Triacylglycerol Biosynthesis Research Articles

Thwarting Metabolic Dysfunction-Associated Fatty Liver Disease (MAFLD) with Common Bean: Dose- and Sex-Dependent Protection against Hepatic Steatosis.

Hepatic steatosis signifies onset of metabolic dysfunction-associated fatty liver disease (MAFLD) caused by disrupted metabolic homeostasis compromising liver function. Regular consumption of common beans reduces the risk of metabolic impairment, but its effective dose, the impact of biological sex, and underlying mechanisms of action are unknown. We fed female and male C57BL6/J mice with obesogenic yet isocaloric diets containing 0%, 17.5%, 35%, and 70% of total dietary protein derived from cooked whole common beans. Liver tissue was collected for histopathology, lipid quantification, and RNA-seq analyses. Beans qualitatively and quantitatively diminished hepatic fat deposition at the 35% dose in female and 70% dose in male mice. Bean-induced differentially expressed genes (DEGs) most significantly mapped to hepatic steatosis and revealed dose-responsive inhibition of de novo lipogenesis markers (Acly, Acaca, Fasn, Elovl6, Scd1, etc.) and triacylglycerol biosynthesis, activation of triacylglycerol degradation, and downregulation of sterol regulatory element-binding transcription factor 1 (SREBF1) signaling. Upregulated fatty acid β-oxidation was more prominent in females, while suppression of Cd36-mediated fatty acid uptake-in males. Sex-dependent bean effects also involved DEGs patterns downstream of peroxisome proliferator-activated receptor α (PPARα) and MLX-interacting protein-like (MLXIPL). Therefore, biological sex determines amount of common bean in the diet required to prevent hepatic lipid accumulation.

Transmembrane Protein 68 Functions as an MGAT and DGAT Enzyme for Triacylglycerol Biosynthesis.

Triacylglycerol (TG) biosynthesis is an important metabolic process for intracellular storage of surplus energy, intestinal dietary fat absorption, attenuation of lipotoxicity, lipid transportation, lactation and signal transduction in mammals. Transmembrane protein 68 (TMEM68) is an endoplasmic reticulum (ER)-anchored acyltransferase family member of unknown function. In the current study we show that overexpression of TMEM68 promotes TG accumulation and lipid droplet (LD) formation in a conserved active sites-dependent manner. Quantitative targeted lipidomic analysis showed that diacylglycerol (DG), free fatty acid (FFA) and TG levels were increased by TMEM68 expression. In addition, TMEM68 overexpression affected the levels of several glycerophospholipids, such as phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, as well as sterol ester contents. TMEM68 exhibited monoacylglycerol acyltransferase (MGAT) and diacylglycerol acyltransferase (DGAT) activities dependent on the conserved active sites in an in vitro assay. The expression of lipogenesis genes, including DGATs, fatty acid synthesis-related genes and peroxisome proliferator-activated receptor γ was upregulated in TMEM68-overexpressing cells. These results together demonstrate for the first time that TMEM68 functions as an acyltransferase and affects lipogenic gene expression, glycerolipid metabolism and TG storage in mammalian cells.

LsSpt23p is a regulator of triacylglycerol synthesis in the oleaginous yeast Lipomyces starkeyi

