TAG Accumulation Research Articles

Metabolically engineered rice biomass and grain using genes associated with lipid pathway show high level of oil content.

Increasing lipid content using metabolic engineering methods in different parts of plant, including, leaves and stem can be considered as an innovative platform for achieving more energy and biofuel in more green habits. Two key enzymes, including, diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT) catalyze the final step of TAG assembly. WRINKLED1 (WRI1) is one of the important transcription factors which regulate the fatty acid biosynthesis network and TAG accumulation by balancing carbon flux between carbohydrates and lipids. In addition, oleosin encoding gene (OLE) can protect TAGs from degradation by packing into oil bodies.In the current study, four important genes involved in TAG assembly and protection (i.e., AtDGAT1 and AtPDAT, AtWRI1, and AtOle) were overexpressed under a constitutive promoter in rice crop. TAG content of transgenic seeds increased significantly (P ≤ 0.05) by 26% in compared with those of control plants. Oleic and palmitic acid contents were significantly increased by 28% (from 32 to 41) and 27% (11 to 14) in seeds of transgenic plants in compared with controls, respectively. Our results showed an increase in the total grain and leaf oil contents by 70% (from 1.1 to 1.87%) and 22.5% (from 1.88 to 2.3%) in the metabolically engineered lines, respectively. This is the first report of transformation in rice for enhancing oil content and energy density in its seeds and vegetative parts. Such metabolically engineered crops would be cultivated for production much more oils in seeds and straw for food and biodiesel consequently.

Effects of dietary carbohydrate sources on lipid metabolism and SUMOylation modification in the liver tissues of yellow catfish.

Dysregulation in hepatic lipid synthesis by excess dietary carbohydrate intake is often relevant with the occurrence of fatty liver; therefore, the thorough understanding of the regulation of lipid deposition and metabolism seems crucial to search for potential regulatory targets. In the present study, we examined TAG accumulation, lipid metabolism-related gene expression, the enzyme activities of lipogenesis-related enzymes, the protein levels of transcription factors or genes involving lipogenesis in the livers of yellow catfish fed five dietary carbohydrate sources, such as glucose, maize starch, sucrose, potato starch and dextrin, respectively. Generally speaking, compared with other carbohydrate sources, dietary glucose promoted TAG accumulation, up-regulated lipogenic enzyme activities and gene expressions, and down-regulated mRNA expression of genes involved in lipolysis and small ubiquitin-related modifier (SUMO) modification pathways. Further studies found that sterol regulatory element binding protein 1 (SREBP1), a key transcriptional factor relevant to lipogenic regulation, was modified by SUMO1. Mutational analyses found two important sites for SUMOylation modification (K254R and K264R) in SREBP1. Mutant SREBP lacking lysine 264 up-regulated the transactivation capacity on an SREBP-responsive promoter. Glucose reduced the SUMOylation level of SREBP1 and promoted the protein expression of SREBP1 and its target gene stearoyl-CoA desaturase 1 (SCD1), indicating that SUMOylation of SREBP1 mediated glucose-induced hepatic lipid metabolism. Our study elucidated the molecular mechanism of dietary glucose increasing hepatic lipid deposition and found that the SREBP-dependent transactivation was regulated by SUMO1 modification, which served as a new target for the transcriptional programmes governing lipid metabolism.

High level accumulation of EPA and DHA in field-grown transgenic Camelina - a multi-territory evaluation of TAG accumulation and heterogeneity.

The transgene‐directed accumulation of non‐native omega‐3 long chain polyunsaturated fatty acids in the seed oil of Camelina sativa (Camelina) was evaluated in the field, in distinct geographical and regulatory locations. A construct, DHA2015.1, containing an optimal combination of biosynthetic genes, was selected for experimental field release in the UK, USA and Canada, and the accumulation of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) determined. The occurrence of these fatty acids in different triacylglycerol species was monitored and found to follow a broad trend irrespective of the agricultural environment. This is a clear demonstration of the stability and robust nature of the transgenic trait for omega‐3 long chain polyunsaturated fatty acids in Camelina. Examination of non‐seed tissues for the unintended accumulation of EPA and DHA failed to identify their presence in leaf, stem, flower, anther or capsule shell material, confirming the seed‐specific accumulation of these novel fatty acids. Collectively, these data confirm the promise of GM plant‐based sources of so‐called omega‐3 fish oils as a sustainable replacement for oceanically derived oils.

