100 research outputs found
Identification of a Δ4‐desaturase from the microalga <i>Ostreococcus lucimarinus</i>
No full textVery long chain PUFA (VLCPUFA) like DHA are essential and health-beneficial components of the human diet. Due to a shortfall in VLCPUFA supply, research is ongoing to establish VLCPUFA production in heterologous systems like for example oilseed plants. For this purpose it is crucial to identify the required enzymes from primary producers of VLCPUFA. Here, we describe a cDNA from the microalga Ostreococcus lucimarinus coding for a Delta 4-fatty acid desaturase. It exhibits a cytochrome b(5) domain fused to its amino terminus and three histidine boxes that are typically found in front-end desaturases. Heterologous expression of the partly codon-optimized version of the cDNA in yeast revealed that the encoded protein catalyzes the desaturation of (n-3)- as well as (n-6)-substrates with a preference for VLCPUFA. In yeast it localized at the endoplasmic reticulum (ER) membrane and analysis of the product distribution into different lipid classes suggested that the enzyme most likely acts in a lipid-dependent manner. Practical applications: The identified Delta 4-desaturase may be useful for the production of DHA in transgenic oleaginous organisms like annual oilseed crops
seed oil
No full textSeed oil composed of wax esters with long-chain monoenoic acyl moieties represents a high-value commodity for industry. Such plant-derived sperm oil-like liquid wax esters are biodegradable and can have excellent properties for lubrication. In addition, wax ester oil may represent a superior substrate for biodiesel production. In this study, we demonstrate that the low-input oil seed crop Camelina sativa can serve as a biotechnological platform for environmentally benign wax ester production. Two biosynthetic steps catalysed by a fatty alcohol-forming acyl-CoA reductase (FAR) and a wax ester synthase (WS) are sufficient to achieve wax ester accumulation from acyl-CoA substrates. To produce plant-derived sperm oil-like liquid wax esters, the WS from Mus musculus (MmWS) or Simmondsia chinensis (ScWS) were expressed in combination with the FAR from Mus musculus (MmFAR1) or Marinobacter aquaeolei (MaFAR) in seeds of Arabidopsis thaliana and Camelina sativa. The three analysed enzyme combinations Oleo3:mCherry:MmFAR1 Delta c/Oleo3:EYFP:MmWS, Oleo3:mCherry:MmFAR1 Delta c/ScWS and MaFAR/ScWS showed differences in the wax ester molecular species profiles and overall biosynthetic performance. By expressing MaFAR/ScWS in Arabidopsis or Camelina up to 59% or 21% of the seed oil TAGs were replaced by wax esters, respectively. This combination also yielded wax ester molecular species with highest content of monounsaturated acyl moieties. Expression of the enzyme combinations in the Arabidopsis fae1 fad2 mutant background high in oleic acid resulted in wax ester accumulation enriched in oleyl oleate (18:1/18:1 > 60%), suggesting that similar values may be obtained with a Camelina high oleic acid line.EU [FP7-KBBE-2007-1
Production of wax esters in plant seed oils by oleosomal cotargeting of biosynthetic enzymes
No full textWax esters are neutral lipids exhibiting desirable properties for lubrication. Natural sources have traditionally been whales. Additionally some plants produce wax esters in their seed oil. Currently there is no biological source available for long chain length monounsaturated wax esters that are most suited for industrial applications. This study aimed to identify enzymatic requirements enabling their production in oilseed plants. Wax esters are generated by the action of fatty acyl-CoA reductase (FAR), generating fatty alcohols and wax synthases (WS) that esterify fatty alcohols and acyl-CoAs to wax esters. Based on their substrate preference, a FAR and a WS from Mus musculus were selected for this study (MmFAR1 and Mm WS). Mm WS resides in the endoplasmic reticulum (ER), whereas MmFAR1 associates with peroxisomes. The elimination of a targeting signal and the fusion to an oil body protein yielded variants of MmFAR1 and Mm WS that were cotargeted and enabled wax ester production when coexpressed in yeast or Arabidopsis. In the fae1 fad2 double mutant, rich in oleate, the cotargeted variants of MmFAR1 and Mm WS enabled formation of wax esters containing >65% oleyl-oleate. The data suggest that cotargeting of unusual biosynthetic enzymes can result in functional interplay of heterologous partners in transgenic plants.-Heilmann, M., T. Iven, K. Ahmann, E. Hornung, S. Stymne, and I. Feussner. Production of wax esters in plant seed oils by oleosomal cotargeting of biosynthetic enzymes. J. Lipid Res. 2012. 53: 2153-2161
Determination of content and fatty acid composition of unlabeled phosphoinositide species by thin-layer chromatography and gas chromatography
