a major constituent of gallic acid (11.23 mg%), γ-tocopherols (17.74 mg%), cycloartenylferulate (1.7 mg%), and squalene (1 g%). Squalene was further recovered (98%), purified (99.9%), and confirmed through 1H and 13C NMR. The in vitro antioxidant activities recorded by using 2,2-diphenyl-1-picrylhydrazyl (EC50 = 6 mg mL-1), ferric reducing antioxidant power (361.85 mM of Trolox Eq./100 g) and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (EC50 = 56.19 mg mL-1) scavenging activity. In vitro cytotoxicity assessed on 3T3-L1 showed good cell survival without any toxicity (upto 400 µg mL-1). B. rubra seed oil has proven nutraceuticals and antioxidant potentials with least toxicity which can be recommended for functional foods applications.

Selection and validation of appropriate reference genes for quantitative real-time PCR analysis in Salvia hispanica.

Quantitative real-time polymerase chain reaction (qRT-PCR) has become the most popular choice for gene expression studies. For accurate expression analysis, it is pertinent to select a stable reference gene to normalize the data. It is now known that the expression of internal reference genes varies considerably during developmental stages and under different experimental conditions. For Salvia hispanica, an economically important oilseed crop, there are no reports of stable reference genes till date. In this study, we chose 13 candidate reference genes viz. Actin11 (ACT), Elongation factor 1-alpha (EF1-α), Eukaryotic translation initiation factor 3E (ETIF3E), alpha tubulin (α-TUB), beta tubulin (ß-TUB), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Cyclophilin (CYP), Clathrin adaptor complex (CAC), Serine/threonine-protein phosphatase 2A (PP2A), FtsH protease (FtsH), 18S ribosomal RNA (18S rRNA), S-adenosyl methionine decarboxylase (SAMDC) and Rubisco activase (RCA) and the expression levels of these genes were assessed in a diverse set of tissue samples representing vegetative stages, reproductive stages and various abiotic stress treatments. Two of the widely used softwares, geNorm and Normfinder were used to evaluate the expression stabilities of these 13 candidate reference genes under different conditions. Results showed that GAPDH and CYP expression remain stable throughout in the different abiotic stress treatments, CAC and PP2A expression were relatively stable under reproductive stages and α-TUB, PP2A and ETIF3E were found to be stably expressed in vegetative stages. Further, the expression levels of Diacylglycerol acyltransferase (DGAT1), a key enzyme in triacylglycerol synthesis was analyzed to confirm the validity of reference genes identified in the study. This is the first systematic study of selection of reference genes in S. hispanica, and will benefit future expression studies in this crop.

A distinct DGAT with sn-3 acetyltransferase activity that synthesizes unusual, reduced-viscosity oils in Euonymus and transgenic seeds.

Endosperm and embryo tissues from the seeds of Euonymus alatus (Burning Bush) accumulate high levels of 3-acetyl-1,2-diacyl-sn-glycerols (acTAGs) as their major storage lipids. In contrast, the aril tissue surrounding the seed produces long-chain triacylglycerols (lcTAGs) typical of most other organisms. The presence of the sn-3 acetyl group imparts acTAGs with different physical and chemical properties, such as a 30% reduction in viscosity, compared to lcTAGs. Comparative transcriptome analysis of developing endosperm and aril tissues using pyrosequencing technology was performed to isolate the enzyme necessary for the synthesis of acTAGs. An uncharacterized membrane-bound O-acyltransferase (MBOAT) family member was the most abundant acyltransferase in the endosperm but was absent from the aril. Expression of this MBOAT in yeast resulted in the accumulation of acTAGs but not lcTAG; hence, the enzyme was named EaDAcT (Euonymus alatus diacylglycerol acetyltransferase). Yeast microsomes expressing EaDAcT possessed acetyl-CoA diacylglycerol acetyltransferase activity but lacked long-chain acyl-CoA diacylglycerol acyltransferase activity. Expression of EaDAcT under the control of a strong, seed-specific promoter in Arabidopsis resulted in the accumulation of acTAGs, up to 40 mol % of total TAG in the seed oil. These results demonstrate the utility of deep transcriptional profiling with multiple tissues as a gene discovery strategy for low-abundance proteins. They also show that EaDAcT is the acetyltransferase necessary and sufficient for the production of acTAGs in Euonymus seeds, and that this activity can be introduced into the seeds of other plants, allowing the evaluation of these unusual TAGs for biofuel and other applications.