Maternal conjugated linoleic acid consumption prevented TAG alterations induced by a high-fat diet in male adult rat offspring.

Maternal nutritional programming by a high-fat (HF) diet is related to hepatic lipid accumulation and steatosis in offspring. Conjugated linoleic acid (CLA) might ameliorate impaired hepatic lipid homoeostasis; therefore, the aim was to investigate the potential preventive effect of maternal CLA consumption on TAG metabolism alterations induced by HF diets in adult male rat offspring receiving or not receiving CLA. Female Wistar rats were fed a control (C) diet, HF diet or HF diet supplemented with CLA (HF+CLA) for 4 weeks before mating and throughout pregnancy and lactation. After weaning, for 9 weeks, male offspring of C or HF rats continued with the same diets as their mothers (C/C or HF/HF groups, respectively) and male offspring of HF+CLA rats were fed HF or HF+CLA diets (HF+CLA/HF or HF+CLA/HF+CLA groups, respectively). Nutritional parameters, serum and liver TAG levels, the TAG secretion rate (TAG-SR) and the activities as well as gene expression of key hepatic enzymes involved in TAG regulation were assessed. The most interesting results were that maternal CLA decreased epididymal white adipose tissue weight and prevented serum and liver TAG accumulation induced by a HF diet in adult male offspring receiving or not receiving CLA. The prevention of liver steatosis in HF+CLA/HF+CLA and HF+CLA/HF offspring was associated with an increased hepatic TAG-SR. Overall, this study provides evidence that maternal CLA consumption programmes TAG regulation and in this way contributes to lowering lipid levels in tissues and preventing liver steatosis in particular.

The Deletion of AMPK β1 Worsens Glucose Tolerance and Liver Lipid Accumulation, but Doesn’t Impact the Metabolic Effects of Olanzapine in Male Mice

Second generation antipsychotic (SGA) medications, like olanzapine (OLZ), are an important component in the pharmacological management of many conditions including schizophrenia. These drugs, have been linked with a neuroendocrine signaling axis that, leads to weight gain, however, peripheral mechanisms and predictors are currently poorly understood. 5’AMP activated protein kinase (AMPK) is a ubiquitously expressed, energy‐sensing enzyme and significant associations between polymorphisms in AMPK subunit genes and OLZ‐induced weight gain have been demonstrated. Conversely, others have suggested that the central activation of AMPK mediates the deleterious metabolic effects of SGAs. The purpose of this study was to determine if reductions in AMPK activity, primarily in the liver, would impact the chronic effects of OLZ treatment. We hypothesized that the absence of AMPK β1, a subunit which is primarily found in the liver, at least in mice, would worsen the metabolic side effects of OLZ. To address this question, we housed male AMPK β1 knockout (KO) and wildtype (WT) mice at thermoneutrality (29°C) and fed them either a control diet (HFD, 45% kcal from fat) or a diet supplemented with OLZ (HFD+OLZ, 50mg/kg diet) for 10 weeks. OLZ treatment did not increase food intake or weight gain to a greater extent than the control diet in either genotype. There was a main effect of the OLZ containing diet to increase heat production, TEE and fat oxidation during the light and dark cycles. The OLZ‐fed mice also demonstrated suppressed RER during the dark cycle. Interestingly, the OLZ‐fed KO mice were more physically active during the light cycle than the OLZ‐fed WT mice. The AMPK β1 KO mice were more glucose intolerant than the WT control mice. On the other hand, the AMPK β1 KO male mice on both diets demonstrated lower circulating serum glycerol and NEFA levels but increased liver TAG accumulation. In conclusion, our findings provide evidence that the absence of AMPK β1 does not potentiate the effects of OLZ on weight gain, but worsens liver lipid accumulation and glucose tolerance.Support or Funding InformationThis study was supported by Canadian Institutes of Health Research

Increasing growth and yield by altering carbon metabolism in a transgenic leaf oil crop.

