Biochemical characterization of acyl-CoA:diacylglycerol acyltransferase2 from the diatom Phaeodactylum tricornutum and its potential effect on LC-PUFAs biosynthesis in planta
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Abstract Background Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), belonging to ω-3 long-chain polyunsaturated fatty acids (ω3-LC-PUFAs), are essential components of human diet. They are mainly supplemented by marine fish consumption, although their native producers are oleaginous microalgae. Currently, increasing demand for fish oils is insufficient to meet the entire global needs, which puts pressure on searching for the alternative solutions. One possibility may be metabolic engineering of plants with an introduced enzymatic pathway producing ω3-LC-PUFAs. Result In this study we focused on the acyl-CoA:diacylglycerol acyltransferase2b ( Pt DGAT2b) from the diatom Phaeodactylum tricornutum , an enzyme responsible for triacylglycerol (TAG) biosynthesis via acyl-CoA-dependent pathway. Gene encoding Pt DGAT2b, incorporated into TAG-deficient yeast strain H1246, was used to confirm its activity and conduct biochemical characterization. Pt DGAT2b exhibited a broad acyl-CoA preference with both di-16:0-DAG and di-18:1-DAG, whereas di-18:1-DAG was favored. The highest preference for acyl donors was observed for 16:1-, 10:0- and 12:0-CoA. Pt DGAT2b also very efficiently utilized CoA-conjugated ω-3 LC-PUFAs (stearidonic acid, eicosatetraenoic acid and EPA). Additionally, verification of the potential role of Pt DGAT2b in planta , through its transient expression in tobacco leaves, indicated increased TAG production with its relative amount increasing to 8%. Its co-expression with the gene combinations aimed at EPA biosynthesis led to, beside elevated TAG accumulation, efficient accumulation of EPA which constituted even 25.1% of synthesized non-native fatty acids (9.2% of all fatty acids in TAG pool). Conclusions This set of experiments provides a comprehensive biochemical characterization of DGAT enzyme from marine microalgae. Additionally, this study elucidates that Pt DGAT2b can be used successfully in metabolic engineering of plants designed to obtain a boosted TAG level, enriched not only in ω-3 LC-PUFAs but also in medium-chain and ω-7 fatty acids.Keywords:
Phaeodactylum tricornutum
Metabolic Engineering
Nannochloropsis
The effects of different illumination and one-side versus two-side illumination on the growth,total lipid and chlorophyll a content of Nannochloropsis oculata and Phaeodactylum tricornutum were studied.An increase of the illumination produced an increase of the biomass of Nannochloropsis oculata,and the two-side illumination was superior to one-side illuminalion under the same illumination;the biomass of Phaeodactylurn tricornutum showed little difference under the different illumination.The total lipid content of Nannochloropsis oculata increased,and then decreased with the increase of illumination;the total lipid content of Phaeodactylum tricornutum increased with the increase of illumination.The chlorophyll a contents of Nannochloropsis oculata and Phaeodactylum tricormutum reduced with the increase of total lipid content.The results showed that compared with Phaeodactylum tricornutum,Nannochloropsis oculata could be considered as feedstocks for lipid production.
Phaeodactylum tricornutum
Nannochloropsis
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Microalgae accumulate lipids when exposed to stressful conditions such as nutrient limitation that can be used to generate biofuels. Nitrogen limitation or deprivation is a strategy widely employed to elicit this response. However, this strategy is associated with a reduction in the microalgal growth, leading to overall poor lipid productivities. Here, we investigated the combined effect of a reduced source of nitrogen (ammonium) and super-saturating light intensities on the growth and induction of lipid accumulation in two model but diverse microalgal species, Phaeodactylum tricornutum and Nannochloropsis oceanica. We hypothesized that the lower energy cost of assimilating ammonium would allow the organisms to use more reductant power for lipid biosynthesis without compromising growth and that this would be further stimulated by the effect of high light (1000 µmol m-2 s-1) stress. We studied the changes in growth and physiology of both species when grown in culture media that either contained nitrate or ammonium as the nitrogen source, and an additional medium that contained ammonium with tungsten in place of molybdenum and compared this with growth in media without nitrogen. We focused our investigation on the early stages of exposure to the treatments to correspond to events relevant to induction of lipid accumulation in these two species.At super-saturating light intensities, lipid productivity in P. tricornutum increased twofold when grown in ammonium compared to nitrogen free medium that increased further when tungsten was present in the medium in place of molybdenum. Conversely, N. oceanica growth and physiology was not compromised by the high light intensities used, and the use of ammonium had a negative effect on the lipid productivity, which was even more marked when tungsten was present.Whilst the use of ammonium and super-saturating light intensities in P. tricornutum was revealed to be a good strategy for increasing lipid biosynthesis, no changes in the lipid productivity of N. oceanica were observed, under these conditions. Both results provide relevant direction for the better design of processes to produce biofuels in microalgae by manipulating growth conditions without the need to subject them to genetic engineering manipulation.
Phaeodactylum tricornutum
Nannochloropsis
Light intensity
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Nannochloropsis
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Phaeodactylum tricornutum
Nannochloropsis
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Effects of different nitrogen sources and their concentrations on the growth,total lipids and fatty acids composition of Phaeodactylum tricornutum were studied.The results showed that P.tricornutum growed poorly in cultures without addition of any nitrogen source,but accumulated lipids effectively.The biomass and lipid content of P.tricornutum were promoted simultaneously with more than 3.9 mmol/L of initial ammonium chloride in medium;8.0 mmol/L of urea enhanced the biomass of algae while decreased the lipid content;in the range of tested concentration,sodium nitrate promoted growth of algae and depressed the accumulation of lipid.The fatty acids composition analysis showed that monounsaturated fatty acid(MPUAs) accounted for 45%~50% of the total fatty acids and was less affected by nitrogen sources,thus provided better mobility for biodiesel.The synthesis of C20∶5(n-3)(EPA) and polyunsaturated fatty acid(PUFAs) were increased with addition of nitrogen sources.With 3.9 mmol/L of ammonium chloride,the ratio of EPA and PUFAs to total fatty acids was the highest,20.4% and 26.7%,respectively.
Phaeodactylum tricornutum
Nannochloropsis
Ammonium chloride
Sodium nitrate
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Phaeodactylum tricornutum
Nannochloropsis
Sodium bicarbonate
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Phaeodactylum tricornutum UTEX 640 strain of microalgae was screened under different culture conditions for their capacity to produce eicosapentaenoic acid (EPA) the most abundant polyunsaturated fatty acid (PUFA). In our experiments, the effect of sodium chlorid, nitrogen source, phosphate, initial pH, as well as the CO2 content of the medium on production of the eicosapentaenoic acid (EPA) by P. tricornutum were investigated. The EPA content of biomass was enhanced by the low pH of the medium, with increased concentrations of B17 vitamin and nitrate, and also with decreasing concentrations of sodium chlorid. The EPA is most likely associated with polar (membran) lipids and the role of EPA appears to be involved with membran permeability in microalge. The synthesis of phospholipids, enhances the EPA content of the cells, as expected. The maximum EPA yields were observed under optimum culture condition 43 — 48 mg/g of dry cell weight.
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Phaeodactylum tricornutum
Nannochloropsis
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