No full textRecent advances in research on the physiological roles of phosphoinositides in eukaryotic organisms indicate a need to distinguish molecular phosphoinositide species on the basis of their characteristic head groups as well as their glycerolipid moieties. Accurate identification of phosphoinositide species in biological samples poses an analytical challenge, because structurally similar inositol phosphate head groups must be resolved, as must lipid-associated fatty acids. Although intact phosphoinositide species have been successfully analyzed, such analyses employ state-of-the-art liquid chromatography/mass spectrometry and require expensive equipment not accessible to many researchers. Described here is a cost-efficient and reliable alternative developed by adaptation of a combination of classic methods for lipid analysis, thin-layer chromatography and gas chromatography. (C) 2008 Elsevier Inc. All rights reserved
Identification and characterization of an acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2) gene from the microalga O. tauri
No full textIn order to identify novel genes encoding enzymes involved in the terminal step of triacylglycerol (TAG) formation, a database search was carried out in the genome of the unicellular photoautotrophic green alga Ostreococcus tauri. The search led to the identification of three putative type 2 acyl-CoA:diacylglycerol acyltransferase-like sequences (DGAT; EC 2.3.1.20), and revealed the absence of any homolog to type 1 or type 3 DGAT sequence in the genome of O. tauri. For two of the cDNA sequences (OtDGAT2A and B) enzyme activity was detected by heterologous expression in Saccharomyces cerevisiae mutant strains with impaired TAG metabolism. However, activity of OtDGAT2A was too low for further analysis. Analysis of their amino acid sequences showed that they share limited identity with other DGAT2 from different plant species, such as Ricinus communis and Vernicia fordii with similar to 25 to 30% identity. Lipid analysis of the mutant yeast cells revealed that OtDGAT2B showed broad substrate specificity accepting saturated as well as mono- and poly-unsaturated acyl-CoAs as substrates. (C) 2010 Elsevier Masson SAS. All rights reserved
Wax ester profiling of seed oil by nano-electrospray ionization tandem mass spectrometry
No full textBackground: Wax esters are highly hydrophobic neutral lipids that are major constituents of the cutin and suberin layer. Moreover they have favorable properties as a commodity for industrial applications. Through transgenic expression of wax ester biosynthetic genes in oilseed crops, it is possible to achieve high level accumulation of defined wax ester compositions within the seed oil to provide a sustainable source for such high value lipids. The fatty alcohol moiety of the wax esters is formed from plant-endogenous acyl-CoAs by the action of fatty acyl reductases (FAR). In a second step the fatty alcohol is condensed with acyl-CoA by a wax synthase (WS) to form a wax ester. In order to evaluate the specificity of wax ester biosynthesis, analytical methods are needed that provide detailed wax ester profiles from complex lipid extracts. Results: We present a direct infusion ESI-tandem MS method that allows the semi-quantitative determination of wax ester compositions from complex lipid mixtures covering 784 even chain molecular species. The definition of calibration prototype groups that combine wax esters according to their fragmentation behavior enables fast quantitative analysis by applying multiple reaction monitoring. This provides a tool to analyze wax layer composition or determine whether seeds accumulate a desired wax ester profile. Besides the profiling method, we provide general information on wax ester analysis by the systematic definition of wax ester prototypes according to their collision-induced dissociation spectra. We applied the developed method for wax ester profiling of the well characterized jojoba seed oil and compared the profile with wax ester-accumulating Arabidopsis thaliana expressing the wax ester biosynthetic genes MaFAR and ScWS. Conclusions: We developed a fast profiling method for wax ester analysis on the molecular species level. This method is suitable to screen large numbers of transgenic plants as well as other wax ester samples like cuticular lipid extracts to gain an overview on the molecular species composition. We confirm previous results from APCI-MS and GC-MS analysis, which showed that fragmentation patterns are highly dependent on the double bond distribution between the fatty alcohol and the fatty acid part of the wax ester.European Commissio
SUCROSE TRANSPORTER 5 supplies Arabidopsis embryos with biotin and affects triacylglycerol accumulation
