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Carbocyclic fatty acids in plants: biochemical and molecular genetic characterization of cyclopropane fatty acid synthesis of Sterculiafoetida.

ABSTRACT: Fatty acids containing three-member carbocyclic rings are found in bacteria and plants. Bacteria synthesize cyclopropane fatty acids (CPA-FAs) only by the addition of a methylene group from S-adenosylmethionine to the cis-double bond of monoenoic phospholipid-bound fatty acids. In plants CPA-FAs are usually minor components with cyclopropene fatty acids (CPE-FAs) more abundant. Sterculia foetida seed oil contains 65-78% CPE-FAs, principally sterculic acid. To address carbocyclic fatty acid synthesis in plants, a cDNA library was constructed from developing seeds during the period of maximum oil deposition. About 0.4% of 5,300 expressed sequence tags were derived from one gene, which shared similarities to the bacterial CPA-FA synthase. However, the predicted protein is twice as large as the bacterial homolog and represents a fusion of an FAD-containing oxidase at the N terminus and a methyltransferase at the C terminus. Functional analysis of the isolated full-length cDNA was conducted in tobacco suspension cells where its expression resulted in the accumulation of up to 6.2% dihydrosterculate of total fatty acids. In addition, the dihydrosterculate was specifically labeled by [methyl-(14)C]methionine and by [(14)C]oleic acid in the transgenic tobacco cells. In in vitro assay of S. foetida seed extracts, S-adenosylmethionine served as a methylene donor for the synthesis of dihydrosterculate from oleate. Dihydrosterculate accumulated largely in phosphatidylcholine in both systems. Together, a CPA-FA synthase was identified from S. foetida, and the pathway in higher plants that produce carbocyclic fatty acids was defined as by transfer of C(1) units, most likely from S-adenosylmethionine to oleate.

SUBMITTER: Bao X

PROVIDER: S-EPMC124547 | biostudies-literature | 2002 May

REPOSITORIES: biostudies-literature

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Carbocyclic fatty acids in plants: biochemical and molecular genetic characterization of cyclopropane fatty acid synthesis of Sterculiafoetida.

Bao Xiaoming X Katz Sue S Pollard Mike M Ohlrogge John J

Proceedings of the National Academy of Sciences of the United States of America 20020507 10

Fatty acids containing three-member carbocyclic rings are found in bacteria and plants. Bacteria synthesize cyclopropane fatty acids (CPA-FAs) only by the addition of a methylene group from S-adenosylmethionine to the cis-double bond of monoenoic phospholipid-bound fatty acids. In plants CPA-FAs are usually minor components with cyclopropene fatty acids (CPE-FAs) more abundant. Sterculia foetida seed oil contains 65-78% CPE-FAs, principally sterculic acid. To address carbocyclic fatty acid synth ...[more]

PMID: 11997456

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Project description:BACKGROUND:Cyclopropane fatty acids (CPA) have been found in certain gymnosperms, Malvales, Litchi and other Sapindales. The presence of their unique strained ring structures confers physical and chemical properties characteristic of unsaturated fatty acids with the oxidative stability displayed by saturated fatty acids making them of considerable industrial interest. While cyclopropenoid fatty acids (CPE) are well-known inhibitors of fatty acid desaturation in animals, CPE can also inhibit the stearoyl-CoA desaturase and interfere with the maturation and reproduction of some insect species suggesting that in addition to their traditional role as storage lipids, CPE can contribute to the protection of plants from herbivory. RESULTS:Three genes encoding cyclopropane synthase homologues GhCPS1, GhCPS2 and GhCPS3 were identified in cotton. Determination of gene transcript abundance revealed differences among the expression of GhCPS1, 2 and 3 showing high, intermediate and low levels, respectively, of transcripts in roots and stems; whereas GhCPS1 and 2 are both expressed at low levels in seeds. Analyses of fatty acid composition in different tissues indicate that the expression patterns of GhCPS1 and 2 correlate with cyclic fatty acid (CFA) distribution. Deletion of the N-terminal oxidase domain lowered GhCPS's ability to produce cyclopropane fatty acid by approximately 70%. GhCPS1 and 2, but not 3 resulted in the production of cyclopropane fatty acids upon heterologous expression in yeast, tobacco BY2 cell and Arabidopsis seed. CONCLUSIONS:In cotton GhCPS1 and 2 gene expression correlates with the total CFA content in roots, stems and seeds. That GhCPS1 and 2 are expressed at a similar level in seed suggests both of them can be considered potential targets for gene silencing to reduce undesirable seed CPE accumulation. Because GhCPS1 is more active in yeast than the published Sterculia CPS and shows similar activity when expressed in model plant systems, it represents a strong candidate gene for CFA accumulation via heterologous expression in production plants.