SummaryEngineering high biomass plants that produce oil (triacylglycerol or TAG) in vegetative rather than seed‐related tissues could help meet our growing demand for plant oil. Several studies have already demonstrated the potential of this approach by creating transgenic crop and model plants that accumulate TAG in their leaves and stems. However, TAG synthesis may compete with other important carbon and energy reserves, including carbohydrate production, and thereby limit plant growth. The aims of this study were thus: first, to investigate the effect of TAG accumulation on growth and development of previously generated high leaf oil tobacco plants; and second, to increase plant growth and/or oil yields by further altering carbon fixation and partitioning. This study showed that TAG accumulation varied with leaf and plant developmental stage, affected leaf carbon and nitrogen partitioning and reduced the relative growth rate and final biomass of high leaf oil plants. To overcome these growth limitations, four genes related to carbon fixation (encoding CBB cycle enzymes SBPase and chloroplast‐targeted FBPase) or carbon partitioning (encoding sucrose biosynthetic enzyme cytosolic FBPase and lipid‐related transcription factor DOF4) were overexpressed in high leaf oil plants. In glasshouse conditions, all four constructs increased early growth without affecting TAG accumulation while chloroplast‐targeted FBPase and DOF4 also increased final biomass and oil yields. These results highlight the reliance of plant growth on carbon partitioning, in addition to carbon supply, and will guide future attempts to improve biomass and TAG accumulation in transgenic leaf oil crops.

Dietary protein insufficiency: an important consideration in fatty liver disease?

Dietary protein insufficiency has been linked to excessive TAG storage and non-alcoholic fatty liver disease (NAFLD) in developing countries. Hepatic TAG accumulation following a low-protein diet may be due to altered peroxisomal, mitochondrial and gut microbiota function. Hepatic peroxisomes and mitochondria normally mediate metabolism of nutrients to provide energy and substrates for lipogenesis. Peroxisome biogenesis and activities can be modulated by odd-chain fatty acids (OCFA) and SCFA that are derived from gut bacteria, for example, propionate and butyrate. Also produced during amino acid metabolism by peroxisomes and mitochondria, propionate and butyrate concentrations correlate inversely with risk of obesity, insulin resistance and NAFLD. In this horizon-scanning review, we have compiled available evidence on the effects of protein malnutrition on OCFA production, arising from loss in mitochondrial, peroxisomal and gut microbiota function, and its association with lipid accumulation in the liver. The methyl donor amino acid composition of dietary protein is an important contributor to liver function and lipid storage; the presence and abundance of dietary branched-chain amino acids can modulate the composition and metabolic activity of the gut microbiome and, on the other hand, can affect protective OCFA and SCFA production in the liver. In preclinical animal models fed with low-protein diets, specific amino acid supplementation can ameliorate fatty liver disease. The association between low dietary protein intake and fatty liver disease is underexplored and merits further investigation, particularly in vulnerable groups with dietary protein restriction in developing countries.

Directed evolution of a bacterial WS/DGAT acyltransferase: improving tDGAT from Thermomonospora curvata.

Some bacteria belonging to the actinobacteria and proteobacteria groups can accumulate neutral lipids expressing enzymes of the wax ester synthase/acyl coenzyme A: diacylglycerol acyltransferase (WS/DGAT) family. tDGAT is a WS/DGAT-like enzyme from Thermomonospora curvata able to produce TAGs and WEs when heterologously expressed in Escherichia coli. In this study, a protocol for the directed evolution of bacterial lipid-producing enzymes based on fluorimetry is developed and tested. tDGAT has been successfully evolved towards the improvement of TAG production with an up to 2.5 times increase in TAG accumulation. Mutants with no ability to produce TAGs but able to accumulate waxes were also selected during the screening. The localization of the mutations that enhance TAG production in the outer surface of tDGAT points out possible new mechanisms that contribute to the activity of this family of enzymes. This Nile red-based high throughput screening provides an evolution platform for other WS/DGAT-like enzymes.

Fish oil supplementation during adolescence attenuates metabolic programming of perinatal maternal high-fat diet in adult offspring.

