Targeted engineering of camelina and pennycress seeds for ultrahigh accumulation of acetyl-TAG.

Acetyl-TAG (3-acetyl-1,2-diacylglycerol), unique triacylglycerols (TAG) possessing an acetate group at the -3 position, exhibit valuable properties, such as reduced viscosity and freezing points. Previous attempts to engineer acetyl-TAG production in oilseed crops did not achieve the high levels found in naturally producing seeds. Here, we demonstrate the successful generation of camelina and pennycress transgenic lines accumulating nearly pure acetyl-TAG at 93 mol% and 98 mol%, respectively.

Metabolic Flux Analysis to Increase Oil in Seeds.

Ensuring an adequate food supply and enough energy to sustainably support future global populations will require enhanced productivity from plants. Oilseeds can help address these needs; but the fatty acid composition of seed oils is not always optimal, and higher yields are required to meet growing demands. Quantitative approaches including metabolic flux analysis can provide insights on unexpected metabolism (i.

Arabidopsis diacylglycerol acyltransferase1 mutants require fatty acid desaturation for normal seed development.

Diacylglycerol acyltransferase1 (DGAT1) is the major enzyme that synthesizes triacylglycerols (TAG) during Arabidopsis seed development. Mutant dgat1 seeds possess low oil content in addition to a high polyunsaturated fatty acid (PUFA) composition. Two genes encoding endoplasmic reticulum localized desaturase enzymes, fatty acid desaturase2 (FAD2) and fatty acid desaturase3 (FAD3), were upregulated in both dgat1-1 and dgat1-2 developing seeds.

Mechanisms of Spirodela polyrhiza tolerance to FGD wastewater-induced heavy-metal stress: Lipidomics, transcriptomics, and functional validation.

Unlike terrestrial angiosperm plants, the freshwater aquatic angiosperm duckweed (Spirodela polyrhiza) grows directly in water and has distinct responses to heavy-metal stress. Plantlets accumulate metabolites, including lipids and carbohydrates, under heavy-metal stress, but how they balance metabolite levels is unclear, and the gene networks that mediate heavy-metal stress responses remain unknown. Here, we show that heavy-metal stress induced by flue gas desulfurization (FGD) wastewater reduces chlorophyll contents, inhibits growth, reduces membrane lipid biosynthesis, and stimulates membrane lipid degradation in S.

High Nutritional Quality of Human-Induced Pluripotent Stem Cell-Generated Proteins through an Advanced Scalable Peptide Hydrogel 3D Suspension System.

Cell-cultured protein technology has become increasingly attractive due to its sustainability and climate benefits. The aim of this study is to determine the nutritional quality of the human-induced pluripotent stem cell (hiPSC)-cultured proteins in an advanced 3D peptide hydrogel system for the highly efficient production of cell-cultured proteins. Our previous study demonstrated a PGmatrix peptide hydrogel for the 3D embedded culture of long-term hiPSC maintenance and expansion (PGmatrix-hiPSC (PG-3D)), which showed significantly superior pluripotency when compared with traditional 2D cell culture on Matrigel and/or Vitronectin and other existing 3D scaffolding systems such as Polyethylene glycol (PEG)-based hydrogels.

Comparison of TLC, HPLC, and direct-infusion ESI-MS methods for the identification and quantification of diacylglycerol molecular species.

Diacylglycerols (DAGs) are anabolic precursors to membrane lipid and storage triacylglycerol biosynthesis, metabolic intermediates of lipid catabolism, and potent cellular signaling molecules. The different DAG molecular species that accumulate over development or in different tissues reflect the changing aspects of cellular lipid metabolism. Consequently, an accurate determination of DAG molecular species in biological samples is essential to understand various metabolic processes and their diagnostic relevance.

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.

Lipidomic Analysis of Arabidopsis T-DNA Insertion Lines Leads to Identification and Characterization of C-Terminal Alterations in FATTY ACID DESATURASE 6.