The oleaginous yeast Lipomyces starkeyi has considerable potential in industrial application, since it can accumulate a large amount of triacylglycerol (TAG), which is produced from sugars under nitrogen limitation condition. However, the regulation of lipogenesis in L. starkeyi has not been investigated in depth. In this study, we compared the genome sequences of wild-type and mutants with increased TAG productivity, and identified a regulatory protein, LsSpt23p, which contributes to the regulation of TAG synthesis in L. starkeyi. L. starkeyi mutants overexpressing LsSPT23 had increased TAG productivity compared with the wild-type strain. Quantitative real-time PCR analysis showed that LsSpt23p upregulated the expression of GPD1, which encodes glycerol 3-phosphate dehydrogenase; the Kennedy pathway genes SCT1, SLC1, PAH1, DGA1, and DGA2; the citrate-mediated acyl-CoA synthesis pathway-related genes ACL1, ACL2, ACC1, FAS1, and FAS2; and OLE1, which encodes ∆9 fatty acid desaturase. Chromatin immunoprecipitation-quantitative PCR assays indicated that LsSpt23p acts as a direct regulator of SLC1 and PAH1, all the citrate-mediated acyl-CoA synthesis pathway-related genes, and OLE1. These results indicate that LsSpt23p regulates TAG synthesis. Phosphatidic acid is a common substrate of phosphatidic acid phosphohydrolase, which is used for TAG synthesis, and phosphatidate cytidylyltransferase 1 for phospholipid synthesis in the Kennedy pathway. LsSpt23p directly regulated PAH1 but did not affect the expression of CDS1, suggesting that the preferred route of carbon is the Pah1p-mediated TAG synthesis pathway under nitrogen limitation condition. The present study contributes to understanding the regulation of TAG synthesis, and will be valuable in future improvement of TAG productivity in oleaginous yeasts. KEY POINTS: LsSpt23p was identified as a positive regulator of TAG biosynthesis LsSPT23 overexpression enhanced TAG biosynthesis gene expression and TAG production LsSPT23M1108T overexpression mutant showed fivefold higher TAG production than control.

Acyl-CoA-dependent and acyl-CoA-independent avocado acyltransferases positively influence oleic acid content in nonseed triacylglycerols.

In higher plants, acyl-CoA:diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT) catalyze the terminal step of triacylglycerol (TAG) synthesis in acyl-CoA-dependent and -independent pathways, respectively. Avocado (Persea americana) mesocarp, a nonseed tissue, accumulates significant amounts of TAG (~70% by dry weight) that is rich in heart-healthy oleic acid (18:1). The oil accumulation stages of avocado mesocarp development coincide with high expression levels for type-1 DGAT (DGAT1) and PDAT1, although type-2 DGAT (DGAT2) expression remains low. The strong preference for oleic acid demonstrated by the avocado mesocarp TAG biosynthetic machinery represents lucrative biotechnological opportunities, yet functional characterization of these three acyltransferases has not been explored to date. We expressed avocado PaDGAT1, PaDGAT2, and PaPDAT1 in bakers' yeast and leaves of Nicotiana benthamiana. PaDGAT1 complemented the TAG biosynthesis deficiency in the quadruple mutant yeast strain H1246, and substantially elevated total cellular lipid content. In vitro enzyme assays showed that PaDGAT1 prefers oleic acid compared to palmitic acid (16:0). Both PaDGAT1 and PaPDAT1 increased the lipid content and elevated oleic acid levels when expressed independently or together, transiently in N. benthamiana leaves. These results indicate that PaDGAT1 and PaPDAT1 prefer oleate-containing substrates, and their coordinated expression likely contributes to sustained TAG synthesis that is enriched in oleic acid. This study establishes a knowledge base for future metabolic engineering studies focused on exploitation of the biochemical properties of PaDGAT1 and PaPDAT1.

Functional analysis of alternative castor bean DGAT enzymes.

The diversity of diacylglycerol acyltransferases (DGATs) indicates alternative roles for these enzymes in plant metabolism besides triacylglycerol (TAG) biosynthesis. In this work, we functionally characterized castor bean (Ricinus communis L.) DGATs assessing their subcellular localization, expression in seeds, capacity to restore triacylglycerol (TAG) biosynthesis in mutant yeast and evaluating whether they provide tolerance over free fatty acids (FFA) in sensitive yeast. RcDGAT3 displayed a distinct subcellular localization, located in vesicles outside the endoplasmic reticulum (ER) in most leaf epidermal cells. This enzyme was unable to restore TAG biosynthesis in mutant yeast; however, it was able to outperform other DGATs providing higher tolerance over FFA. RcDAcTA subcellular localization was associated with the ER membranes, resembling RcDGAT1 and RcDGAT2, but it failed to rescue the long-chain TAG biosynthesis in mutant yeast, even with fatty acid supplementation. Besides TAG biosynthesis, our results suggest that RcDGAT3 might have alternative functions and roles in lipid metabolism.

