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1.
PLoS One ; 16(8): e0256625, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34432852

RESUMO

Although docosahexaenoic acid (DHA), an important dietary omega-3 polyunsaturated fatty acid (PUFA), is at present primarily sourced from marine fish, bioengineered crops producing DHA may offer a more sustainable and cost-effective source. DHA has been produced in transgenic oilseed crops, however, DHA in seed oil primarily occupies the sn-1/3 positions of triacylglycerol (TAG) with relatively low amounts of DHA in the sn-2 position. To increase the amount of DHA in the sn-2 position of TAG and in seed oil, putative lysophosphatidic acid acyltransferases (LPAATs) were identified and characterized from the DHA-producing alga Schizochytrium sp. and from soybean (Glycine max). The affinity-purified proteins were confirmed to have LPAAT activity. Expression of the Schizochytrium or soybean LPAATs in DHA-producing Arabidopsis expressing the Schizochytrium PUFA synthase system significantly increased the total amount of DHA in seed oil. A novel sensitive band-selective heteronuclear single quantum coherence (HSQC) NMR method was developed to quantify DHA at the sn-2 position of glycerolipids. More than two-fold increases in sn-2 DHA were observed for Arabidopsis lines expressing Schizochytrium or soybean LPAATs, with one Schizochytrium LPAAT driving DHA accumulation in the sn-2 position to 61% of the total DHA. Furthermore, expression of a soybean LPAAT led to a redistribution of DHA-containing TAG species, with two new TAG species identified. Our results demonstrate that transgenic expression of Schizochytrium or soybean LPAATs can increase the proportion of DHA at the sn-2 position of TAG and the total amount of DHA in the seed oil of a DHA-accumulating oilseed plant. Additionally, the band-selective HSQC NMR method that we developed provides a sensitive and robust method for determining the regiochemistry of DHA in glycerolipids. These findings will benefit the advancement of sustainable sources of DHA via transgenic crops such as canola and soybean.


Assuntos
Aciltransferases/metabolismo , Proteínas de Algas/metabolismo , Arabidopsis/genética , Ácidos Docosa-Hexaenoicos/metabolismo , Óleos de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Sementes/metabolismo , Triglicerídeos/metabolismo , Aciltransferases/química , Aciltransferases/genética , Aciltransferases/isolamento & purificação , Sequência de Aminoácidos , Genes de Plantas , Homozigoto , Espectroscopia de Ressonância Magnética , Filogenia , Plantas Geneticamente Modificadas
2.
Nat Biotechnol ; 34(8): 881-7, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27398790

RESUMO

Dietary omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), docosahexaenoic acid (DHA, C22:6) and eicosapentaenoic acid (EPA, C20:5) are usually derived from marine fish. Although production of both EPA and DHA has been engineered into land plants, including Arabidopsis, Camelina sativa and Brassica juncea, neither has been produced in commercially relevant amounts in a widely grown crop. We report expression of a microalgal polyketide synthase-like PUFA synthase system, comprising three multidomain polypeptides and an accessory enzyme, in canola (Brassica napus) seeds. This transgenic enzyme system is expressed in the cytoplasm, and synthesizes DHA and EPA de novo from malonyl-CoA without substantially altering plastidial fatty acid production. Furthermore, there is no significant impact of DHA and EPA production on seed yield in either the greenhouse or the field. Canola oil processed from field-grown grain contains 3.7% DHA and 0.7% EPA, and can provide more than 600 mg of omega-3 LC-PUFAs in a 14 g serving.


