Optimization of the biomass production of oil algae Chlorella minutissima UTEX2341

High production cost is a major obstacle to the extensive use of microalgae biodiesel. To cut the cost and achieve higher biomass productivity, Chlorella minutissima UTEX2341 was cultured under photoheterotrophic conditions. With the carbon, nitrogen and phosphorus concentration of 26.37, 2.61 and 0.03 g L?1 d?1 respectively, a maximum biomass productivity of 1.78 g L?1 d?1 was obtained, which was 59 times more than that cultured under autotrophic condition. The lipid productivity reached 0.29 g L?1 d?1, which was 11.9 times higher than the highest value reported by Oh et al. (2010). The conversion rate of microalgae lipids to FAME was found to be elevated from 45.65% to 62.97% and the FAME productivity increased from 1.16 to 180.68 mg L?1 d?1 after the optimization. 94% of the fatty acid of C. minutissima UTEX2341 was found to be composed of palmitic, oleic, linoleic and γ linoleic and the unsaturated fatty acids were the main parts (79.42%).     

Proteomic profiling of oil bodies isolated from the unicellular green microalga Chlamydomonas reinhardtii: with focus on proteins involved in lipid metabolism

Oil bodies are sites of energy and carbon storage in many organisms including microalgae. As a step toward deciphering oil accumulation mechanisms in algae, we used proteomics to analyze purified oil bodies from the model microalga Chlamydomonas reinhardtii grown under nitrogen deprivation. Among the 248 proteins (≥ 2 peptides) identified by LC-MS/MS, 33 were putatively involved in the metabolism of lipids (mostly acyl-lipids and sterols). Compared with a recently reported Chlamydomonas oil body proteome, 19 new proteins of lipid metabolism were identified, spanning the key steps of the triacylglycerol synthesis pathway and including a glycerol-3-phosphate acyltransferase (GPAT), a lysophosphatidic acid acyltransferase (LPAT) and a putative phospholipid:diacylglycerol acyltransferase (PDAT). In addition, proteins putatively involved in deacylation/reacylation, sterol synthesis, lipid signaling and lipid trafficking were found to be associated with the oil body fraction. This data set thus provides evidence that Chlamydomonas oil bodies are not only storage compartments but also are dynamic structures likely to be involved in processes such as oil synthesis, degradation and lipid homeostasis. The proteins identified here should provide useful targets for genetic studies aiming at increasing our understanding of triacyglycerol synthesis and the role of oil bodies in microalgal cell functions.     

Activities of enzymes of phospholipid and fatty acid synthesis in fetal and adult rat type II pneumocytes

Although differentiated fetal and adult type II pneumocytes are ultrastructurally similar, it is not known whether there are metabolic differences between them. We measured the activities of selected enzymes of phospholipid and fatty acid synthesis in fetal and adult rat type II cells, in late gestation fetal rat lung explants and in intact lung from rat fetuses of comparable gestational age. The activity of 1-acylglycerophosphocholine acyltransferase was significantly greater in adult type II cells than in fetal type II cells, fetal explants or intact fetal lung. The activity of CDP diacylglycerol:glycerol-3-phosphate 3-phosphatidyltransferase was similar in fetal and adult type II cells, but significantly lower in explants and intact fetal lung. There was a significant positive correlation between the percentage of alveolar epithelial cells in the cultures and tissue studied and CDP diacylglycerol:glycerol-3-phosphate 3-phosphatidyltransferase activity. This suggests that the previously reported correlation between phosphatidylglycerol synthesis and the percentage of alveolar epithelial cells in various lung culture systems may be related to the activity of this enzyme. Phosphatidylglycerol synthesis and CDP diacylglycerol:glycerol-3-phosphate 3-phosphatidyltransferase activity may be metabolic markers of type II cells, whereas the acyltransferase activity may be an indicator of type II cell maturation.     

Triacylglycerol biosynthesis in bovine liver and subcutaneous adipose tissue.

