Metabolic engineering of omega-3 long chain polyunsaturated fatty acids in plants using different ∆6- and ∆5-desaturases co-expressed with LPCAT from the marine diatom Phaeodactylum tricornutum.

Continuous research on obtaining an even more efficient production of very long-chain polyunsaturated fatty acids (VLC-PUFAs) in plants remains one of the main challenges of scientists working on plant lipids. Since crops are not able to produce these fatty acids due to the lack of necessary enzymes, genes encoding them must be introduced exogenously from native organisms producing VLC-PUFAs. In this study we reported, in tobacco leaves, the characterization of three distinct ∆6-desaturases from diatom Phaeodactylum tricornutum, fungi Rhizopus stolonifer and microalge Osterococcus tauri and two different ∆5-desaturases from P. tricornutum and single-celled saprotrophic eukaryotes Thraustochytrium sp. The in planta agroinfiltration of essential ∆6-desaturases, ∆6-elongases and ∆5-desaturases allowed for successful introduction of eicosapentaenoic acid (20:5∆5,8,11,14,17) biosynthesis pathway. However, despite the desired, targeted production of ω3-fatty acids we detected the presence of ω6-fatty acids, indicating and confirming previous results that all tested desaturases are not specifically restricted to neither ω3- nor ω6-pathway. Nevertheless, the additional co-expression of acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) from Phaeodactylum tricornutum boosted the proportion of ω3-fatty acids in newly synthesized fatty acid pools. For the most promising genes combinations the EPA content reached at maximum 1.4% of total lipid content and 4.5% of all fatty acids accumulated in the TAG pool. Our results for the first time describe the role of LPCAT enzyme and its effectiveness in alleviating a bottleneck called 'substrate dichotomy' for improving the transgenic production of VLC-PUFAs in plants.

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.

Phospholipid:diacylglycerol acyltransferase1-overexpression stimulates lipid turnover, oil production and fitness in cold-grown plants.

BACKGROUND: Extensive population growth and climate change accelerate the search for alternative ways of plant-based biomass, biofuel and feed production. Here, we focus on hitherto unknow, new promising cold-stimulated function of phospholipid:diacylglycerol acyltransferase1 (PDAT1) - an enzyme catalyzing the last step of triacylglycerol (TAG) biosynthesis. RESULT: Overexpression of AtPDAT1 boosted seed yield by 160% in Arabidopsis plants exposed to long-term cold compared to standard conditions. Such seeds increased both their weight and acyl-lipids content. This work also elucidates PDAT1's role in leaves, which was previously unclear. Aerial parts of AtPDAT1-overexpressing plants were characterized by accelerated growth at early and vegetative stages of development and by biomass weighing three times more than control. Overexpression of PDAT1 increased the expression of SUGAR-DEPENDENT1 (SDP1) TAG lipase and enhanced lipid remodeling, driving lipid turnover and influencing biomass increment. This effect was especially pronounced in cold conditions, where the elevated synergistic expression of PDAT1 and SDP1 resulted in double biomass increase compared to standard conditions. Elevated phospholipid remodeling also enhanced autophagy flux in AtPDAT1-overexpresing lines subjected to cold, despite the overall diminished autophagy intensity in cold conditions. CONCLUSIONS: Our data suggest that PDAT1 promotes greater vitality in cold-exposed plants, stimulates their longevity and boosts oilseed oil production at low temperature.