Project description:Saturated mid-chain branched fatty acids (SMCBFAs) are widely used in the petrochemical industry for their high oxidative stability and low melting temperature. Dihydrosterculic acid (DHSA) is a cyclopropane fatty acid (CPA) that can be converted to SMCBFA via hydrogenation, and therefore oils rich in DHSA are a potential feedstock for SMCBFA. Recent attempts to produce DHSA in seed oil by recombinant expression of cyclopropane fatty acid synthases (CPFASes) resulted in decreased oil content and poor germination or low DHSA accumulation. Here we explored the potential for plant vegetative tissue to produce DHSA by transiently expressing CPFAS enzymes in leaf. When CPFASes from plant and bacterial origin were transiently expressed in Nicotiana benthamiana leaf, it accumulated up to 1 and 3.7% DHSA in total fatty acid methyl ester (FAME), respectively, which increased up to 4.8 and 11.8%, respectively, when the N. benthamiana endogenous oleoyl desaturase was silenced using RNA interference (RNAi). Bacterial CPFAS expression produced a novel fatty acid with a cyclopropane ring and two carbon-carbon double bonds, which was not seen with plant CPFAS expression. We also observed a small but significant additive effect on DHSA accumulation when both plant and bacterial CPFASes were co-expressed, possibly due to activity upon different oleoyl substrates within the plant cell. Lipidomics analyses found that CPFAS expression increased triacylglycerol (TAG) accumulation relative to controls and that DHSA was distributed across a range of lipid species, including diacylglycerol and galactolipids. DHSA and the novel CPA were present in phosphatidylethanolamine when bacterial CPFAS was expressed in leaf. Finally, when plant diacylglycerol acyltransferase was coexpressed with the CPFASes DHSA accumulated up to 15% in TAG. This study shows that leaves can readily produce and accumulate DHSA in leaf oil. Our findings are discussed in line with current knowledge in leaf oil production for a possible route to DHSA production in vegetative tissue.

Project description:Enterococcus faecalis, a multiple antibiotic-resistant Gram-positive bacterium, has emerged as a serious nosocomial pathogen. Here, we used a genetic approach to characterize the strategies used by E. faecalis to fulfill its requirements for endogenous fatty acid (FA) synthesis in vitro and in vivo. The type II fatty acid synthesis (FASII) pathway is encoded by two operons and two monocistronic genes. Expression of all of these genes is repressed by exogenous FAs, which are incorporated into the E. faecalis membrane and modify its composition. Deletion of nine genes of the 12-gene operon abolished growth in an FA-free medium. Addition of serum, which is lipid rich, restored growth. Interestingly, the E. faecalis membrane contains cyclic fatty acids that modify membrane properties but that are unavailable in host serum. The cfa gene that encodes the cyclopropanation process is located in a locus independent of the FASII genes. Its deletion did not alter growth under the conditions tested, but yielded bacteria devoid of cyclic FAs. No differences were observed between mice infected with wild-type (WT) or with FASII or cyclopropanation mutant strains, in terms of bacterial loads in blood, liver, spleen, or kidneys. We conclude that in E. faecalis, neither FASII nor cyclopropanation enzymes are suitable antibiotic targets. IMPORTANCE Membrane lipid homeostasis is crucial for bacterial physiology, adaptation, and virulence. Fatty acids are constituents of the phospholipids that are essential membrane components. Most bacteria incorporate exogenous fatty acids into their membranes. Enterococcus faecalis has emerged as a serious nosocomial pathogen that is responsible for urinary tract infections, bacteremia, and endocarditis and is intrinsically resistant to numerous antibiotics. E. faecalis synthesizes saturated and unsaturated fatty acids, as well as cyclic fatty acids that are not found in the human host. Here, we characterized mutant strains deficient in fatty acid synthesis and modification using genetic, biochemical, and in vivo approaches. We conclude that neither the fatty acid synthesis pathway nor the cyclopropanation enzyme are suitable targets for E. faecalis antibiotic development.

Dataset Information

Carbocyclic fatty acids in plants: biochemical and molecular genetic characterization of cyclopropane fatty acid synthesis of Sterculiafoetida.

Publications

Carbocyclic fatty acids in plants: biochemical and molecular genetic characterization of cyclopropane fatty acid synthesis of Sterculiafoetida.

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