1. Adipose tissue from Angus and Brahman steers incubated with [1-14C]palmitate in the absence and presence of glucose exhibited a greater rate of lipid production than liver (P < 0.05). 2. Homogenates of adipose tissue used in the glycerol-3-phosphate acyltransferase assay exhibited a greater glycerolipid specific activity (nmol lipid/mg protein/30 min) when compared to liver (P < 0.05). 3. The inverse was true for liver homogenates when calculated for tissue activity (nmol lipid/g tissue/30 min). 4. Lysophosphatidate was produced in greater (P < 0.05) amounts than all other glycerolipids in the glycerol-3-phosphate acyltransferase assay. 5. The activity of phosphatidate phosphohydrolase in liver homogenates displayed greater rates than their respective adipose tissue homogenates. 6. Diacylglycerol acyltransferase activity was greater in adipose tissue homogenates compared to liver homogenates.     

Redundant systems of phosphatidic acid biosynthesis via acylation of glycerol-3-phosphate or dihydroxyacetone phosphate in the yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae lipid particles harbor two acyltransferases, Gat1p and Slc1p, which catalyze subsequent steps of acylation required for the formation of phosphatidic acid. Both enzymes are also components of the endoplasmic reticulum, but this compartment contains additional acyltransferase(s) involved in the biosynthesis of phosphatidic acid (K. Athenstaedt and G. Daum, J. Bacteriol. 179:7611-7616, 1997). Using the gat1 mutant strain TTA1, we show here that Gat1p present in both subcellular fractions accepts glycerol-3-phosphate and dihydroxyacetone phosphate as a substrate. Similarly, the additional acyltransferase(s) present in the endoplasmic reticulum can acylate both precursors. In contrast, yeast mitochondria harbor an enzyme(s) that significantly prefers dihydroxyacetone phosphate as a substrate for acylation, suggesting that at least one additional independent acyltransferase is present in this organelle. Surprisingly, enzymatic activity of 1-acyldihydroxyacetone phosphate reductase, which is required for the conversion of 1-acyldihydroxyacetone phosphate to 1-acylglycerol-3-phosphate (lysophosphatidic acid), is detectable only in lipid particles and the endoplasmic reticulum and not in mitochondria. In vivo labeling of wild-type cells with [2-3H, U-14C]glycerol revealed that both glycerol-3-phosphate and dihydroxyacetone phosphate can be incorporated as a backbone of glycerolipids. In the gat1 mutant and the 1-acylglycerol-3-phosphate acyltransferase slc1 mutant, the dihydroxyacetone phosphate pathway of phosphatidic acid biosynthesis is slightly preferred as compared to the wild type. Thus, mutations of the major acyltransferases Gat1p and Slc1p lead to an increased contribution of mitochondrial acyltransferase(s) to glycerolipid synthesis due to their substrate preference for dihydroxyacetone phosphate.     

Characterization of the Glycerolipid Composition and Biosynthetic Capacity of Pea Root Plastids

The glycerolipid composition of pea (Pisum sativum L.) root plastids and their capacity to synthesize glycerolipids from [UL-14C]glycerol-3-phosphate were determined. Pea root plastids primarily consist of monogalactosyldiacylglycerol, triacylglycerol, phosphatidylcholine, digalactosyldiacylglycerol, and diacylglycerol. Maximum rates of total glycerolipid biosynthesis were obtained in the presence of 2.4 mM glycerol-3-phosphate, 15 mM KHCO3, 0.2 mM sodium-acetate, 0.5 mM each of NADH and NADPH, 0.05 mM coenzyme A, 2 mM MgCl2, 1 mM ATP, 0.1 M Bis-Tris propane (pH 7.5), and 0.31 M sorbitol. Glycerolipid biosynthesis was completely dependent on exogenously supplied ATP, coenzyme A, and a divalent cation, whereas the remaining cofactors improved their activity from 1.3- to 2.4-fold. Radioactivity from glycerol-3-phosphate was recovered predominantly in phosphatidic acid, phosphatidylglycerol, diacylglycerol, and triacylglycerol with lesser amounts in phosphatidylcholine and monoacylglycerol. The proportions of the various radiolabeled lipids that accumulated were dependent on the pH and the concentration of ATP and glycerol-3-phosphate. The data presented indicate that pea root plastids can synthesize almost all of their component glycerolipids and that glycerolipid biosynthesis is tightly coupled to de novo fatty acid biosynthesis. pH and the availability of ATP may have important roles in the regulation of lipid biosynthesis at the levels of phosphatidic acid phosphatase and in the reactions that are involved in phosphatidylglycerol and triacylglycerol biosynthesis.     