Newly identified essential amino acids affecting peanut (Arachis hypogaea L.) DGAT2 enzyme activity

Triacylglycerols is the major storage lipid in most crop seeds. As the key enzyme catalyzing the final step of triacylglycerols biosynthesis, the activity of diacylglycerol acyltransferases directly related to oil content. It has been shown that certain amino acids are very important for enzyme activity, one amino acid variation will greatly change the enzyme activity. In this study, we identified three amino acid point mutations that affect the Arachis hypogaea diacylglycerol acyltransferase 2 enzyme activity, T107M, K251R and L316P. According to the three amino acid variations, three single-nucleotide-mutant sequences of Arachis hypogaea diacylglycerol acyltransferase 2a were constructed and transformed into yeast strain H1246 for function verification. Results showed that T107M and K251R could change the fatty acid content and composition of the transformed yeast strains, whereas L316P led to the loss of enzyme activity. By analyzing the 2D and 3D structures of the three variants, we found that the changes of spatial structure of T107M, K251R and L316P caused the changes of the enzyme activity. Our study could provide a theoretical basis for changing the enzyme activity of DGAT by genetic engineering, and provide a new idea for increasing the oil content of the crops.

Multiomics Approach Captures Hepatic Metabolic Network Altered by Chronic Ethanol Administration.

Using a multiplatform and multiomics approach, we identified metabolites, lipids, proteins, and metabolic pathways that were altered in the liver after chronic ethanol administration. A functional enrichment analysis of the multiomics dataset revealed that rats treated with ethanol experienced an increase in hepatic fatty acyl content, which is consistent with an initial development of steatosis. The nuclear magnetic resonance spectroscopy (NMR) and liquid chromatography-mass spectrometry (LC-MS) metabolomics data revealed that the chronic ethanol exposure selectively modified toxic substances such as an increase in glucuronidation tyramine and benzoyl; and a depletion in cholesterol-conjugated glucuronides. Similarly, the lipidomics results revealed that ethanol decreased diacylglycerol, and increased triacylglycerol, sterol, and cholesterol biosynthesis. An integrated metabolomics and lipidomics pathway analysis showed that the accumulation of hepatic lipids occurred by ethanol modulation of the upstream lipid regulatory pathways, specifically glycolysis and glucuronides pathways. A proteomics analysis of lipid droplets isolated from control EtOH-fed rats and a subsequent functional enrichment analysis revealed that the proteomics data corroborated the metabolomic and lipidomic findings that chronic ethanol administration altered the glucuronidation pathway.

CGI-58 Protein Acts as a Positive Regulator of Triacylglycerol Accumulation in Phaeodactylum tricornutum.

Comparative gene identification-58 (CGI-58) is an activating protein of triacylglycerol (TAG) lipase. It has a variety of catalytic activities whereby it may play different roles in diverse organisms. In this study, a homolog of CGI-58 in Phaeodactylum tricornutum (PtCGI-58) was identified. PtCGI-58 was localized in mitochondria by GFP fusion protein analysis, which is different from the reported subcellular localization of CGI-58 in animals and plants. Respectively, PtCGI-58 overexpression resulted in increased neutral lipid content and TAG accumulation by 42-46% and 21-32%. Likewise, it also increased the relative content of eicosapentaenoic acid (EPA), and in particular, the EPA content in TAGs almost doubled. Transcript levels of genes involved in de novo fatty acid synthesis and mitochondrial β-oxidation were significantly upregulated in PtCGI-58 overexpression strains compared with wild-type cells. Our findings suggest that PtCGI-58 may mediate the breakdown of lipids in mitochondria and the recycling of acyl chains derived from mitochondrial β-oxidation into TAG biosynthesis. Moreover, this study potentially illuminates new functions for CGI-58 in lipid homeostasis and provides a strategy to enrich EPA in algal TAGs.