Mass-spectrometry-based screening of lipid extracts of wounded and unwounded leaves from a collection of 364 Arabidopsis thaliana T-DNA insertion lines produced lipid profiles that were scored on the number and significance of their differences from the leaf lipid profiles of wild-type plants. The analysis identified Salk_109175C, which displayed alterations in leaf chloroplast glycerolipid composition, including a decreased ratio between two monogalactosyldiacylglycerol (MGDG) molecular species, MGDG(18:3/16:3) and MGDG(18:3/18:3). Salk_109175C has a confirmed insertion in the At5g64790 locus; the insertion did not co-segregate with the recessive lipid phenotype in the F2 generation of a wild-type (Columbia-0) × Salk_109175C cross.

Manufacturing specialized wax esters in plants.

Biologically produced wax esters can fulfil different industrial purposes. These functionalities almost drove the sperm whale to extinction from hunting. After the ban on hunting, there is a niche in the global market for biolubricants with properties similar to spermaceti.

Suppression of Improves Soybean Seed Composition by Increasing Oil and Reducing Undigestible Oligosaccharides.

In developing soybean seeds, carbon is partitioned between oil, protein and carbohydrates. Here, we demonstrate that suppression of lipase-mediated turnover of triacylglycerols (TAG) during late seed development increases fatty acid content and decreases the presence of undigestible oligosaccharides. During late stages of embryo development, the fatty acid content of soybean seed decreases while the levels of the oligosaccharides raffinose and stachyose increase.

The tail of chlorophyll: Fates for phytol.

Understanding the pathways involved in chlorophyll breakdown provides a molecular map to the color changes observed in plant life on a global scale each fall. Surprisingly, little is known about the fate of phytol, chlorophyll's 20-carbon branched-chain tail, during this process. A recent study from Gutbrod et al.

Temporal changes in metabolism late in seed development affect biomass composition.

The negative association between protein and oil production in soybean (Glycine max) seed is well-documented. However, this inverse relationship is based primarily on the composition of mature seed, which reflects the cumulative result of events over the course of soybean seed development and therefore does not convey information specific to metabolic fluctuations during developmental growth regimes. In this study, we assessed maternal nutrient supply via measurement of seed coat exudates and metabolite levels within the cotyledon throughout development to identify trends in the accumulation of central carbon and nitrogen metabolic intermediates.

Expression of a high-activity diacylglycerol acetyltransferase results in enhanced synthesis of acetyl-TAG in camelina seed oil.

Acetyl-triacylglycerols (acetyl-TAG) contain an acetate group in the sn-3 position instead of the long-chain fatty acid present in regular triacylglycerol (TAG). The acetate group confers unique physical properties such as reduced viscosity and a lower freezing point to acetyl-TAG, providing advantages for use as emulsifiers, lubricants, and 'drop-in' biofuels. Previously, the synthesis of acetyl-TAG in the seeds of the oilseed crop camelina (Camelina sativa) was achieved through the heterologous expression of the diacylglycerol acetyltransferase gene EaDAcT, isolated from Euonymus alatus seeds that naturally accumulate high levels of acetyl-TAG.

Pennycress, carbon wise: labeling experiments reveal how pennycress seeds efficiently incorporate carbon into biomass.

This article comments on: . Non-conventional pathways enable pennycress ( L.) embryos to achieve high biosynthetic efficiency.

The Plastid Lipase PLIP1 Is Critical for Seed Viability in Mutant Seed.

In developing Arabidopsis () seeds, the synthesis of triacylglycerol (TAG) is mediated primarily by the acyl-CoA-dependent enzyme diacylglycerol acyltransferase1 (DGAT1). In the absence of DGAT1 activity, phospholipid:diacylglycerol acyltransferase (PDAT1) plays an important role in TAG synthesis, consistent with the higher-than-expected oil content and altered fatty acid composition of seed. Transcript profiling of developing wild type (Columbia-0) and mutant seed identified 602 differentially expressed genes.

Functional diversity of glycerolipid acylhydrolases in plant metabolism and physiology.

