Caenorhabditis elegans Δ12-Desaturase FAT-2 Is a Bifunctional Desaturase Able to Desaturate a Diverse Range of Fatty Acid Substrates at the Δ12 and Δ15 Positions

Zhou, Xue-Rong; Green, Allan; Singh, Surinder P.

doi:10.1074/jbc.m111.266114

Cited by 72 publications

(67 citation statements)

References 16 publications

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“…Δ9,Δ12,Δ15-16:3 also has been identified as an initially undiscerned product of Δ15-FAD from the zygomycete fungus Mortierella alpina [21] and Δ12-FAD from the nematode Caenorhabditis elegans [20].…”

Section: Discussionmentioning

confidence: 99%

Δ12-Fatty Acid Desaturase from Candida parapsilosis Is a Multifunctional Desaturase Producing a Range of Polyunsaturated and Hydroxylated Fatty Acids

et al. 2014

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Numerous Δ12-, Δ15- and multifunctional membrane fatty acid desaturases (FADs) have been identified in fungi, revealing great variability in the enzymatic specificities of FADs involved in biosynthesis of polyunsaturated fatty acids (PUFAs). Here, we report gene isolation and characterization of novel Δ12/Δ15- and Δ15-FADs named CpFad2 and CpFad3, respectively, from the opportunistic pathogenic yeast Candida parapsilosis. Overexpression of CpFad3 in Saccharomyces cerevisiae strains supplemented with linoleic acid (Δ9,Δ12-18:2) and hexadecadienoic acid (Δ9,Δ12-16:2) leads to accumulation of Δ15-PUFAs, i.e., α-linolenic acid (Δ9,Δ12,Δ15-18:3) and hexadecatrienoic acid with an unusual terminal double bond (Δ9,Δ12,Δ15-16:3). CpFad2 produces a range of Δ12- and Δ15-PUFAs. The major products of CpFad2 are linoleic and hexadecadienoic acid (Δ9,Δ12-16:2), accompanied by α-linolenic acid and hexadecatrienoic acid (Δ9,Δ12,Δ15-16:3). Using GC/MS analysis of trimethylsilyl derivatives, we identified ricinoleic acid (12-hydroxy-9-octadecenoic acid) as an additional product of CpFad2. These results demonstrate that CpFAD2 is a multifunctional FAD and indicate that detailed analysis of fatty acid derivatives might uncover a range of enzymatic selectivities in other Δ12-FADs from budding yeasts (Ascomycota: Saccharomycotina).

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Section: Discussionmentioning

confidence: 99%

Δ12-Fatty Acid Desaturase from Candida parapsilosis Is a Multifunctional Desaturase Producing a Range of Polyunsaturated and Hydroxylated Fatty Acids

et al. 2014

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“…Fatty acid methyl esters (FAMEs) of total lipids (equivalent to 10 mg dry weight) were produced by incubating extracted lipid in 1 N methanolic HCl (Supelco, Bellefonte, PA) at 80 °C for 3 h. FAMEs were analysed by an Agilent 7890A gas chromatograph coupled with flame ionization detector (GC‐FID, Agilent Technologies, Palo Alto, CA), on a BPX70 column (30 m, 0.25 mm inner diameter, 0.25 μm film thickness, SGE) essentially as described previously (Zhou et al ., ), except the column temperature programme. The column temperature was programmed as an initial temperature at 100 °C holding for 3 min, ramping to 240 °C at a rate of 7 °C/min and holding for 1 min.…”

Section: Methodsmentioning

confidence: 99%

A reconfigured Kennedy pathway which promotes efficient accumulation of medium‐chain fatty acids in leaf oils

Reynolds

Taylor²,

Cullerne

et al. 2017

Plant Biotechnology Journal

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SummaryMedium‐chain fatty acids (MCFA, C6‐14 fatty acids) are an ideal feedstock for biodiesel and broader oleochemicals. In recent decades, several studies have used transgenic engineering to produce MCFA in seeds oils, although these modifications result in unbalance membrane lipid profiles that impair oil yields and agronomic performance. Given the ability to engineer nonseed organs to produce oils, we have previously demonstrated that MCFA profiles can be produced in leaves, but this also results in unbalanced membrane lipid profiles and undesirable chlorosis and cell death. Here we demonstrate that the introduction of a diacylglycerol acyltransferase from oil palm, EgDGAT1, was necessary to channel nascent MCFA directly into leaf oils and therefore bypassing MCFA residing in membrane lipids. This pathway resulted in increased flux towards MCFA rich leaf oils, reduced MCFA in leaf membrane lipids and, crucially, the alleviation of chlorosis. Deep sequencing of African oil palm (Elaeis guineensis) and coconut palm (Cocos nucifera) generated candidate genes of interest, which were then tested for their ability to improve oil accumulation. Thioesterases were explored for the production of lauric acid (C12:0) and myristic (C14:0). The thioesterases from Umbellularia californica and Cinnamomum camphora produced a total of 52% C12:0 and 40% C14:0, respectively, in transient leaf assays. This study demonstrated that the introduction of a complete acyl‐CoA‐dependent pathway for the synthesis of MFCA‐rich oils avoided disturbing membrane homoeostasis and cell death phenotypes. This study outlines a transgenic strategy for the engineering of biomass crops with high levels of MCFA rich leaf oils.

