Trialkylsulfonium- and Trialkylselenoniumhydroxides for the Pyrolytic Alkylation of Acidic Compounds

Butte, W.; Eilers, J.; Kirsch, M.

doi:10.1080/00032718208069519

Cited by 78 publications

(50 citation statements)

References 4 publications

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“…To analyze the FAs of single seeds, we prepared fatty acid methyl esters (FAMEs) by incubating the seeds with 0.2 M trimethylsulfonium hydroxide in methanol (Butte et al, 1982). To similarly analyze bulk seeds, FAMEs were prepared by incubation in 0.5 mL BCl 3 for 1 h at 80°C, extracting them with 1 mL of hexane, and then drying under N 2 .…”

Section: Fa Analysismentioning

confidence: 99%

Metabolic Engineering of Seeds Can Achieve Levels of ω-7 Fatty Acids Comparable with the Highest Levels Found in Natural Plant Sources

Nguyen

Mishra²,

Whittle³

et al. 2010

Plant Physiology

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Plant oils containing v-7 fatty acids (FAs; palmitoleic 16:1D 9 and cis-vaccenic 18:1D 11 ) have potential as sustainable feedstocks for producing industrially important octene via metathesis chemistry. Engineering plants to produce seeds that accumulate high levels of any unusual FA has been an elusive goal. We achieved high levels of v-7 FA accumulation by systematic metabolic engineering of Arabidopsis (Arabidopsis thaliana). A plastidial 16:0-ACP desaturase has been engineered to convert 16:0 to 16:1D 9 with specificity .100-fold than that of naturally occurring paralogs, such as that from cat's claw vine (Doxantha unguis-cati). Expressing this engineered enzyme (Com25) in seeds increased v-7 FA accumulation from ,2% to 14%. Reducing competition for 16:0-ACP by down-regulating the b-ketoacyl-ACP synthase II 16:0 elongase further increased accumulation of v-7 FA to 56%. The level of 16:0 exiting the plastid without desaturation also increased to 21%. Coexpression of a pair of fungal 16:0 desaturases in the cytosol reduced the 16:0 level to 11% and increased v-7 FA to as much as 71%, equivalent to levels found in Doxantha seeds.There is increasing interest in the use of plant oils as renewable sources of industrial chemical feedstocks Carlsson, 2009). Recent developments in olefin metathesis have demonstrated that long-chain monoene FA from vegetable oils can be efficiently split into the corresponding short-chain a-olefin and v-alkenoic acids (Rybak et al., 2008;Meier, 2009). Thus, ethenolytic metathesis of v-7 fatty acids (FAs), such as palmitoleic or cis-vaccenic acids, yields 1-octene and 9-decenoate. 1-Octene is a high-demand feedstock with a global consumption of over half a million tons per year that is primarily used as a comonomer in the expanding production of linear low density polyethylene. It is mainly synthesized from petroleumderived ethylene via oligomerization to yield a complex mixture of a-olefins or from coal-derived syngas (Anonymous, 2007).A plant oil containing high (.70%) content of v-7 FA would represent a new and sustainable feedstock for 1-octene production. Several natural plant oil sources of v-7 FA have been reported, e.g. milkweed (Asclepias syriaca) accumulates approximately 25% v-7 FA (comprising approximately 10% 16:1D 9 and approximately 15% 18:1D 11 ) and cat's claw vine (Doxantha unguis-cati) accumulates approximately 72% v-7 FA (comprising approximately 55% 16:1D 9 and approximately 17% 18:1D 11 ), but the low yields and poor agronomic properties of these plants preclude their commercial use for v-7 FA production. Isolation of the D 9 -16:0-ACP desaturases genes responsible for the production of v-7 FA from milkweed and Doxantha (Cahoon et al., 1997 presented an opportunity for their heterologous expression and v-7 FA production in transgenic crops. However, heterologous expression of the milkweed desaturase in Arabidopsis (Arabidopsis thaliana) failed to produce detectable v-7 FA, and when the Doxantha desaturase was expressed in Brassica napus, it resulted in the accumul...