Camelina cytosol-localized diacylglycerol acyltransferase 3 contributes to the accumulation of seed storage oils

In plants, triacylglycerol (TAG) is a form of seed storage oil that can provide a source of carbon and energy for seed germination and seedling development. Among three diacylglycerol acyltransferases (DGAT1, DGAT2, and DGAT3) that synthesize TAG, DGAT3 is involved in TAG synthesis in the cytosol, however, its biological function is still unclear. In this study, three CsDGAT3 homoeologs ( CsDGAT3–1 , CsDGAT3–2 , and CsDGAT3–3 ) showed higher expression levels than CsDGAT1 in developing seeds of camelina ( Camelina sativa ). These were predominantly expressed in developing seeds, and the CsDGAT3 proteins were localized to the cytosol in the epidermal cells of Nicotiana benthamiana leaves. Seed weight and total seed fatty acid levels of transgenic Arabidopsis ( Arabidopsis thaliana ) seeds overexpressing CsDGAT3–2 or CsDGAT3–3 were approximately 13% and 11% higher, respectively, than those of nontransgenic seeds. In particular, the levels of C18:3 and C20:1 increased, while levels of C18:1 and C18:2 decreased. In CsDGAT3 -knockout camelina, as compared to wild type, the seed weights and total fatty acid levels were about 7% and 19% lower, and the C18:3 and C20:1 levels, specifically, were reduced. We detected no significant differences in total seed fatty acid levels or composition between the Arabidopsis dgat1 mutant and the dgat1 mutant overexpressing CsDGAT3–3 . Taken together, cytosol-localized DGAT3 contributes to the accumulation of seed storage oil and prefers the acylation of C18:3, which is the most abundant fatty acid in camelina seed oils. This finding can be applied to the development of oilseed plants with enhanced seed storage oils containing high levels of omega-3 fatty acids. • Camelina DGAT3 participates in TAG biosynthesis via the cytosolic DGAT-dependent pathway in seeds. • Camelina DGAT3 shows a preference for C18:3 and C20:1 as acyl-CoA substrates. • Cytosolic DGAT3-mediated TAG biosynthesis contributes to the accumulation of storage oils in seeds. • DGAT3 is a novel genetic resource to enhance seed oil contents with high omega-3 fatty acids levels.

Flashing lights affect the photophysiology and expression of carotenoid and lipid synthesis genes in Nannochloropsis gaditana

Nannochloropsis gaditana is a promising microalga for biotechnology. One of the strategies to stimulate its full potential in metabolite production is exposure to flashing lights. Here, we report how N. gaditana adapts to different flashing light regimes (5, 50, and 500 Hz) by changing its cellular physiology and the relative expression of genes related to critical cellular functions. We analyzed the differential mRNA abundance of genes related to photosynthesis, nitrogen assimilation and biosynthesis of chlorophyll, carotenoids, lipids, fatty acids and starch. Analysis of photosynthetic efficiency and high mRNA abundance of photoprotection genes supported the inference that excess excitation energy provided by light absorbance during photosynthesis was produced under low frequency flashing lights and was dissipated by photopigments via the xanthophyll-cycle. Increased relative expression levels of genes related to the synthesis of carotenoids and chlorophyll confirmed the accumulation of photopigments previously observed at low frequency flashing lights. Higher differential mRNA abundance of genes related to the triacylglycerol biosynthesis were observed at lower frequency flashing lights, possibly triggered by a poor nitrogen assimilation caused by low mRNA abundance of a nitrate reductase gene. This study advances a new understanding of algal physiology and metabolism leading to improved cellular performance and metabolite production.

Diacylglycerol Acyltransferase 3(DGAT3) Is Responsible for the Biosynthesis of Unsaturated Fatty Acids in Vegetative Organs of Paeonia rockii.