Most current knowledge about plant lipid metabolism has focused on the biosynthesis of lipids and their transport between different organelles. However, lipid composition changes during development and in response to environmental cues often go beyond adjustments of lipid biosynthesis. When lipids have to be removed to adjust the extent of membranes during down regulation of photosynthesis, or lipid composition has to be adjusted to alter the biophysical properties of membranes, or lipid derived chemical signals have to be produced, lipid-degrading enzymes come into play.

Molecular tools enabling pennycress (Thlaspi arvense) as a model plant and oilseed cash cover crop.

Thlapsi arvense L. (pennycress) is being developed as a profitable oilseed cover crop for the winter fallow period throughout the temperate regions of the world, controlling soil erosion and nutrients run-off on otherwise barren farmland. We demonstrate that pennycress can serve as a user-friendly model system akin to Arabidopsis that is well-suited for both laboratory and field experimentation.

Towards the synthetic design of camelina oil enriched in tailored acetyl-triacylglycerols with medium-chain fatty acids.

The ability to manipulate expression of key biosynthetic enzymes has allowed the development of genetically modified plants that synthesise unusual lipids that are useful for biofuel and industrial applications. By taking advantage of the unique activities of enzymes from different species, tailored lipids with a targeted structure can be conceived. In this study we demonstrate the successful implementation of such an approach by metabolically engineering the oilseed crop Camelina sativa to produce 3-acetyl-1,2-diacyl-sn-glycerols (acetyl-TAGs) with medium-chain fatty acids (MCFAs).

Review: Metabolic engineering of unusual lipids in the synthetic biology era.

The plant kingdom produces a variety of fatty acid structures, many of which possess functional groups useful for industrial applications. The species that produce these unusual fatty acids are often not suitable for large scale commercial production. The ability to create genetically modified plants, together with emerging synthetic biology approaches, offers the potential to develop alternative oil seed crops capable of producing high levels of modified lipids.

Membrane topology and identification of key residues of EaDAcT, a plant MBOAT with unusual substrate specificity.

Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) catalyzes the transfer of an acetyl group from acetyl-CoA to the sn-3 position of diacylglycerol to form 3-acetyl-1,2-diacyl-sn-glycerol (acetyl-TAG). EaDAcT belongs to a small, plant-specific subfamily of the membrane bound O-acyltransferases (MBOAT) that acylate different lipid substrates. Sucrose gradient density centrifugation revealed that EaDAcT colocalizes to the same fractions as an endoplasmic reticulum (ER)-specific marker.

Metabolic engineering of to produce a reduced viscosity oil from lignocellulose.

Background: Acetyl-triacylglycerols (acetyl-TAGs) are unusual triacylglycerol (TAG) molecules that contain an -3 acetate group. Compared to typical triacylglycerol molecules (here referred to as long chain TAGs; lcTAGs), acetyl-TAGs possess reduced viscosity and improved cold temperature properties, which may allow direct use as a drop-in diesel fuel. Their different chemical and physical properties also make acetyl-TAGs useful for other applications such as lubricants and plasticizers.

Defining the extreme substrate specificity of diacylglycerol acetyltransferase, an unusual membrane-bound -acyltransferase.

diacylglycerol acetyltransferase (DAcT) synthesizes the unusually structured 3-acetyl-1,2-diacylglycerols (acetyl-TAG) found in the seeds of a few plant species. A member of the membrane-bound -acyltransferase (MBOAT) family, DAcT transfers the acetyl group from acetyl-CoA to -1,2-diacylglycerol (DAG) to produce acetyl-TAG. assays demonstrated that the enzyme is also able to utilize butyryl-CoA and hexanoyl-CoA as acyl donors, though with much less efficiency compared with acetyl-CoA.

Rapid Quantification of Low-Viscosity Acetyl-Triacylglycerols Using Electrospray Ionization Mass Spectrometry.

Acetyl-triacylglycerols (acetyl-TAG) possess an sn-3 acetate group, which confers useful chemical and physical properties to these unusual triacylglycerols (TAG). Current methods for quantification of acetyl-TAG are time consuming and do not provide any information on the molecular species profile. Electrospray ionization mass spectrometry (ESI-MS)-based methods can overcome these drawbacks.