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Section: Fa Analysismentioning

confidence: 99%

Metabolic Engineering of Seeds Can Achieve Levels of ω-7 Fatty Acids Comparable with the Highest Levels Found in Natural Plant Sources

Nguyen

Mishra²,

Whittle³

et al. 2010

Plant Physiology

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“…Fatty acid methyl esters (FAMEs) were reextracted with 2 ml of hexane and dried under N 2 . FAMEs of single Arabidopsis seeds were prepared (24). FAMEs were analyzed by using an HP5890 gas chromatograph (Hewlett-Packard) fitted with a 60-m ϫ 250-m SP-2340 capillary column (Supelco).…”

Section: Materials Andmentioning

confidence: 99%

Switching desaturase enzyme specificity by alternate subcellular targeting

Heilmann

Pidkowich²,

Girke³

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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The functionality, substrate specificity, and regiospecificity of enzymes typically evolve by the accumulation of mutations in the catalytic portion of the enzyme until new properties arise. However, emerging evidence suggests enzyme functionality can also be influenced by metabolic context. When the plastidial Arabidopsis 16:0⌬ 7 desaturase FAD5 (ADS3) was retargeted to the cytoplasm, regiospecificity shifted 70-fold, ⌬ 7 to ⌬ 9 . Conversely, retargeting of two related cytoplasmic 16:0⌬ 9 Arabidopsis desaturases (ADS1 and ADS2) to the plastid, shifted regiospecificity Ϸ25-fold, ⌬ 9 to ⌬ 7 . All three desaturases exhibited ⌬ 9 regiospecificity when expressed in yeast, with desaturated products found predominantly on phosphatidylcholine. Coexpression of each enzyme with cucumber monogalactosyldiacylglycerol (MGDG) synthase in yeast conferred ⌬ 7 desaturation, with 16:1⌬ 7 accumulating specifically on the plastidial lipid MGDG. Positional analysis is consistent with ADS desaturation of 16:0 on MGDG. The lipid headgroup acts as a molecular switch for desaturase regiospecificity. FAD5 ⌬ 7 regiospecificity is thus attributable to plastidial retargeting of the enzyme by addition of a transit peptide to a cytoplasmic ⌬ 9 desaturase rather than the numerous sequence differences within the catalytic portion of ADS enzymes. The MGDG-dependent desaturase activity enabled plants to synthesize 16:1⌬ 7 and its abundant metabolite, 16:3⌬ 7,10,13 . Bioinformatics analysis of the Arabidopsis genome identified 239 protein families that contain members predicted to reside in different subcellular compartments, suggesting alternative targeting is widespread. Alternative targeting of bifunctional or multifunctional enzymes can exploit eukaryotic subcellular organization to create metabolic diversity by permitting isozymes to interact with different substrates and thus create different products in alternate compartments.M etabolic diversity in living systems arises primarily from biotransformations catalyzed by enzymes; indeed life itself depends on the specificity of enzymes. The unique functionality, substrate specificity, and regiospecificity of an enzyme typically evolves by the gradual accumulation of changes in the catalytic portion of the enzyme until new properties arise. However, there is an emerging body of evidence suggesting that an enzyme's functional characteristics can also be affected by its metabolic context and that temporal and spatial dynamics of enzyme interactions can be important determinants of enzyme functionality (1). Examples include the ''rewiring'' of mitogen-activated protein kinase pathways on alternative scaffolds (2); the localization-dependent interactions of transcription factors with RNA polymerase in the nucleus (reviewed in ref. 3); the interchangeable tissue-specific roles of the transcription factors WER and GL1 in plant epidermal hair development (4); the modification of laccase͞peroxidase activity in the extracellular matrix by specific dirigent proteins (reviewed in ref. 5); the altered gati...