Assuntos
Brassica napus/metabolismo , Ácidos Docosa-Hexaenoicos/química , Melhoramento Genético/métodos , Microalgas/fisiologia , Óleos de Plantas/metabolismo , Policetídeo Sintases/metabolismo , Brassica napus/genética , Ácidos Docosa-Hexaenoicos/isolamento & purificação , Ácidos Docosa-Hexaenoicos/metabolismo , Óleos de Plantas/análise , Óleos de Plantas/química , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Policetídeo Sintases/genética , Engenharia de Proteínas/métodos , Óleo de Brassica napus
3.
Lipids ; 44(7): 621-30, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19495823

RESUMO

Schizochytrium produces long chain polyunsaturated fatty acids (PUFAs) via a PUFA synthase. Targeted mutagenesis of one gene of this synthase was conducted to confirm PUFA synthase function and determine its metabolic necessity. The resulting mutants were auxotrophic and required supplementation with PUFAs. In vivo labeling experiments with radioactive fatty acids demonstrated the presence of several elongase and desaturase activities associated with the standard pathway of PUFA synthesis. However, this system was missing a critical Delta12 desaturase activity and was therefore not capable of synthesizing PUFAs from the 16- or 18-carbon saturated fatty acid products of the fatty acid synthase. Because Schizochytrium uses a PUFA synthase system for the production of PUFAs, the existence of a partial desaturase-elongase system (if not a simple vestige) is suggested to be either a scavenging mechanism for intermediate fatty acids prematurely released by the PUFA synthase or for PUFAs found in the organism's native environment.


Assuntos
Diatomáceas/metabolismo , Ácidos Graxos Insaturados/biossíntese , Metabolismo dos Lipídeos/genética , Redes e Vias Metabólicas/genética , Células Cultivadas , Clonagem Molecular , Diatomáceas/genética , Ácido Graxo Sintase Tipo II/genética , Organismos Geneticamente Modificados , Oxirredução
4.
Plant Physiol Biochem ; 47(6): 472-8, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19272783

RESUMO

In marine bacteria and some thraustochytrids (marine stramenopiles) long-chain polyunsaturated fatty acids (LC-PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are produced de novo by PUFA synthases. These large, multi-domain enzymes carry out the multitude of individual reactions required for conversion of malonyl-CoA to the final LC-PUFA products. Here we report on the release of fatty acids from the PUFA synthase found in Schizochytrium, a thraustochytrid that has been developed as a commercial source for DHA-enriched biomass and oil. Data from in vitro activity assays indicate that the PUFAs are released from the enzyme as free fatty acids (FFAs). Addition of ATP and Mg(2+) to in vitro assays facilitates appearance of radiolabel from (14)C-malonyl-CoA in a triacylglycerol fraction, suggesting the involvement of acyl-CoA synthetases (ACS). Furthermore, addition of triascin C, an inhibitor of ACSs, to the assays blocks this conversion. When the Schizochytrium PUFA synthase is expressed in Escherichia coli, the products of the enzyme accumulate as FFAs, suggesting that the thioesterase activity required for fatty acid release is an integral part of the PUFA synthase.


Assuntos
Acetato-CoA Ligase/metabolismo , Ácidos Docosa-Hexaenoicos/metabolismo , Ácido Graxo Sintase Tipo II/metabolismo , Ácidos Graxos não Esterificados/biossíntese , Ácidos Graxos Insaturados/metabolismo , Malonil Coenzima A/metabolismo , Oomicetos/metabolismo , Tioléster Hidrolases/metabolismo , Trifosfato de Adenosina/metabolismo , Inibidores Enzimáticos/metabolismo , Magnésio/metabolismo , Oomicetos/enzimologia , Oomicetos/genética , Óleos de Plantas/metabolismo , Triglicerídeos/metabolismo
5.
J Am Chem Soc ; 130(20): 6336-7, 2008 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-18444614

RESUMO

Acyl carrier protein (ACP) plays an essential role in fatty acid and polyketide biosynthesis, and most of the fatty acid synthases (FASs) and polyketide synthases (PKSs) known to date are characterized with a single ACP for each cycle of chain elongation. Polyunsaturated fatty acid (PUFA) biosynthesis is catalyzed by the PUFA synthase, and all PUFA synthases known to date contain tandem ACPs (ranging from 5 to 9). Using the Pfa PUFA synthase from Shewanella japonica as a model system, we report here that these tandem ACPs are functionally equivalent regardless of their physical location within the PUFA synthase subunit, but the total number of ACPs controls the overall PUFA titer. These findings set the stage to interrogate other domains and subunits of PUFA synthase for their roles in controlling the final PUFA products and could potentially be exploited to improve PUFA production.