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“…FAMEs were extracted with 300 μL of hexane, and analysed by GC with Agilent 7890A GC on a 30-m BPX70 column essentially as described before [77] except that the ramping program changed to initial temperature at 150°C, holding 1 min, ramping 3°C/min to 210°C, the 50°C/min to 240°C for a final holding 2 min. Confirming double bond positions in the FAME by 2,4-dimethyloxazoline (DMOX) modification and GC-MS analysis were carried out same as previously described [77], except with a Shimadzu GC-MS QP2010 Plus on a 30-m BPX70 column. The column temperature was programmed as an initial temperature at 150°C for 1 min, ramping at 5°C/min to 200°C then 10°C/min to 240°C with holding for 5 min.…”

Section: Methodsmentioning

confidence: 99%

A large and functionally diverse family of Fad2 genes in safflower (Carthamus tinctoriusL.)

et al. 2013

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BackgroundThe application and nutritional value of vegetable oil is highly dependent on its fatty acid composition, especially the relative proportion of its two major fatty acids, i.e oleic acid and linoleic acid. Microsomal oleoyl phosphatidylcholine desaturase encoded by FAD2 gene is known to introduce a double bond at the Δ12 position of an oleic acid on phosphatidylcholine and convert it to linoleic acid. The known plant FAD2 enzymes are encoded by small gene families consisting of 1-4 members. In addition to the classic oleate Δ12-desaturation activity, functional variants of FAD2 that are capable of undertaking additional or alternative acyl modifications have also been reported in a limited number of plant species. In this study, our objective was to identify FAD2 genes from safflower and analyse their differential expression profile and potentially diversified functionality.ResultsWe report here the characterization and functional expression of an exceptionally large FAD2 gene family from safflower, and the temporal and spatial expression profiles of these genes as revealed through Real-Time quantitative PCR. The diversified functionalities of some of the safflower FAD2 gene family members were demonstrated by ectopic expression in yeast and transient expression in Nicotiana benthamiana leaves. CtFAD2-1 and CtFAD2-10 were demonstrated to be oleate desaturases specifically expressed in developing seeds and flower head, respectively, while CtFAD2-2 appears to have relatively low oleate desaturation activity throughout the plant. CtFAD2-5 and CtFAD2-8 are specifically expressed in root tissues, while CtFAD2-3, 4, 6, 7 are mostly expressed in the cotyledons and hypocotyls in young safflower seedlings. CtFAD2-9 was found to encode a novel desaturase operating on C16:1 substrate. CtFAD2-11 is a tri-functional enzyme able to introduce a carbon double bond in either cis or trans configuration, or a carbon triple (acetylenic) bond at the Δ12 position.ConclusionsIn this study, we isolated an unusually large FAD2 gene family with 11 members from safflower. The seed expressed FAD2 oleate Δ12 desaturase genes identified in this study will provide candidate targets to manipulate the oleic acid level in safflower seed oil. Further, the divergent FAD2 enzymes with novel functionality could be used to produce rare fatty acids, such as crepenynic acid, in genetically engineered crop plants that are precursors for economically important phytoalexins and oleochemical products.

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Caenorhabditis elegans Δ12-Desaturase FAT-2 Is a Bifunctional Desaturase Able to Desaturate a Diverse Range of Fatty Acid Substrates at the Δ12 and Δ15 Positions

Cited by 72 publications

References 16 publications

Δ12-Fatty Acid Desaturase from Candida parapsilosis Is a Multifunctional Desaturase Producing a Range of Polyunsaturated and Hydroxylated Fatty Acids

Δ12-Fatty Acid Desaturase from Candida parapsilosis Is a Multifunctional Desaturase Producing a Range of Polyunsaturated and Hydroxylated Fatty Acids

A reconfigured Kennedy pathway which promotes efficient accumulation of medium‐chain fatty acids in leaf oils

A large and functionally diverse family of Fad2 genes in safflower (Carthamus tinctoriusL.)

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