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“…Fatty acid methyl esters were prepared by homogenization of single seeds in 10 l of trimethylsulfoniumhydroxide͞methanol (27) in a 100-l autosampler vial. After a 15-min reaction at room temperature, samples were dried, resuspended in 35 l of hexane, and analyzed with a Hewlett-Packard 5890 GC fitted with a 30-m ϫ 320-m (i.d.)…”

Section: Characterization Of the Substrate Specificity Of Mutants Of mentioning

confidence: 99%

Substrate-dependent mutant complementation to select fatty acid desaturase variants for metabolic engineering of plant seed oils

Cahoon

Shanklin

2000

Proc. Natl. Acad. Sci. U.S.A.

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We demonstrate that naturally occurring C14 and C16-specific acylacyl carrier protein (ACP) desaturases from plants can complement the unsaturated fatty acid (UFA) auxotrophy of an Escherichia coli fabA͞fadR mutant. Under the same growth conditions, C 18-specific ⌬ 9 -stearoyl (18:0)-ACP desaturases are unable to complement the UFA auxotrophy. This difference most likely results from the presence of sufficient substrate pools of C 14 and C16 acyl-ACPs but a relative lack of C 18 acyl-ACP pools in E. coli to support the activities of the plant fatty acid desaturase. Based on this, a substrate-dependent selection system was devised with the use of the E. coli UFA auxotroph to isolate mutants of the castor ⌬ 9 -18:0-ACP desaturase that display enhanced specificity for C 14 and C16 acyl-ACPs. Using this selection system, a number of desaturase variants with altered substrate specificities were isolated from pools of randomized mutants. These included several G188L mutant isolates, which displayed a 15-fold increase in specific activity with 16:0-ACP relative to the wild-type castor ⌬ 9 -18:0-ACP desaturase. Expression of this mutant in Arabidopsis thaliana resulted in the accumulation of unusual monounsaturated fatty acids to amounts of >25% of the seed oil. The bacterial selection system described here thus provides a rapid means of isolating variant fatty acid desaturase activities for modification of seed oil composition.A cyl-acyl carrier protein (ACP)-desaturases (DES) are a family of soluble enzymes that are associated with the synthesis of monounsaturated fatty acids in plants (1). These enzymes catalyze the insertion of a double bond into saturated fatty acids bound to ACP in the plastids of plant cells, and thus serve as one of the primary determinants of the unsaturated fatty acid content of plant membranes and seed storage oils. The most widely occurring member of this family is the ⌬ 9 -stearoyl (18:0 ‡ )-ACP DES, which introduces the double bond of oleic acid (1). In addition, a number of other acyl-ACP DES have been identified that are associated with the synthesis of unusual monounsaturated fatty acids and have altered substrate and regio-specifities relative to the ⌬ 9 -18:0-ACP DES. Variant acyl-ACP DES identified to date include the ⌬ 4 -palmitoyl (16:0)-ACP DES from Umbelliferae seed (2, 3), the ⌬ 6 -16:0-ACP DES from black-eyed Susan vine (Thunbergia alata) seed (4), the ⌬ 9 -myristoyl (14:0)-ACP DES from geranium (Pelargonium xhortorum) trichomes (5), and ⌬ 9 -16:0-ACP DES from cat's claw (Doxantha unguis-cati) (6) and milkweed (Asclepias syriaca) (7) seeds. These enzymes share Ն60% amino acid sequence identity with the ⌬ 9 -18:0-ACP DES.This collection of structurally related, but functionally diverged, enzymes provides a tool for understanding how acyl-ACP DES recognize the chain length of substrates and position the placement of double bonds. Characterization of these properties also has been greatly aided by information from the crystal structure of the castor ⌬ 9 -18:0-ACP DES (8). From the...

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Trialkylsulfonium- and Trialkylselenoniumhydroxides for the Pyrolytic Alkylation of Acidic Compounds

Cited by 78 publications

References 4 publications

Metabolic Engineering of Seeds Can Achieve Levels of ω-7 Fatty Acids Comparable with the Highest Levels Found in Natural Plant Sources

Metabolic Engineering of Seeds Can Achieve Levels of ω-7 Fatty Acids Comparable with the Highest Levels Found in Natural Plant Sources

Switching desaturase enzyme specificity by alternate subcellular targeting

Substrate-dependent mutant complementation to select fatty acid desaturase variants for metabolic engineering of plant seed oils

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