Assuntos
Proteína de Transporte de Acila/metabolismo , Ácidos Graxos Insaturados/biossíntese , Ácidos Docosa-Hexaenoicos/metabolismo , Ácido Eicosapentaenoico/biossíntese , Escherichia coli/enzimologia , Escherichia coli/genética , Ácido Graxo Sintase Tipo II/biossíntese , Ácido Graxo Sintase Tipo II/genética , Ácido Graxo Sintase Tipo II/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Shewanella/enzimologia , Shewanella/genética , Shewanella/metabolismo
6.
Biochim Biophys Acta ; 1554(3): 192-201, 2002 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-12160992

RESUMO

Properties of the Photosystem II (PSII) complex were examined in the wild-type (control) strain of the cyanobacterium Synechocystis PCC 6803 and its site-directed mutant D1-His252Leu in which the histidine residue 252 of the D1 polypeptide was replaced by leucine. This mutation caused a severe blockage of electron transfer between the PSII electron acceptors Q(A) and Q(B) and largely inhibited PSII oxygen evolving activity. Strong illumination induced formation of a D1-cytochrome b-559 adduct in isolated, detergent-solubilized thylakoid membranes from the control but not the mutant strain. The light-induced generation of the adduct was suppressed after prior modification of thylakoid proteins either with the histidine modifier platinum-terpyridine-chloride or with primary amino group modifiers. Anaerobic conditions and the presence of radical scavengers also inhibited the appearance of the adduct. The data suggest that the D1-cytochrome adduct is the product of a reaction between the oxidized residue His(252) of the D1 polypeptide and the N-terminal amino group of the cytochrome alpha subunit. As the rate of the D1 degradation in the control and mutant strains is similar, formation of the adduct does not seem to represent a required intermediary step in the D1 degradation pathway.


Assuntos
Cianobactérias/metabolismo , Grupo dos Citocromos b/química , Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteína do Fotossistema II , Sequência de Aminoácidos , Histidina , Luz , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Subunidades Proteicas
7.
Arch Biochem Biophys ; 401(1): 11-20, 2002 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-12054482

RESUMO

The recently described enzyme, polyenoic fatty acid isomerase (PFI), from the marine alga Ptilota filicina J. Argardh has been analyzed with respect to its protein structure and an associated cofactor. The enzyme was purified to homogeneity (as judged by SDS-PAGE and silver staining). By sedimentation equilibrium ultracentrifugation the mass of the native enzyme was estimated to be 125 kDa. The N-terminal peptide sequence derived from this protein was used to isolate two very similar cDNA clones encoding novel 500-amino acid proteins, both with calculated molecular masses of 55.9 kDa and pIs of 4.87. The data predict translation of a preprotein containing a signal peptide of 21 amino acids that is removed during maturation. Deglycosylation assays demonstrate that native PFI from P. filicina is a glycoprotein. The purified protein is chromophoric with a flavin-like UV spectrum and sequence analysis reveals the presence of a flavin-binding motif near the mature N-terminus. Heterologous expression of active PFI in Arabidopsis, using one of the cDNA clones, was successful as evidenced by conversion of arachidonic acid to a conjugated triene in an in vitro assay of the transgenic plant tissues.


Assuntos
Isomerases de Ligação Dupla Carbono-Carbono/química , Rodófitas/enzimologia , Sequência de Aminoácidos , Arabidopsis/genética , Sequência de Bases , Isomerases de Ligação Dupla Carbono-Carbono/genética , Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Clonagem Molecular , Sondas de DNA/genética , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Glicosilação , Dados de Sequência Molecular , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rodófitas/genética
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