Metabolic engineering of fatty acids for breeding of new oilseed crops: strategies, problems and first results

Drexler, Hjördis H.S.; Spiekermann, Patricia; Meyer, Astrid; Domergue, Frédéric; Zank, T.; Sperling, Petra; Abbadi, Amine; Heinz, Ernst

doi:10.1078/0176-1617-01025

Cited by 87 publications

(55 citation statements)

References 153 publications

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“…This has also been reinforced by results in transgenic plants producing some modified fatty acids (mFAs), such as those containing hydroxy, epoxy, conjugated diene, or cyclopropane groups. These mFAs are synthesized by the FAD2-related enzymes acting on the sn-2 acyl groups on PC (Drexler et al, 2003;Jaworski and Cahoon, 2003;Lu et al, 2006;Napier and Graham, 2010). Despite high levels of mFAs occurring in many native species, attempts to produce high yields of mFAs in a crop species, or in Arabidopsis as a model, have largely been unsuccessful (Jaworski and Cahoon, 2003;Lu et al, 2006;Lu and Kang, 2008).…”

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confidence: 99%

The Phosphatidylcholine Diacylglycerol Cholinephosphotransferase Is Required for Efficient Hydroxy Fatty Acid Accumulation in Transgenic Arabidopsis

2012

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We previously identified an enzyme, phosphatidylcholine diacylglycerol cholinephosphotransferase (PDCT), that plays an important role in directing fatty acyl fluxes during triacylglycerol (TAG) biosynthesis. The PDCT mediates a symmetrical interconversion between phosphatidylcholine (PC) and diacylglycerol (DAG), thus enriching PC-modified fatty acids in the DAG pool prior to forming TAG. We show here that PDCT is required for the efficient metabolism of engineered hydroxy fatty acids in Arabidopsis (Arabidopsis thaliana) seeds. When a fatty acid hydroxylase (FAH12) from castor (Ricinus communis) was expressed in Arabidopsis seeds, the PDCT-deficient mutant accumulated only about half the amount of hydroxy fatty acids compared with that in the wild-type seeds. We also isolated a PDCT from castor encoded by the RcROD1 (Reduced Oleate Desaturation1) gene. Seed-specific coexpression of this enzyme significantly increased hydroxy fatty acid accumulation in wild type-FAH12 and in a previously produced transgenic Arabidopsis line coexpressing a castor diacylglycerol acyltransferase 2. Analyzing the TAG molecular species and regiochemistry, along with analysis of fatty acid composition in TAG and PC during seed development, indicate that PDCT acts in planta to enhance the fluxes of fatty acids through PC and enrich the hydroxy fatty acids in DAG, and thus in TAG. In addition, PDCT partially restores the oil content that is decreased in FAH12-expressing seeds. Our results add a new gene in the genetic toolbox for efficiently engineering unusual fatty acids in transgenic oilseeds.

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The Phosphatidylcholine Diacylglycerol Cholinephosphotransferase Is Required for Efficient Hydroxy Fatty Acid Accumulation in Transgenic Arabidopsis

2012

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“…Interestingly, different principles of transformations have been found in these genera. While higher plants use exclusively polyunsaturated C 18 fatty acids for the production of oxylipins, animals and algae rely predominantly on the transformation of polyunsaturated C 20 fatty acids (3,6), which are not ubiquitously found in the plant kingdom (7). Moreover, the formation of volatile short chain aldehydes relies on the combined action of LOX and HPL species in higher plants, whereas animals and algae seem to be more flexible, because they may use either the LOX/HPL system or specific LOX forms alone (6,8,9).…”

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A Multifunctional Lipoxygenase with Fatty Acid Hydroperoxide Cleaving Activity from the Moss Physcomitrella patens