'Diacylglycerol acyltransferase (DGAT)' acts as a key rate-limiting enzyme that catalyzes the final step of the de novo biosynthesis of triacylglycerol (TAG). The study was to characterize the function of the DGAT3 gene in Paeonia rockii, which is known for its accumulation of high levels of unsaturated fatty acids (UFAs). We identified a DGAT3 gene which encodes a soluble protein that is located within the chloroplasts of P. rockii. Functional complementarity experiments in yeast demonstrated that PrDGAT3 restored TAG synthesis. Linoleic acid (LA, C18:2) and α-linolenic acid (ALA, C18:3) are essential unsaturated fatty acids that cannot be synthesized by the human body. Through the yeast lipotoxicity test, we found that the yeast cell density was largely increased by adding exogenous LA and, especially, ALA to the yeast medium. Further ectopic transient overexpression in Nicotiana benthamiana leaf tissue and stable overexpression in Arabidopsis thaliana indicated that PrDGAT3 significantly enhanced the accumulation of the TAG and UFAs. In contrast, we observed a significant decrease in the total fatty acid content and in several major fatty acids in PrDGAT3-silenced tree peony leaves. Overall, PrDGAT3 is important in catalyzing TAG synthesis, with a substrate preference for UFAs, especially LA and ALA. These results suggest that PrDGAT3 may have practical applications in improving plant lipid nutrition and increasing oil production in plants.

Improved assembly and annotation of the sesame genome.

Sesame (Sesamum indicum L.) is an important oilseed crop that produces abundant seed oil and has a pleasant flavor and high nutritional value. To date, several Illumina-based genome assemblies corresponding to different sesame genotypes have been published and widely used in genetic and genomic studies of sesame. However, these assemblies consistently showed low continuity with numerous gaps. Here, we reported a high-quality, reference-level sesame genome assembly by integrating PacBio high-fidelity sequencing and Hi-C technology. Our updated sesame assembly was 309.35 Mb in size with a high chromosome anchoring rate (97.54%) and contig N50 size (13.48 Mb), which were better than previously published genomes. We identified 163.38 Mb repetitive elements and 24,345 high-confidence protein-coding genes in the updated sesame assembly. Comparative genomic analysis showed that sesame shared an ancient whole-genome duplication event with two Lamiales species. A total of 2,782 genes were tandemly duplicated. We also identified several genes that were likely involved in fatty acid and triacylglycerol biosynthesis. Our improved sesame assembly and annotation will facilitate future genetic studies and genomics-assisted breeding of sesame.

Design and application of a kinetic model of lipid metabolism in Saccharomyces cerevisiae

Lipid biosynthesis plays a vital role in living cells and has been increasingly engineered to overproduce various lipid-based chemicals. However, owing to the tightly constrained and interconnected nature of lipid biosynthesis, both understanding and engineering of lipid metabolism remain challenging, even with the help of mathematical models. Here we report the development of a kinetic metabolic model of lipid metabolism in Saccharomyces cerevisiae that integrates fatty acid biosynthesis, glycerophospholipid metabolism, sphingolipid metabolism, storage lipids, lumped sterol synthesis, and the synthesis and transport of relevant target-chemicals, such as fatty acids and fatty alcohols. The model was trained on lipidomic data of a reference S. cerevisiae strain, single knockout mutants, and lipid overproduction strains reported in literature. The model was used to design mutants for fatty alcohol overproduction and the lipidomic analysis of the resultant mutant strains coupled with model-guided hypothesis led to discovery of a futile cycle in the triacylglycerol biosynthesis pathway. In addition, the model was used to explain successful and unsuccessful mutant designs in metabolic engineering literature. Thus, this kinetic model of lipid metabolism can not only enable the discovery of new phenomenon in lipid metabolism but also the engineering of mutant strains for overproduction of lipids.