Perinatal maternal high-fat diet (HFD) increases susceptibility to obesity and fatty liver diseases in adult offspring, which can be attenuated by the potent hypolipidaemic action of fish oil (FO), an n-3 PUFA source, during adult life. Previously, we described that adolescent HFD offspring showed resistance to FO hypolipidaemic effects, although FO promoted hepatic molecular changes suggestive of reduced lipid accumulation. Here, we investigated whether this FO intervention only during the adolescence period could affect offspring metabolism in adulthood. Then, female Wistar rats received isoenergetic, standard (STD: 9 % fat) or high-fat (HFD: 28·6 % fat) diet before mating, and throughout pregnancy and lactation. After weaning, male offspring received the standard diet; and from 25 to 45 d old they received oral administration of soyabean oil or FO. At 150 d old, serum and hepatic metabolic parameters were evaluated. Maternal HFD adult offspring showed increased body weight, visceral adiposity, hyperleptinaemia and decreased hepatic pSTAT3/STAT3 ratio, suggestive of hepatic leptin resistance. FO intake only during the adolescence period reduced visceral adiposity and serum leptin, regardless of maternal diet. Maternal HFD promoted dyslipidaemia and hepatic TAG accumulation, which was correlated with reduced hepatic carnitine palmitoyl transferase-1a content, suggesting lipid oxidation impairment. FO intake did not change serum lipids; however, it restored hepatic TAG content and hepatic markers of lipid oxidation to STD offspring levels. Therefore, we concluded that FO intake exclusively during adolescence programmed STD offspring and reprogrammed HFD offspring male rats to a healthier metabolic phenotype in adult life, reducing visceral adiposity, serum leptin and hepatic TAG content in offspring adulthood.

Transcriptomic and lipidomic analysis of an EPA-containing Nannochloropsis sp. PJ12 in response to nitrogen deprivation

To understand genes involved in neutral lipid accumulation upon nitrogen deprivation (ND) in a novel isolate of Nannochloropsis sp. PJ12, we performed comparative transcriptomic and lipidomic analyses of cells under ND and NR (nitrogen replete) conditions. Transcriptomic profiling indicated that, while enzymes involved in TCA cycle in PJ12 under ND condition were upregulated compared to that under NR condition, those involved in Calvin cycle and glycolysis under ND condition were downregulated. Furthermore, we showed that enzymes involved in fatty acid synthesis and glycerolipid synthesis were downregulated but not β-oxidation. Lipidomic profiling indicated that, while the level of neutral lipids in ND cells was increased compared to that of NR cells, level of photosynthetic membrane-lipids DGDG and PG was decreased. Taken together, our analysis indicated that TAG accumulation is attributed to the modification of membrane lipids derived primarily from “prokaryotic” pathway and secondarily from “eukaryotic” pathway based on the 16:X or 18:X fatty acid at the sn2 position of the glycerol backbone. We propose that two-phase (NR-ND) growth is ideal for biomass and biofuel production because ND reduces cell growth rate due to the loss of photosynthetic membrane and decreased quantum yield.

Различие влияния липополисахарида E. coli , олеиновой кислоты и этомоксира на экспрессию белка ADRP в эпителиальных клетках

Intracellularly neutral lipids are deposited in lipid bodies (LB) – special organelles composed of triglyceride or cholesterol esters core surrounded by a phospholipid monolayer. LBs play a central role in cellular lipid metabolism. The biogenesis of LB and consumption of neutral lipids are controlled by proteins of the PAT family, which are expressed on the surface of LB and regulate lipids storage and degradation providing the interaction of LB with mitochondria and access of lipases to their substrates. The ADRP (adipose differentiation-related protein), a member of the PAT family, is a quantitatively major LB surface protein expressed in many cell types. The goal of the present work was to study the involvement of ADRP in the formation of LB and intracellular TAG accumulation under the action of different stimuli –lipopolysaccharide (LPS), exogenous oleic acid and etomoxir, an inhibitor of carnitine-palmitoyltransferase 1. The experiments were performed on epithelial cells isolated from the mucosal surface of the frog urinary bladder. Incubation of the cells for 21 hours with E. coli LPS (25 μg / ml), oleic acid (50 μM) and etomoxir (100 μM) led to a significant increase in the number and size of LBs and an increase in intracellular TAG accumulation. Despite the fact that all the substances used effectively stimulated LB growth, only incubation with LPS was accompanied by a sharp increase in the expression of ADRP protein, whereas in the presence of etomoxir or oleic acid the expression of this protein did not change. The obtained results indicate that the molecular mechanisms underlying the increase in LB formation and TAG accumulation under the action of different stimuli in the same cell type can be differed in relation to the involvement of LB surface proteins.