Isolation of a gene encoding a 1,2-diacylglycerol-sn-acetyl-CoA acetyltransferase from developing seeds of Euonymus alatus.

1,2-Diacyl-3-acetyl-sn-glycerols (ac-TAG) are unusual triacylglycerols that constitute the major storage lipid in the seeds of Euonymus alatus (Burning Bush). These ac-TAGs have long-chain acyl groups esterified at both the sn-1 and sn-2 positions of glycerol. Cell-free extracts of developing seeds of E. alatus contain both long-chain acyl-CoA and acetyl-CoA sn-1,2-diacylglycerol acyltransferase (DGAT) activity. We have isolated a gene from developing seeds of Euonymus alatus that shows a very high sequence similarity to the members of the DGAT1 gene family (i.e. related to acyl-CoA:cholesterol acyltransferases). This Euonymus DGAT1 gene, when expressed in wild type yeast, results in a 5-fold enhancement of long-chain triacylglycerol (lc-TAG) accumulation, as well as the appearance of low levels of ac-TAG. Hydrogenated ac-TAG molecular species were identified by gas chromatography-mass spectrometry. Microsomes isolated from this transformed yeast show diacylglycerol:acetyl-CoA acetyltransferase activity, which is about 40-fold higher than that measured in microsomes prepared from yeast transformed with the empty vector or with the Arabidopsis thaliana DGAT1 gene. The specific activity of this microsomal acetyltransferase activity is of the same order of magnitude as the microsomal long-chain DGAT activities measured for yeast lines transformed with the empty vector or either the Arabidopsis or Euonymus DGAT1 genes. Despite this, ac-TAG accumulation in yeast transformed with the Euonymus DGAT1 gene was very low (0.26% of lc-TAG), whereas lc-TAG accumulation was enhanced. Possible reasons for this anomaly are discussed. Expression of the Euonymus DGAT1-like gene in yeast lines where endogenous TAG synthesis has been deleted confirmed that the gene product has both long-chain acyl- and acetyltransferase activity.

Acetyl coenzyme A concentrations in plant tissues.

Despite the importance of acetyl coenzyme A in many facets of metabolism and the availability of methods for estimation of its concentration, data for acetyl-CoA concentrations in plant tissues have been very scarce. A method using reversed phase HPLC for the quantitative estimation of acetyl-CoA was applied to a variety of plant tissues. In three different developing oilseeds the bulk acetyl-CoA concentration ranged from 5 to 25 nmoles/g fresh weight. In Arabidopsis thaliana leaves it was 5 nmoles/g fresh weight, and in Spinacia oleracea leaves 6.8 nmoles/g fresh weight. Immediate quenching of the harvested tissue in liquid nitrogen is needed to obtain high recoveries of acetyl-CoA.

Isolation of lysophosphatidic acid phosphatase from developing peanut cotyledons.

The soluble fraction of immature peanut (Arachis hypogaea) was capable of dephosphorylating [(3)H]lysophosphatidic acid (LPA) to generate monoacylglycerol (MAG). The enzyme responsible for the generation of MAG, LPA phosphatase, has been identified in plants and purified by successive chromatography separations on octyl-Sepharose, Blue Sepharose, Superdex-75, and heparin-agarose to apparent homogeneity from developing peanuts. This enzyme was purified 5,048-fold to a final specific activity of 858 nmol min(-1) mg(-1). The enzyme has a native molecular mass of approximately 39 kD determined by gel filtration and migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a subunit molecular mass of 39 +/- 1.5 kD. The K(m) values for oleoyl-, stearoyl-, and palmitoyl-sn-glycerol-3-phosphate were determined to be 28.6, 39.3, and 47.9 microM, respectively. The LPA phosphatase was specific to LPA and did not utilize any other substrate such as glycerol-3-phosphate, phosphatidic acid, or p-nitrophenylphosphate. The enzyme activity was stimulated by the low concentrations of detergents such as Triton X-100 and octylglucoside. Cations had no effect on the enzyme activity. Fatty acids, sphingosine, and sphingomyelin at low concentrations stimulated the enzyme activity. The identification of LPA phosphatase in plants demonstrates the existence of MAG biosynthetic machinery in plants.