Change in Oil Composition and the Major Fatty Acids and Triacylglycerol Biosynthesis Genes in Drupe of Selected Olive Cultivars during Growing Season; A Two Years Study

AbstractUncovering the relationships between the content and compositions of olive oil, environmental factor, and developmental stages can lead to the effective cultivation choices and maximizing productivity. Fatty acid contents of 4 olive cultivars are screened at 5 developmental stages during 2 cropping seasons. Moreover, the transcript abundance of 4 fatty acids and triacylglycerol biosynthesis genes are evaluated in 2 cultivars displaying distinct oil content and composition. Thought, all studied cultivars display a rise in oil content and a decline in oleic/linoleic acid as maturity advances, the fatty acid content, however changes in a cultivar‐dependent manner. In contrast to other cultivars, “Mari” shows an increasing trend for oleic acid and fruit RH, and steady state pattern for linoleic acid during fruit development. All these characteristics result in a unique oil quality in “Mari”. Accordingly, there are slight differences in the oil related‐attributes in 2 cropping years at different sampling dates which are also cultivar dependent. The expression patterns of oil biosynthesis genes are well‐paralleled with oil accumulation and compositions in the studied olive cultivars; as OeSAD2 and OeDGAT2 experience a significant higher expression in “Mari”, while “Shengeh” has higher levels for OeFAD2‐2 and OeDGAT1 during most sampling times.Practical Applications: According to these data, the lower expression of OeFAD2‐2, as well as, substantially higher expression of OeSAD2 and OeDGAT2 are suggested as the major players for higher oil and oleic acid production in “Mari”. Results obtained from this study improve knowledge about oil biosynthesis in olive drupe in relation to fruit ripening and environmental factors. These data are useful for both growers and experts enabling them to make more appropriate planting and harvest decisions, as well as, to design suitable breeding programs to achieve olive improvement objectives.

Anatomical, physiological, biochemical and molecular responses of Eucalyptus spp. under water deficit conditions and characteristics of Tunisian arid species: an overview

The genus Eucalyptus occurs in a large range of environmental conditions, mainly arid/semi-arid areas. It includes species with high capacity to survive with extremely low water potential. For that, our review shows an important effect of drought on Eucalyptus spp. plants’ resistance mechanisms and management strategies. In fact, water stress acts directly on growth, productivity, yield, it affects also response to pests and diseases, disturbs wood formation and essential oil yield. However, the general patterns of response to water stress varied among species, genotypes, hybrids and clones. To assume, reducing water loss in eucalyptus species is manifested by reducing leaves area, reducing gas exchange, increasing water uptake thanks to a prolific and deep root systems. A greater accumulation of osmolytes that gives rise to osmotic adjustment including carbohydrates especially cyclitols and quercitol, other amino acids and organic acids, also some proteins which play a vital role in sustaining cellular functions under drought conditions. More than that, water stress increases mainly the levels of pigments, chlorophyll fluorescence parameters, malondialdehyde (MDA), abscisic acid (ABA) and the biosynthesis of triacylglycerols (TAGs) in Eucalyptus species.

Genome-wide analysis of the WSD family in sunflower and functional identification of HaWSD9 involvement in wax ester biosynthesis and osmotic stress.