莱茵衣藻磷脂二脂酰甘油酰基转移酶3在三酰甘油合成中的功能研究

为研究磷脂二脂酰甘油酰基转移酶（PDAT）在三酰甘油合成中的功能,克隆了莱茵衣藻（Chlamydomonas reinhardtii） PDAT同源基因CrPDAT3干涉片段,通过构建CrPDAT3 RNAi 干涉载体并转化莱茵衣藻,对CrPDAT3基因有效沉默,结果显示转基因藻株生长减缓,油脂含量下降14.65%-45.15%,说明CrPDAT3对油脂合成起到重要的作用。研究结果对于该基因应用于微藻油脂的遗传改良将起到重要作用。       

Oil biosynthesis in microsomal membrane preparations from Mortierella alpina

Microsomal membrane preparations from Mortierella alpina catalysed the conversion of sn-glycerol 3-phosphate and [(14)C]oleoyl-CoA to radioactive phosphatidic acid, diacylglycerol and triacylglycerol. Experiments with lysophosphatidic acid and [(14)C]oleoyl-CoA gave a similar pattern of radioactivity in the complex lipids. The specific activity of lysophosphatidate acyltransferase was almost eight times greater than sn-glycerol-3-phosphate acyltransferase, indicating that the first acylation step was limiting in oil assembly in the microsomal membranes. Little conversion of radioactive oleate into phosphatidylcholine occurred, suggesting that triacylglycerol assembly and its relationship to phosphatidylcholine metabolism differed to that found in oilseeds.     

Biosynthesis of phosphatidic acid in lipid particles and endoplasmic reticulum of Saccharomyces cerevisiae.

Lipid particles of the yeast Saccharomyces cerevisiae harbor two enzymes that stepwise acylate glycerol-3-phosphate to phosphatidic acid, a key intermediate in lipid biosynthesis. In lipid particles of the s1c1 disruptant YMN5 (M. M. Nagiec et al., J. Biol. Chem. 268:22156-22163, 1993) acylation stops after the first step, resulting in the accumulation of lysophosphatidic acid. Two-dimensional gel electrophoresis confirmed that S1c1p is a component of lipid particles. Lipid particles of a second mutant strain, TTA1 (T. S. Tillman and R. M. Bell, J. Biol. Chem. 261:9144-9149, 1986), which harbors a point mutation in the GAT gene, are essentially devoid of glycerol-3-phosphate acyltransferase activity in vitro. Synthesis of phosphatidic acid is reconstituted by combining lipid particles from YMN5 and TTA1. These results indicate that two distinct enzymes are necessary for phosphatidic acid synthesis in lipid particles: the first step, acylation of glycerol-3-phosphate, is catalyzed by a putative Gat1p; the second step, acylation of lysophosphatidic acid, requires S1c1p. Surprisingly, YMN5 and TTA1 mutants grow like the corresponding wild types because the endoplasmic reticulum of both mutants has the capacity to form a reduced but significant amount of phosphatidic acid. As a consequence, an s1c1 gat1 double mutant is also viable. Lipid particles from this double mutant fail completely to acylate glycerol-3-phosphate, whereas endoplasmic reticulum membranes harbor residual enzyme activities to synthesize phosphatidic acid. Thus, yeast contains at least two independent systems of phosphatidic acid biosynthesis.     

