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1.
J Agric Food Chem ; 67(35): 9851-9857, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31418561

RESUMO

Arachidonic acid (ARA, C20:4) is a typical ω-6 polyunsaturated fatty acid with special functions. Using Yarrowia lipolytica as an unconventional chassis, we previously showed the performance of the Δ-6 pathway in ARA production. However, a significant increase in the Δ-9 pathway has rarely been reported. Herein, the Δ-9 pathway from Isochrysis galbana was constructed via pathway engineering, allowing us to synthesize ARA at 91.5 mg L-1. To further improve the ARA titer, novel enzyme fusions of Δ-9 elongase and Δ-8 desaturase were redesigned in special combinations containing different linkers. Finally, with the integrated pathway engineering and synthetic enzyme fusion, a 29% increase in the ARA titer, up to 118.1 mg/L, was achieved using the reconstructed strain RH-4 that harbors the rigid linker (GGGGS). The results show that the combined pathway and protein engineering can significantly facilitate applications of Y. lipolytica.


Assuntos
Ácido Araquidônico/biossíntese , Engenharia Metabólica , Yarrowia/genética , Yarrowia/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Glucose/metabolismo , Haptófitas/enzimologia
2.
Environ Microbiol ; 20(11): 4157-4169, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30246477

RESUMO

Dimethylsulfide (DMS), a dominant organic sulfur species in the surface ocean, may act as a signalling molecule and contribute to mutualistic interactions between bacteria and marine algae. These proposed functions depend on the DMS concentration in the vicinity of microorganisms. Here, we modelled the DMS enrichment at the surface of DMS-releasing marine algal cells as a function of DMS production rate, algal cell radius and turbulence. Our results show that the DMS concentration at the surface of unstressed phytoplankton with low DMS production rates can be enriched by <1 nM, whereas for mechanically stressed algae with high activities of the enzyme DMSP-lyase (a coccolithophore and a dinoflagellate) DMS cell surface enrichments can reach ~10 nM, and could potentially reach µM levels in large cells. These DMS enrichments are much higher than the median DMS concentration in the surface ocean (1.9 nM), and thus may attract and support the growth of bacteria living in the phycosphere. The bacteria in turn may provide photoactive iron chelators (siderophores) that enhance algal iron uptake and provide algal growth factors such as auxins and vitamins. The present study highlights new insights on the extent and impact of microscale DMS enrichments at algal surfaces, thereby contributing to our understanding of the potential chemoattractant and mutualistic roles of DMS in marine microorganisms.


Assuntos
Haptófitas/metabolismo , Fitoplâncton/metabolismo , Sulfetos/metabolismo , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Bactérias/metabolismo , Liases de Carbono-Enxofre/genética , Liases de Carbono-Enxofre/metabolismo , Diatomáceas/enzimologia , Diatomáceas/genética , Diatomáceas/metabolismo , Dinoflagelados/enzimologia , Dinoflagelados/genética , Dinoflagelados/metabolismo , Ecossistema , Haptófitas/enzimologia , Haptófitas/genética , Ferro/metabolismo , Fitoplâncton/enzimologia , Fitoplâncton/genética , Água do Mar/microbiologia , Água do Mar/parasitologia , Sideróforos/metabolismo , Sulfetos/análise
3.
Sci Rep ; 8(1): 11230, 2018 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-30046151

RESUMO

Alkenones are unusual long-chain neutral lipids that were first identified in oceanic sediments. Currently they are regarded as reliable palaeothermometers, since their unsaturation status changes depending on temperature. These molecules are synthesised by specific haptophyte algae and are stored in the lipid body as the main energy storage molecules. However, the molecular mechanisms that regulate the alkenone biosynthetic pathway, especially the low temperature-dependent desaturation reaction, have not been elucidated. Here, using an alkenone-producing haptophyte alga, Tisochrysis lutea, we show that the alkenone desaturation reaction is catalysed by a newly identified desaturase. We first isolated two candidate desaturase genes and found that one of these genes was drastically upregulated in response to cold stress. Gas chromatographic analysis revealed that the overexpression of this gene, named as Akd1 finally, increased the conversion of di-unsaturated C37-alkenone to tri-unsaturated molecule by alkenone desaturation, even at a high temperature when endogenous desaturation is efficiently suppressed. We anticipate that the Akd1 gene will be of great help for elucidating more detailed mechanisms of temperature response of alkenone desaturation, and identification of active species contributing alkenone production in metagenomic and/or metatranscriptomic studies in the field of oceanic biogeochemistry.


