Biochemical characterization of acyl-CoA:diacylglycerol acyltransferase2 from the diatom Phaeodactylum tricornutum and its potential effect on LC-PUFAs biosynthesis in planta.

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 (PtDGAT2b) from the diatom Phaeodactylum tricornutum, an enzyme responsible for triacylglycerol (TAG) biosynthesis via acyl-CoA-dependent pathway. Gene encoding PtDGAT2b, incorporated into TAG-deficient yeast strain H1246, was used to confirm its activity and conduct biochemical characterization. PtDGAT2b 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. PtDGAT2b also very efficiently utilized CoA-conjugated ω-3 LC-PUFAs (stearidonic acid, eicosatetraenoic acid and EPA). Additionally, verification of the potential role of PtDGAT2b 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 PtDGAT2b 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.

Biorefining of rapeseed meal: A new and sustainable strategy for improving Cr(VI) biosorption on residual wastes from agricultural byproducts after phenolic extraction.

Phenolic recovery from agricultural byproducts has been highlighted due to their health-promoting bioactivities. However, uncontrolled discard of residues after extraction process would induce environmental pollution and bioresource waste. In this study, biorefining of phenolic-rich rapeseed meal (RSM) and its defatted sample (dRSM) was attempted by holistic utilization of phenolic extract and residue separately. Phenolic removal could significantly improve residues' Cr(VI) adsorption capacities by about 21%, which presented extended physical surface and more released functional groups. Moreover, simulating raw material by remixing 3% separated phenolic extracts or main component sinapic acid therein with corresponding residues further improved about 12% adsorption efficiencies. These indicated that the different present forms of phenolics had opposite effects on Cr(VI) removal. While natural conjugational form inhibited hosts' biosorption, free form had enhanced functions for either extract or residue. Four optimal adsorption parameters (pH, adsorbent dosage, contact time and initial Cr(VI) concentration), three kinetic (pseudo-first order, pseudo-second order and intra-particle diffusion) models and two isotherms (Langmuir and Freundlich) were used to reveal the adsorption process. The maximum Cr(VI) adsorption capacity on residues could reach about 100 mg/g, which was superior to that of most biosorbents derived from agricultural byproducts, even some biochar. Together with the residues' advantages with everlasting capacity after 3 adsorption-desorption cycles and excellent abilities for adsorbing multiple co-existed metal ions (Cr(VI), Cd(II), Cu(II), Pb(II), Ni(II) and Zn(II)), phenolic recovery was first proved to be a new and sustainable strategy for modifying biosorbents from agricultural byproducts with zero waste.

Improvement of Omega-3 Docosahexaenoic Acid Production by Marine Dinoflagellate Crypthecodinium cohnii Using Rapeseed Meal Hydrolysate and Waste Molasses as Feedstock.

Rapeseed meal and waste molasses are two important agro-industrial by-products which are produced in large quantities. In this study, solid state fermentation and fungal autolysis were performed to produce rapeseed meal hydrolysate (RMH) using fungal strains of Aspergillus oryzae, Penicillium oxalicum and Neurospora crassa. The hydrolysate was used as fermentation feedstock for heterotrophic growth of microalga Crypthecodinium cohnii that produce docosahexaenoic acid (DHA). The addition of waste molasses as a supplementary carbon source greatly increased the biomass and DHA yield. In the batch fermentations using media composed of diluted RMH (7%) and 1-9% waste molasses, the highest biomass concentration and DHA yield reached 3.43 g/L and 8.72 mg/L, respectively. The algal biomass produced from RMH and molasses medium also had a high percentage of DHA (22-34%) in total fatty acids similar to that of commercial algal biomass. RMH was shown to be rich in nitrogen supply comparable to the commercial nitrogen feedstock like yeast extract. Using RMH as sole nitrogen source, waste molasses excelled other carbon sources and produced the highest concentration of biomass. This study suggests that DHA production of the marine dinoflagellate C. cohnii could be greatly improved by concomitantly using the cheap by-products rapeseed meal hydrolysate and molasses as alternative feedstock.

Characterization of three Δ9-fatty acid desaturases with distinct substrate specificity from an oleaginous fungus Cunninghamella echinulata.

In oleaginous fungus Cunninghamella echinulata, Δ9-fatty acid desaturase introduces the first double bond into a saturated fatty acid. Three distinct genes, designated as d9dma, d9dmb and d9dmc, all encoding putative Δ9-fatty acid desaturases were isolated from this strain. The predicted proteins showed 79-87 % identity to other fungal Δ9-fatty acid desaturases. They all contain three conserved histidine boxes, C-terminal cytochrome b 5 fusion and four transmembrane domains characteristic of Δ9-desaturase. Each putative Δ9-desaturase gene from C. echinulata was able to complement the ole1 mutation in Saccharomyces cerevisiae L8-14C through heterologous expression. Analysis of the fatty acid composition of the transgenic yeast revealed that the conversion rates of 16:0 and 18:0 by D9DMA were obviously higher than those of D9DMB and D9DMC. In addition, D9DMA, D9DMB and D9DMC all had a substrate preference for 18:0 compared with 16:0. Of interest, D9DMA could saturate 12:0, 14:0, 16:0, 17:0, 18:0 and 20:0, while D9DMB saturated 14:0, 16:0, 17:0, 18:0 and 20:0. We also noticed that the transcriptional level of d9dma in C. echinulata was stimulated by cell growth but not by decline in temperature. In contrast, expression of d9dmb and d9dmc was regulated by neither cell growth nor decline in temperature in this strain.

Triacylglycerol accumulation and change in fatty acid content of four marine oleaginous microalgae under nutrient limitation and at different culture ages.

Alteration of lipid biosynthesis is one of important biochemical changes when oleaginous microalgae grow under varied environmental conditions. The effects of culture age and nutrient limitation on triacylglycerol (TAG) accumulation and fatty acid content were investigated in four eicosapentaenoic acid (EPA)-rich marine microalgae. The amounts of TAGs in Chaetoceros sp., Phaeodactylum tricornutum and Nannochloropsis oculata increased sharply from day 4 to day 11, and then the former two remained nearly unchanged while the latter declined gradually during the batch culture. In contrast, no marked increase in TAG accumulation was observed in Pavlova viridis during the culture. Changes in total fatty acid (TFA) content mirrored those observed for TAG accumulation, while the EPA content reached a maximum generally at day 7 or 11 in the range of 11 - 32 mg g(-1) dry cell weight (DCW) and then declined. Nitrogen limitation led to a gradual increase in the amounts of TAGs from N. oculata pronouncedly but almost no change in other three species. The TFA content of the cultures after 5 days of nitrogen limitation was nearly twice that after 1 day in Chaetoceros sp., P. tricornutum and P. viridis, while the lowest increase (220 - 283 mg g(-1) DCW) was observed in N. oculata. TAGs increased gradually under phosphorus limitation in all four species but not sharply compared with that under nitrogen limitation in N. oculata. The TFA content increased gradually under phosphorus limitation and after 5 days of phosphorus limitation it was 1.5 - 2 times that after 1 day. The EPA content was generally not significantly affected by nitrogen or phosphorus limitation. Culture age and nutrient limitation could be useful variables for optimizing TAG accumulation and fatty acid content with potential for biodiesel production.