Increasing lipid production using an NADP+-dependent malic enzyme from Rhodococcus jostii.

The occurrence of NADP+-dependent malic enzymes (NADP+-MEs) in several Rhodococcus strains was analysed. The NADP+-ME number in Rhodococcus genomes seemed to be a strain-dependent property. Total NADP+-ME activity increased by 1.8- and 2.6-fold in the oleaginous Rhodococcus jostii RHA1 and Rhodococcus opacus PD630 strains during cultivation under nitrogen-limiting conditions. Total NADP+-ME activity inhibition by sesamol resulted in a significant decrease of the cellular biomass and lipid production in oleaginous rhodococci. A non-redundant ME coded by the RHA1_RS44255 gene located in a megaplasmid (pRHL3) of R. jostii RHA1 was characterized and its heterologous expression in Escherichia coli resulted in a twofold increase in ME activity in an NADP+-dependent manner. The overexpression of RHA1_RS44255 in RHA1 and PD630 strains grown on glucose promoted an increase in total NADP+-ME activity and an up to 1.9-foldincrease in total fatty acid production without sacrificing cellular biomass. On the other hand, its expression in Rhodococcus fascians F7 grown on glycerol resulted in a 1.3-1.4-foldincrease in total fatty acid content. The results of this study confirmed the contribution of NADP+-MEs to TAG accumulation in oleaginous rhodococci and the utility of these enzymes as an alternative approach to increase bacterial oil production from different carbon sources.

Structural and functional insights into putative TAG accumulating hydrolase protein (Rv1179c) of Mycobacterium tuberculosis H37Rv

Mycobacterium tuberculosis persistence in the host is one of the major challenges towards treating tuberculosis. A dedicated set of lipid metabolism genes enable it to resist anti-tuberculosis drugs. Host TAG accumulation is one such property displayed by Mycobacterium. We analyzed expression profile of mycobacterial genes under stress condition inducing TAG accumulation. TAG accumulation was accompanied by overexpression of Rv1179c. Structural analysis of Rv1179c suggests hydrolase like activity of Rv1179c. Hydrolases are involved in host lipid import and TAG accumulation.

Dissociation of Systemic Glucose Homeostasis from Triacylglyceride Accumulation by Reduced Acsl6 Expression in Skeletal Muscle

Long chain acyl-CoA synthetase (ACSL) is an enzyme that activates fatty acids before they are further metabolized. ACSL6 is the one of main ACSL isoforms exclusively expressed in skeletal muscle, but the consequences of the suppression of this gene in systemic glucose homeostasis has yet to be reported. Hence, we investigated the roles of ACSL6 gene in glucose tolerance and TAG distribution in physiological conditions. Eight-week-old male C57BL/6J mice were administered with control or Acsl6 siRNAs and then fed with either AIN-93 control diet or high fat diet. At seven days after the first siRNA injection, oral glucose tolerance tests and TAG quantification were performed. In vivo administration of Acsl6 siRNA decreased Acsl6 expression only in skeletal muscle under AIN-93 or a high fat diet. However Acsl6 siRNA injection to animals increased TAG accumulation in the liver without the change of Acsl6 expression. Atelocollagen mediated Acsl6 suppression enhanced whole-body glucose tolerance coinciding with decreased TAG accumulation in skeletal muscle of mice fed an AIN-93 diet. However, the improved glucose tolerance by Acsl6 reduction was ablated by high fat diet. Moreover reduced Acsl6 did not alter the phosphorylation of insulin signaling proteins in skeletal muscle. These results suggest that Acsl6 reduction in skeletal muscle enhances glucose homeostasis and dissociates the insulin responses from TAG accumulation in skeletal muscle.