The wax esters are important cuticular wax composition that cover the outer surface of plant organs and play a critical role in protection and energy metabolism. Wax ester synthesis in plant is catalyzed by a bifunctional wax ester synthase/acyl-CoA: diacylglycerol acyltransferase (WSD). Sunflower (Helianthus annuus L.) is an important oil crop in the world; however, little is known about WSD in sunflower. In this study, we identified and performed a functional analysis of twelve HaWSD genes from sunflower genome. Tissue-specific expression revealed that 12 HaWSD genes were differentially expressed in various organs and tissues of sunflower, except seeds. HaWSD genes were highly induced by salinity, drought, cold, and abscisic acid (ABA) in sunflower. To ascertain their function, HaWSD9, with highly expressed levels in stems and leaves, was cloned and expressed in a yeast mutant defective in triacylglycerol (TAG) biosynthesis. HaWSD9 complemented the phenotype by producing wax ester but not TAG in vivo, indicating that it functions as a wax ester synthase. Subcellular localization analysis indicated that HaWSD9 was located in the endoplasmic reticulum (ER). Heterologous introduction of HaWSD9 into Arabidopsis wsd1 mutant exhibited increased epicuticular wax crystals and cuticular wax contents on the stems. As compared with the wsd1 mutant, HaWSD9 overexpressing transgenic Arabidopsis showed less cuticle permeability, chlorophyll leaching and water loss rate. Further analysis showed that the HaWSD9 transgenics enhanced tolerance to ABA, mannitol, drought and salinity, and maintained higher leaf relative water content (RWC) than the wsd1 mutant under drought stress, suggesting that HaWSD9 play an important physiological role in stress response as well as wax synthase. These results contribute to understanding the function of HaWSD genes in wax ester synthesis and stress tolerance in sunflower.

Applications and prospects of genome editing in plant fatty acid and triacylglycerol biosynthesis.

Triacylglycerol (TAG), which is a neutral lipid, has a structure in which three molecules of fatty acid (FA) are ester-bonded to one molecule of glycerol. TAG is important energy source for seed germination and seedling development in plants. Depending on the FA composition of the TAG, it is used as an edible oil or industrial material for cosmetics, soap, and lubricant. As the demand for plant oil is rising worldwide, either the type of FA must be changed or the total oil content of various plants must be increased. In this review, we discuss the regulation of FA metabolism by Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, a recent genome-editing technology applicable to various plants. The development of plants with higher levels of oleic acid or lower levels of very long-chain fatty acids (VLCFAs) in seeds are discussed. In addition, the current status of research on acyltransferases, phospholipases, TAG lipases, and TAG synthesis in vegetative tissues is described. Finally, strategies for the application of CRISPR/Cas9 in lipid metabolism studies are mentioned.

Fatty acid export (FAX) proteins contribute to oil production in the green microalga Chlamydomonas reinhardtii.

In algae and land plants, transport of fatty acids (FAs) from their site of synthesis in the plastid stroma to the endoplasmic reticulum (ER) for assembly into acyl lipids is crucial for cellular lipid homeostasis, including the biosynthesis of triacylglycerol (TAG) for energy storage. In the unicellular green alga Chlamydomonas reinhardtii, understanding and engineering of these processes is of particular interest for microalga-based biofuel and biomaterial production. Whereas in the model plant Arabidopsis thaliana, FAX (fatty acid export) proteins have been associated with a function in plastid FA-export and hence TAG synthesis in the ER, the knowledge on the function and subcellular localization of this protein family in Chlamydomonas is still scarce. Among the four FAX proteins encoded in the Chlamydomonas genome, we found Cr-FAX1 and Cr-FAX5 to be involved in TAG production by functioning in chloroplast and ER membranes, respectively. By in situ immunolocalization, we show that Cr-FAX1 inserts into the chloroplast envelope, while Cr-FAX5 is located in ER membranes. Severe reduction of Cr-FAX1 or Cr-FAX5 proteins by an artificial microRNA approach results in a strong decrease of the TAG content in the mutant strains. Further, overexpression of chloroplast Cr-FAX1, but not of ER-intrinsic Cr-FAX5, doubled the content of TAG in Chlamydomonas cells. We therefore propose that Cr-FAX1 in chloroplast envelopes and Cr-FAX5 in ER membranes represent a basic set of FAX proteins to ensure shuttling of FAs from chloroplasts to the ER and are crucial for oil production in Chlamydomonas.