Inhibition by ozone of the acylation of glycerol 3-phosphate in mitochondria and microsomes from rat lung

The synthesis of lipids from [U-¹⁴C]glycerol 3-phosphate by mitochondrial or microsomal fractions from rat lung was inhibited by ozone. The susceptible reaction was the first acylation of glycerol 3-phosphate. Enzymes unaffected by the ozone exposure included: acyl-CoA thioesterase, acyl-CoA thiokinase, acyl-CoA:acylglycerol 3-phosphate acyltransferase, acyl-CoA:diacylglycerol acyltransferase, and acyl-CoA:acylglycerophosphocholine acyltransferase. The effect of ozone on lipid synthesis is closely comparable to the inhibition by N-ethylmaleimide suggesting that the effect of ozone is the oxidation of enzyme sulfhydryl groups. There was no indication of lipid oxidation caused by ozone and no indication of the production of a stable toxic compound.     

Soybean phospholipid dependent reductions in triacylglycerol concentration and synthesis in the liver of fasted-refed rats.

The effect of dietary soybean phospholipid on the activities of hepatic triacylglycerol-synthesizing enzymes was compared with soybean oil in fasted-refed rats. Soybean oil at the dietary level corresponding to 20% but not at 5% fatty acid level (21.2 and 5.3% on weight bases, respectively) significantly decreased liver microsomal diacylglycerol acyltransferase activities measured with the endogenous diacylglycerol substrate. Dietary soybean phospholipid even at the dietary level corresponding to 2% fatty acids (3.4% on weight base) significantly decreased the acyltransferase activities measured with endogenous substrate. The dietary phospholipid further decreased the parameter as the dietary level increased, and at the 5% fatty acid level, it was lower than that obtained with soybean oil at 20% fatty acid level. Soybean oil and phospholipid decreased the diacylglycerol acyltransferase activities measured with the saturating concentration of exogenous dioleoylglycerol substrate only when the activities were expressed in terms of total activity (mumol/min per liver) but to much lesser extents. Dietary phospholipid compared to the oil profoundly decreased not only hepatic triacylglycerol but also microsomal diacylglycerol levels. It was indicated that the availability of microsomal diacylglycerol as the substrate for diacylglycerol transferase is the critical determinant in regulating hepatic triacylglycerol synthesis and concentration in this experimental situation. Alterations in the activities of microsomal glycerol 3-phosphate acyltransferase and of the enzymes in fatty acid synthesis could account for the phospholipid-dependent decrease in the microsomal concentration of this intermediate in triacylglycerol synthesis.     

The initial step of glycerolipid metabolism in Leishmania major promastigotes involves a single glycerol-3-phosphate acyltransferase enzyme important for the synthesis of triacylglycerol but not essential for virulence

The synthesis of the major phospholipids, including those that play an essential role in Leishmania virulence, initiates with the acylation of glycerol-3-phosphate and dihydroxyacetonephosphate at the sn-1 position by glycerol-3-phosphate and dihydroxyacetonephosphate acyltransferases respectively. In this study, we show that Leishmania major promastigotes express a single glycerol-3-phosphate acyltransferase activity important for triacylglycerol synthesis but not essential for virulence. The encoding gene, LmGAT, expressed in yeast results in full complementation of the lethality of a mutant, gat1Deltagat2Delta, lacking glycerol-3-phosphate activity. Biochemical analyses revealed that LmGAT is a low-affinity glycerol-3-phosphate acyltransferase and exhibits higher specific activity with unsaturated long fatty acyl-CoA donors. A L. major null mutant, Deltalmgat/Deltalmgat, was created and a thorough analysis of its lipid composition was performed. Deletion of LmGAT resulted in a complete loss of Leishmania glycerol-3-phosphate acyltransferase activity and a major reduction in triacylglycerol synthesis. Consistent with the specificity of LmGAT for glycerol-3-phosphate but not dihydroxyacetonephosphate, Deltalmgat/Deltalmgat mutant expressed normal levels of the ether-lipid derivatives and virulence factors, lipophosphoglycan and GPI-anchored proteins, gp63, and its virulence was not affected in mice.     