The \u0394F/Fm\u2032-guided supply of nitrogen in culture medium facilitates sustainable production of TAG in Nannochloropsis oceanica IMET1

BackgroundTriacylglycerol (TAG) from photosynthetic microalgae is a sustainable feedstock for biodiesel production. Physiological stress triggers microalgal TAG accumulation. However excessive physiological stress will impair the photosynthesis system seriously thus decreasing TAG productivity because of the low biomass production. Hence, it is critical to quantitatively and timely monitor the degree of the stress while the microalgal cells growing so that the optimal TAG productivity can be obtained.ResultsThe lack of an on-line monitored indicator has limited our ability to gain knowledge of cellular “health status” information regarding high TAG productivity. Therefore, to monitor the degree of nitrogen stress of the cells, we investigated the correlation between the photosynthetic system II (PS II) quantum yield and the degree of stress based on the high relevancy between photosynthetic reduction and nitrogen stress-induced TAG accumulation in microalgal cells. ΔF/Fm′, which is the chlorophyll fluorescence parameter that reflects the effective capability of PS II, was identified to be a critical factor to indicate the degree of stress of the cells. In addition, the concept of a nitrogen stress index has been defined to quantify the degree of stress. Based on this index and by monitoring ΔF/Fm′ and guiding the supply of nitrogen in culture medium to maintain a stable degree of stress, a stable and efficient semi-continuous process for TAG production has been established.ConclusionThe results indicate that the semi-continuous cultivation process with a controlled degree of stress by monitoring the ΔF/Fm′ indicator will have a significant impact on microalgal TAG production, especially for the outdoor controllable cultivation of microalgae on a large scale.

Simultaneous increase in cellular content and volumetric concentration of lipids in Bracteacoccus bullatus cultivated at reduced nitrogen and phosphorus concentrations

Manipulation of the nutrient concentration is an inexpensive and efficient method for increasing lipid and TAG accumulation in algal cells. However, high volumetric production requires finding a proper balance between the decrease of biomass production and the increase in the total lipid content. We isolated a strain of green microalga Bracteacoccus bullatus and increased its lipid content from 17 to 59% of biomass dry weight by manipulating of nitrogen and phosphorus content in the medium. The 10-fold reduction of the nitrogen and phosphorus concentration in the medium was the most efficient method of the lipid induction compared to nutrient deplete and high nutrient conditions. The oleic (48–64% mass of total fatty acids) and linoleic (14–24% mass of total fatty acids) acids dominated in the fatty acid profile, thus making this strain a suitable candidate for biodiesel production.

Heterogeneous expression of human PNPLA3 triggers algal lipid accumulation and lipid droplet enlargement

Microalgal metabolic engineering holds great promise for algal biofuels. However, identification of the key lipid metabolic target remains challenging due to its complex regulation. In this study, we advocated an alternative strategy that potentially rewired lipid metabolism by unprecedented mechanisms. PNPLA3, a human protein associated with non-alcoholic fatty liver disease (NAFLD), was firstly tested in microalgae for enhancing lipid accumulation. HsPNPLA3 was synthesized with a site mutation (I148M) and expressed in a model diatom Phaeodactylum tricornutum. Heterogeneous HsPNPLA3-I148M was successfully integrated, transcribed and expressed. Lipidomic analyses revealed that HsPNPLA3-I148M significantly elevated TAG content by 1.55-fold in algae, while algal growth and photosynthetic rate were not impaired. Fatty acid profile showed that content of C16:0, C18:1 and C20:4 was increased by 1.43-, 4.18- and 4.3-fold, respectively, which implied that HsPNPLA3-I148M might regulate the fatty acid substrate preference. Overall, the findings demonstrated that human PNPLA3 played a potential role in elevating TAG accumulation by regulating lipogenic enzymes and provide unprecedented insights into its functional significance.

Enhancing the enzymatic saccharification of bamboo shoot shell by sequential biological pretreatment with Galactomyces sp. CCZU11-1 and deep eutectic solvent extraction.

In this study, sequential biological pretreatment (BP) with Galactomyces sp. CCZU11-1 at 30°C for 3days and deep eutectic solvent (DES) choline chloride: oxalic acid (ChCl:OA, 1mol/2mol) extraction at 120°C for 1.5h was used for pretreating BSS. It was found that combination pretreatment could effectively remove xylan and lignin for enhancing enzymatic saccharification. The reducing sugars and glucose from the hydrolysis of 100g/L pretreated BSS with complexed cellulases of Galactomyces sp. CCZU11-1 were obtained in the yields of 81.0% and 74.1%, respectively. The BSS-hydrolyzates had no inhibitory effects on the lipid-accumulating microorganism Bacillus sp. CCZU11-1, and the cell mass and TAG accumulation were 4.8g CDW/L and 2.2g TAG/L, respectively. Fatty acids including palmitic acid (C16:0; 25.3%), palmitoleic acid (C16:1; 24.4%), stearic acid (C18:0; 15.1%), and oleic acid (C18:1; 21.6%) were accumulated in cells. Clearly, this combination pretreatment has high potential application in future.