Lipidome analysis and characterization of Buglossoides arvensis acyltransferases that incorporate polyunsaturated fatty acids into triacylglycerols

Buglossoides arvensis is a burgeoning oilseed crop that contains an unique combination of ω-3 and ω-6 polyunsaturated fatty acids (PUFA), constituting ~80–85% of seed triacylglycerols (TAGs). To uncover the critical TAG biosynthetic pathways contributing for high PUFA accumulation, we performed lipidome of developing seeds and characterized acyltransferases involved in the final step of TAG biosynthesis. During seed development, distribution of lipid molecular species in individual lipid classes showed distinct patterns from an early-stage (6 days after flowering (DAF)) to the middle-stage (12 and 18 DAF) of oil biosynthesis. PUFA-containing TAG species drastically increased from 6 to 12 DAF. The expression profiles of key triacylglycerol biosynthesis genes and patterns of phosphatidylcholine, diacylglycerol and triacylglycerol molecular species during seed development were used to predict the contribution of diacylglycerol acyltransferases (DGAT1 and DGAT2) and phospholipid: diacylglycerol acyltransferases (PDAT1 and PDAT2) to PUFA-rich TAG biosynthesis. Our analysis suggests that DGATs play a crucial role in enriching TAGs with PUFA compared to PDATs. This was further confirmed by fatty acid feeding studies in yeast expressing acyltransferases. BaDGAT2 preferentially incorporated high amounts of PUFAs into TAG, compared to BaDGAT1. Our results provide insight into the molecular mechanisms of TAG accumulation in this plant and identify target genes for transgenic production of SDA in traditional oilseed crops.

Inhibition of monogalactosyldiacylglycerol synthesis by down-regulation of MGD1 leads to membrane lipid remodeling and enhanced triacylglycerol biosynthesis in Chlamydomonas reinhardtii

BackgroundMembrane lipid remodeling involves regulating the physiochemical modification of cellular membranes against abiotic stress or senescence, and it could be a trigger to increase neutral lipid content. In algae and higher plants, monogalactosyldiacylglycerol (MGDG) constitutes the highest proportion of total membrane lipids and is highly reduced as part of the membrane lipid remodeling response under several abiotic stresses. However, genetic regulation of MGDG synthesis and its influence on lipid synthesis has not been studied in microalgae. For development of an industrial microalgae strain showing high accumulation of triacylglycerol (TAG) by promoting membrane lipid remodeling, MGDG synthase 1 (MGD1) down-regulated mutant of Chlamydomonas reinhardtii (Cr-mgd1) was generated and evaluated for its suitability for biodiesel feedstock.ResultsThe Cr-mgd1 showed a 65% decrease in CrMGD1 gene expression level, 22% reduction in MGDG content, and 1.39 and 5.40 times increase in diacylglyceryltrimethylhomoserines (DGTS) and TAG, respectively. The expression levels of most genes related to the decomposition of MGDG (plastid galactoglycerolipid degradation1) and TAG metabolism (diacylglycerol O-acyltransferase1, phospholipid:diacylglycerol acyltransferase, and major lipid droplet protein) were increased. The imbalance of DGDG/MGDG ratio in Cr-mgd1 caused reduced photosynthetic electron transport, resulting in less light energy utilization and increased reactive oxygen species levels. In addition, endoplasmic reticulum stress was induced by increased DGTS levels. Thus, accelerated TAG accumulation in Cr-mgd1 was stimulated by increased cellular stress as well as lipid remodeling. Under high light (HL) intensity (400 µmol photons/m2/s), TAG productivity in Cr-mgd1–HL (1.99 mg/L/d) was 2.71 times higher than that in wild type (WT–HL). Moreover, under both nitrogen starvation and high light intensity, the lipid (124.55 mg/L/d), TAG (20.03 mg/L/d), and maximum neutral lipid (56.13 mg/L/d) productivity were the highest.ConclusionsBy inducing lipid remodeling through the mgd1 gene expression regulation, the mutant not only showed high neutral lipid content but also reached the maximum neutral lipid productivity through cultivation under high light and nitrogen starvation conditions, thereby possessing improved biomass properties that are the most suitable for high quality biodiesel production. Thus, this mutant may help understand the role of MGD1 in lipid synthesis in Chlamydomonas and may be used to produce high amounts of TAG.