Senger

Wichard

Kunze

et al. 2005

Journal of Biological Chemistry

100

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A complex mixture of fatty acid-derived aldehydes, ketones, and alcohols is released upon wounding of the moss Physcomitrella patens. To investigate the formation of these oxylipins at the molecular level we isolated a lipoxygenase from P. patens, which was identified in an EST library by sequence homology to lipoxygenases from plants. Sequence analysis of the cDNA showed that it exhibits a domain structure similar to that of type2 lipoxygenases from plants, harboring an N-terminal import signal for chloroplasts. The recombinant protein was identified as arachidonate 12-lipoxygenase and linoleate 13-lipoxygenase with a preference for arachidonic acid and eicosapentaenoic acid. In contrast to any other lipoxygenase cloned so far, this enzyme exhibited in addition an unusual high hydroperoxidase and also a fatty acid chain-cleaving lyase activity. Because of these unique features the pronounced formation of (2Z)-octen-1-ol, 1-octen-3-ol, the dienal (5Z,8Z,10E)-12-oxo-dodecatrienoic acid and 12-keto eicosatetraenoic acid was observed when arachidonic acid was administered as substrate. 12-Hydroperoxy eicosatetraenoic acid was found to be only a minor product. Moreover, the P. patens LOX has a relaxed substrate tolerance accepting C 18 -C 22 fatty acids giving rise to even more LOX-derived products. In contrast to other lipoxygenases a highly diverse product spectrum is formed by a single enzyme accounting for most of the observed oxylipins produced by the moss. This single enzyme might, in a fast and effective way, be involved in the formation of signal and/or defense molecules thus contributing to the broad resistance of mosses against pathogens.

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“…Using organisms producing VLC-PUFAs such as the fungus Mortierella alpina, the moss Physcomitrella patens, the worm Caenorhaditis elegans, and the diatom Phaeodactylum tricornutum as gene sources, a large collection of sequences coding for elongases and desaturases was created in the last 10 years (reviewed in Ref. 6). Each coding sequence was separately expressed in yeast or plant and the substrate specificity of the encoded enzyme verified so that cDNAs encoding all the enzymatic activities required for DHA synthesis are available.…”

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Acyl Carriers Used as Substrates by the Desaturases and Elongases Involved in Very Long-chain Polyunsaturated Fatty Acids Biosynthesis Reconstituted in Yeast

Domergue

Abbadi

Ott

et al. 2003

Journal of Biological Chemistry

Self Cite

139

146

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The health benefits attributed to very long-chain polyunsaturated fatty acids and the long term goal to produce them in transgenic oilseed crops have led to the cloning of all the genes coding for the desaturases and elongases involved in their biosynthesis. The encoded activities have been confirmed in vivo by heterologous expression, but very little is known about the actual acyl substrates involved in these pathways. Using a ⌬6-elongase and front-end desaturases from different organisms, we have reconstituted in Saccharomyces cerevisiae the biosynthesis of arachidonic acid from exogenously supplied linoleic acid in order to identify these acyl carriers. Acyl-CoA measurements strongly suggest that the elongation step involved in polyunsaturated fatty acids biosynthesis is taking place within the acyl-CoA pool. In contrast, detailed analyses of lipids revealed that the two desaturation steps (⌬5 and ⌬6) occur predominantly at the sn-2 position of phosphatidylcholine when using ⌬5-and ⌬6-desaturases from lower plants, fungi, worms, and algae. The specificity of these ⌬6-desaturases for the fatty acid acylated at this particular position as well as a limiting re-equilibration with the acyl-CoA pool result in the accumulation of ␥-linolenic acid at the sn-2 position of phosphatidylcholine and prevent efficient arachidonic acid biosynthesis in yeast. We confirm by using a similar experimental approach that, in contrast, the human ⌬6-desaturase uses linoleoyl-CoA as substrate, which results in high efficiency of the subsequent elongation step. In addition, we report that ⌬12-desaturases have no specificity toward the lipid polar headgroup or the sn-position.

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Metabolic engineering of fatty acids for breeding of new oilseed crops: strategies, problems and first results

Cited by 87 publications

References 153 publications

The Phosphatidylcholine Diacylglycerol Cholinephosphotransferase Is Required for Efficient Hydroxy Fatty Acid Accumulation in Transgenic Arabidopsis

The Phosphatidylcholine Diacylglycerol Cholinephosphotransferase Is Required for Efficient Hydroxy Fatty Acid Accumulation in Transgenic Arabidopsis

A Multifunctional Lipoxygenase with Fatty Acid Hydroperoxide Cleaving Activity from the Moss Physcomitrella patens

Acyl Carriers Used as Substrates by the Desaturases and Elongases Involved in Very Long-chain Polyunsaturated Fatty Acids Biosynthesis Reconstituted in Yeast

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