Comparative transcriptome analysis of lipid biosynthesis in seeds and non-seed tissues of sea buckthorn

Sea buckthorn (Hippophae L.) is a woody crop with an unusual ability to thrive in very dry, nutrient-poor habitats. Seeds and berry pulp are rich in bioactive compounds with powerful medicinal properties, and are being used worldwide to an increasing extent. However, genomic information is still lacking for this crop, which limits the possibilities of raise the oil quality and yield with advanced breeding program. In this study, (1) sea buckthorn de novo transcriptome libraries for seeds, pulp, leaf, stem and root tissues were first constructed simultaneously, and 62,166 unigenes were assembled. Sixty-six differential expression genes (e-value cutoff <1 × 10−30) were found to be involved in lipid biosynthesis pathway, (2) based on the comparative transcriptome data, we first revealed the differentially expressed genes of oil biosynthesis and accumulation in seeds and berry pulp, and the high unigene expression of source gene ‘GPD1’ (3.61) contributed to synthesis more G3P of TAG precursor in pulp, and the high expression of sink genes ‘DGAT1 and DGAT2’ (14.57 and 12.57, respectively) accelerated high TAG accumulation in pulp, compared to their expressions in seeds (0, 1.24 and 6.85, respectively). The oil content in pulp (36.90%) was also higher than that in seed (14.15%), (3) qRT-PCR verification results showed the expressions of GPD1, DGAT1 and DGAT2 genes (1.46, 0.93 and 2.13 fold change, respectively) in pulp were also higher than those in seeds (0.47, 0.21 and 1.02 fold change, respectively). Our results suggest that the high coordinated expression of source ‘GPD1’ and sink ‘DGAT1 and DGAT2’ genes resulted in strong oil biosynthesis and accumulation in the pulp with high-oil pulp, compared to its seed with low-oil seeds. These transcriptome data provide genomic information for identification of genetic regulation system for sea buckthorn, especially understanding lipid biosynthesis mechanism in seeds and pulp. The detection of these three source and sink genes of lipid biosynthesis provided scientific basis for improving low oil contents in seeds and pulp of sea buckthorn berries.

Heterologous expression of a thermophilic diacylglycerol acyltransferase triggers triglyceride accumulation in Escherichia coli.

Triglycerides (TAGs), the major storage molecules of metabolic energy and source of fatty acids, are produced as single cell oil by some oleogenic microorganisms. However, these microorganisms require strict culture conditions, show low carbon source flexibilities, lack efficient genetic modification tools and in some cases pose safety concerns. TAGs have essential applications such as behaving as a source for added-value fatty acids or giving rise to the production of biodiesel. Hence, new alternative methods are urgently required for obtaining these oils. In this work we describe TAG accumulation in the industrially appropriate microorganism Escherichia coli expressing the heterologous enzyme tDGAT, a wax ester synthase/triacylglycerol:acylCoA acyltranferase (WS/DGAT). With this purpose, we introduce a codon-optimized gene from the thermophilic actinomycete Thermomonospora curvata coding for a WS/DGAT into different E. coli strains, describe the metabolic effects associated to the expression of this protein and evaluate neutral lipid accumulation. We observe a direct relation between the expression of this WS/DGAT and TAG production within a wide range of culture conditions. More than 30% TAGs were detected within the bacterial neutral lipids in 90 minutes after induction. TAGs were observed to be associated with the hydrophobic enzyme while forming round intracytoplasmic bodies, which could represent a bottleneck for lipid accumulation in E. coli. We detected an increase of almost 3-fold in the monounsaturated fatty acids (MUFA) occurring in the recombinant strains. These MUFA were predominant in the accumulated TAGs achieving 46% of the TAG fatty acids. These results set the basis for further research on the achievement of a suitable method towards the sustainable production of these neutral lipids.