Diacylglycerol Acyltransferase in Microsomes and Oil Bodies of Oil Palm Mesocarp

Specific acyltransferase activities were detected in microsomesand oil bodies from palm mesocarp in the presence of exogenouslysophosphatidic acid, diacylglycerol and glycerol-3-phosphate.These activities were 2–3 times higher per mg proteinin oil bodies than in microsomes. Diacylglycerol acyltransferasein both preparations required Mg²⁺, dithiothreitol, gelatineand fat free bovine serum albumin for maximum activity at pH8.5. The enzyme had an apparent Km of 0.18 mM for dipalmitin.In both preparations, the enzyme was active with myristoyl-CoA,palmitoyl-CoA, stearoyl-CoA and oleoyl-CoA. When presented witha mixture of two acyl- CoAs, microsomal enzyme showed no selectivitybut oil body enzyme had a preference for oleoyl-CoA over palmitoyl-CoA. (Received October 11, 1991; Accepted December 27, 1991)     

Effects of levonorgestrel on enzymes responsible for synthesis of triacylglycerols in rat liver

The effects of levonorgestrel treatment (4 micrograms/day per kg body weight 0.75 for 18 days) on rate-limiting enzymes of hepatic triacylglycerol synthesis, namely glycerol-3-phosphate acyltransferase and phosphatidic acid phosphatase were investigated in microsomal, mitochondrial and cytosolic fractions of rat liver. Levonorgestrel treatment resulted in a significant reduction (26%) of hepatic microsomal glycerol-3-phosphate acyltransferase specific activity. Hepatic mitochondrial glycerol-3-phosphate acyltransferase specific activity was unchanged. Levonorgestrel treatment also significantly reduced (by 20%) the specific activity of hepatic microsomal magnesium-independent phosphatidic acid phosphatase. However, magnesium-dependent phosphatic acid phosphatase specific activities in microsomal and cytosolic fractions were unaffected. Cytosolic magnesium-independent phosphatidic acid phosphatase activity was also unchanged. These studies are consistent with the view that levonorgestrel lowers serum triacylglycerol levels, at least in part, by inhibition of the glycerol-3-phosphate acyltransferase (EC 2.3.1.15) step in hepatic triacylglycerol synthesis.     

The regulation of triacylglycerol biosynthesis in cocoa (Theobroma cacao) L.

Developing cocoa cotyledons accumulate initially an unsaturated oil which is particularly rich in oleate and linoleate. However, as maturation proceeds, the characteristic high stearate levels appear in the storage triacylglycerols. In the early stages of maturation, tissue slices of developing cotyledons (105 days post anthesis, dpa) readily accumulate radioactivity from [¹⁴C]acetate into the diacylglycerols and label predominantly palmitate and oleate. In older tissues (130 dpa), by contrast, the triacylglycerols are extensively labelled and, at the same time, there is an increase in the percentage labelling of stearate. Thus, the synthesis of triacylglycerol and the production of stearate are co-ordinated during development. The relative labelling of the phospholipids (particularly phosphatidylcholine) was rather low at both stages of development which contrasts with oil seeds that accumulate a polyunsaturated oil (e.g. safflower). Microsomal membrane preparations from the developing cotyledons readily utilised an equimolar [¹⁴C]acyl-CoA substrate (consisting of palmitate, stearate and oleate) and glycerol 3-phosphate to form phosphatidate, diacylglycerol and triacylglycerol. Analysis of the [¹⁴C]acyl constituents at the sn-1 and sn-2 positions of phosphatidate and diacylglycerol revealed that the first acylase enzyme (glycerol 3-phosphate acyltransferase) selectively utilised palmitate over stearate and excluded oleate, whereas the second acylase (lysophosphatidate acyltransferase) was highly selective for the unsaturated acyl-CoA. On the other hand, the third acylase (diacylglycerol acyltransferase) exhibited an almost equal selectivity for palmitate and stearate. Thus, stearate is preferentially enriched at position sn-3 of triacylglycerol at 120–130 dpa because of the relatively higher selectivity of the diacylglycerol acyltransferase for this fatty acid compared with those of the other two acylation enzymes.Abbreviation dpa days post anthesis We are grateful to Drs. G. Pettipher (Cadbury-Schweppes, Reading, UK), M. End and P. Hadley (Department of Horticulture, University of Reading) for the supply of cocoa pods and to the Agricultural and Food Research Council for financial support. We also wish to thank Dr. S. Stymne (Swedish University of Agricultural Sciences, Uppsala, Sweden) for a generous gift of acyl-CoA substrates.     