No full textThe Arabidopsis SUC5 protein represents a classical sucrose/H+ symporter. Functional analyses previously revealed that SUC5 also transports biotin, an essential co-factor for fatty acid synthesis. However, evidence for a dual role in transport of the structurally unrelated compounds sucrose and biotin in plants was lacking. Here we show that SUC5 localizes to the plasma membrane, and that the SUC5 gene is expressed in developing embryos, confirming the role of the SUC5 protein as substrate carrier across apoplastic barriers in seeds. We show that transport of biotin but not of sucrose across these barriers is impaired in suc5 mutant embryos. In addition, we show that SUC5 is essential for the delivery of biotin into the embryo of biotin biosynthesis-defective mutants (bio1 and bio2). We compared embryo and seedling development as well as triacylglycerol accumulation and fatty acid composition in seeds of single mutants (suc5, bio1 or bio2), double mutants (suc5bio1 and suc5bio2) and wild-type plants. Although suc5 mutants were like the wild-type, bio1 and bio2 mutants showed developmental defects and reduced triacylglycerol contents. In suc5bio1 and suc5bio2 double mutants, developmental defects were severely increased and the triacylglycerol content was reduced to a greater extent in comparison to the single mutants. Supplementation with externally applied biotin helped to reduce symptoms in both single and double mutants, but the efficacy of supplementation was significantly lower in double than in single mutants, showing that transport of biotin into the embryo is lower in the absence of SUC5.German Bundesministerium fur Bildung und Forschung (BMBF) [Fkz 0315429
Anillin facilitates septin assembly to prevent pathological outfoldings of central nervous system myelin
No full textMyelin serves as an axonal insulator that facilitates rapid nerve conduction along axons. By transmission electron microscopy, a healthy myelin sheath comprises compacted membrane layers spiraling around the cross-sectioned axon. Previously we identified the assembly of septin filaments in the innermost non-compacted myelin layer as one of the latest steps of myelin maturation in the central nervous system (CNS) (Patzig et al., 2016). Here we show that loss of the cytoskeletal adaptor protein anillin (ANLN) from oligodendrocytes disrupts myelin septin assembly, thereby causing the emergence of pathological myelin outfoldings. Since myelin outfoldings are a poorly understood hallmark of myelin disease and brain aging we assessed axon/myelin-units in Anln-mutant mice by focused ion beam-scanning electron microscopy (FIB-SEM); myelin outfoldings were three-dimensionally reconstructed as large sheets of multiple compact membrane layers. We suggest that anillin-dependent assembly of septin filaments scaffolds mature myelin sheaths, facilitating rapid nerve conduction in the healthy CNS
Phosphoinositides modulate auxin-dependent transcription by controlling the histone acetyltransferase GCN5 in Arabidopsis
No full textPhosphoinositide (PIs) kommen in eukaryotischen Membranen vor. In Pflanzen sind PIs Regulatoren des zytoplasmatischen Membranverkehrs und des Zytoskeletts. Die Arabidopsis PI-Kinase PIP5K2 lokalisiert im Zellkern und an der Plasmamembran. Bei veränderter Expression von PIP5K2 wurden in Arabidopsis reduzierte oder erhöhte Histon-Acetylierung detektiert. Interaktionstests zwischen PIP5K2 und Histon-modifizierenden Enzymen zeigten eine Interaktion mit der Histon-Acetyltransferase GCN5. Überexpression von PIP5K2 in Protoplasten schwächte die Auxin-vermittelte Aktivierung GCN5-abhängiger Gene ab, was mit verminderter H3-Acetylierung konsistent ist. In vitro hemmten PIs die H3-Acetylierung durch GCN5, was auf einen direkten Einfluss von PIs auf GCN5 hinweist. Eine PI-Bindung von GCN5, speziell an PtdIns3P, die für die nukleäre Lokalisierung von GCN5 erforderlich schien, konnte bestätigt werden. Die Daten weisen darauf hin, dass GCN5 durch PtdIns3P an aktive Transkriptionsstellen rekrutiert werden könnte. PIP5K2 entfernt möglicherweise PtdIns-monophosphate aus dieser Interaktion und beeinflusst so die Transkription.Phosphoinositides (PIs) occur in eukaryotic membranes. In plants, PIs are regulators of cytoplasmic membrane trafficking and cytoskeleton. The Arabidopsis PI-kinase PIP5K2 localizes in the nucleus and at the plasma membrane. Upon changed expression of PIP5K2, reduced or increased degrees of histone acetylation were detected in Arabidopsis. Interaction assays between PIP5K2 and histone-modifying enzymes revealed an interaction with the histone acetyltransferase GCN5. Overexpression of PIP5K2 in protoplasts attenuated auxin-mediated activation of GCN5-dependent genes, consistent with decreased H3 acetylation. In vitro, PIs inhibited H3 acetylation by GCN5, indicating a direct effect of PIs on GCN5. PI binding of GCN5, especially to PtdIns3P, that was required for nuclear localization of GCN5, could be confirmed. The data suggest that GCN5 may be recruited by PtdIns3P to active transcription sites. PIP5K2 may remove PtdIns-monophosphates from this interaction and thus affects transcription
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