Final step of phosphatidic Acid synthesis in pea chloroplasts occurs in the inner envelope membrane

The second enzyme of phosphatidic acid synthesis from glycerol-3-phosphate, 1-acylglycerophospate acyltransferase, was localized to the inner envelope membrane of pea chloroplasts. The activity of this enzyme was measured by both a coupled enzyme assay and a direct enzyme assay. Using the coupled enzyme assay, phosphatidic acid phosphatase was also localized to the inner envelope membrane, although this enzyme has very low activity in pea chloroplasts. The addition of UDP-galactose to unfractionated pea chloroplast envelope preparations did not result in significant conversion of newly synthesized diacylglycerol to monogalactosyldiacylglycerol. Thus, the envelope synthesized phosphatidic acid may not be involved in galactolipid synthesis in pea chloroplasts.     

Phosphatidylcholine deficiency upregulates enzymes of triacylglycerol metabolism in CHO cells

We studied the regulation of triacylglycerol (TAG) metabolism by phosphatidylcholine (PC) in CHO MT58 cells, which are deficient in PC synthesis because of a temperature-sensitive CTP:phosphocholine cytidylyltransferase. At the permissive growth temperature (34 degrees C), these cells contained 49% less TAG and 30% less PC than wild-type CHO K1 cells. Treatment with dipalmitoylphosphatidylcholine normalized both the PC and TAG levels. Despite low TAG levels, the incorporation of [14C]oleate into TAG was increased in CHO MT58 cells. The in vitro de novo synthesis of TAG and the activity of diacylglycerol acyltransferase were 90% and 34% higher, respectively. Two other key enzyme activities in TAG synthesis, acyl-CoA synthetase and mitochondrial glycerol-3-phosphate acyltransferase (GPAT), increased by 48% and 2-fold, respectively, and mitochondrial GPAT mRNA increased by approximately 4-fold. Additionally, TAG hydrolysis was accelerated in CHO MT58 cells, and in vitro lipolytic activity increased by 68%. These studies suggest that a homeostatic mechanism increases TAG synthesis and recycling in response to PC deficiency. TAG recycling produces diacylglycerol and fatty acids that can be substrates for de novo PC synthesis and for lysophosphatidylcholine (lysoPC) acylation. In CHO MT58 cells, in which de novo PC synthesis is blocked, lysoPC acylation with fatty acid originating from TAG may represent the main pathway for generating PC.     

Characterisation of acyltransferases from Synechocystis sp. PCC6803

As phylogenetic ancestors of plant chloroplasts cyanobacteria resemble plastids with respect to lipid and fatty acid composition. These membrane lipids show the typical prokaryotic fatty acid pattern in which the sn-2 position is exclusively esterified by C(16) acyl groups. In the course of de novo glycerolipid biosynthesis this prokaryotic fatty acid pattern is established by the sequential acylation of glycerol-3-phosphate with acyl-ACPs by the activity of different acyltransferases. In silico approaches allowed the identification of putative Synechocystis acyltransferases involved in glycerolipid metabolism. Functional expression studies in Escherichia coli showed that sll1848 codes for a lysophosphatidic acid acyltransferase with a high specificity for 16:0-ACP, whereas slr2060 encodes a lysophospholipid acyltransferase, with a broad acyl-ACP specificity but a strong preference for lysophosphatidyglycerol especially its sn-2 acyl isomer as acyl-acceptor. The generation and analysis of the corresponding Synechocystis knockout mutants revealed that lysophosphatidic acid acyltransferase unlike the lysophospholipid acyltransferase is essential for the vital functions of the cells.