Assuntos
Alcenos/metabolismo , Vias Biossintéticas/genética , Ácidos Graxos Dessaturases/genética , Haptófitas/genética , Catálise , Resposta ao Choque Frio/genética , Regulação Enzimológica da Expressão Gênica/genética , Haptófitas/enzimologia , Haptófitas/fisiologia , Temperatura
4.
Nat Microbiol ; 3(4): 430-439, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29483657

RESUMO

Dimethylsulfoniopropionate (DMSP) is a globally important organosulfur molecule and the major precursor for dimethyl sulfide. These compounds are important info-chemicals, key nutrients for marine microorganisms, and are involved in global sulfur cycling, atmospheric chemistry and cloud formation1-3. DMSP production was thought to be confined to eukaryotes, but heterotrophic bacteria can also produce DMSP through the pathway used by most phytoplankton 4 , and the DsyB enzyme catalysing the key step of this pathway in bacteria was recently identified 5 . However, eukaryotic phytoplankton probably produce most of Earth's DMSP, yet no DMSP biosynthesis genes have been identified in any such organisms. Here we identify functional dsyB homologues, termed DSYB, in many phytoplankton and corals. DSYB is a methylthiohydroxybutryate methyltransferase enzyme localized in the chloroplasts and mitochondria of the haptophyte Prymnesium parvum, and stable isotope tracking experiments support these organelles as sites of DMSP synthesis. DSYB transcription levels increased with DMSP concentrations in different phytoplankton and were indicative of intracellular DMSP. Identification of the eukaryotic DSYB sequences, along with bacterial dsyB, provides the first molecular tools to predict the relative contributions of eukaryotes and prokaryotes to global DMSP production. Furthermore, evolutionary analysis suggests that eukaryotic DSYB originated in bacteria and was passed to eukaryotes early in their evolution.


Assuntos
Cloroplastos/enzimologia , Haptófitas/enzimologia , Metiltransferases/genética , Mitocôndrias/enzimologia , Compostos de Sulfônio/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Diatomáceas/enzimologia , Diatomáceas/genética , Dinoflagelados/enzimologia , Dinoflagelados/genética , Haptófitas/genética , Metiltransferases/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Fitoplâncton/metabolismo
5.
Arch Microbiol ; 200(3): 413-422, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29184974

RESUMO

Lytic viral infection and programmed cell death (PCD) are thought to represent two distinct death mechanisms in phytoplankton, unicellular photoautotrophs that drift with ocean currents. PCD (apoptosis) is mainly brought about by the activation of caspases, a protease family with unique substrate selectivity. Here, we demonstrated that virus infection induced apoptosis of marine coccolithophorid Emiliania huxleyi BOF92 involving activation of metacaspase. E. huxleyi cells exhibited cell death process akin to that of apoptosis when exposed to virus infection. We observed typical hallmarks of apoptosis including cell shrinkage, associated nuclear morphological changes and DNA fragmentation. Immunoblotting revealed that antibody against human active-caspase-3 shared epitopes with a protein of ≈ 23 kDa; whose pattern of expression correlated with the onset of cell death. Moreover, analysis on two-dimensional gel electrophoresis revealed that two spots of active caspase-3 co-migrated with the different isoelectric points. Phosphatase treatment of cytosolic extracts containing active caspases-3 showed a mobility shift, suggesting that phosphorylated form of this enzyme might be present in the extracts. Computational prediction of phosphorylation sites based on the amino acid sequence of E. huxleyi metacaspase showed multiple phosphorylated sites for serine, threonine and tyrosine residues. This is the first report showing that phosphorylation modification of metacaspase in E. huxleyi might be required for certain biochemical and morphological changes during virus induced apoptosis.


Assuntos
Apoptose , Caspases/metabolismo , Vírus Gigantes/fisiologia , Haptófitas/enzimologia , Fitoplâncton/enzimologia , Sequência de Aminoácidos , Caspases/genética , Sequência Conservada , Fragmentação do DNA , Expressão Gênica , Haptófitas/genética , Haptófitas/ultraestrutura , Haptófitas/virologia , Fosforilação , Processamento de Proteína Pós-Traducional
6.
Biotechnol Lett ; 40(3): 577-584, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29288354

RESUMO

OBJECTIVES: To express a Δ6-desaturase gene and produce gamma-linolenic acid (GLA) and stearidonic acid (SDA) in prokaryotic expression system (Escherichia coli), and analyze its substrate specificity in the omega-3 fatty acid biosynthetic pathway. RESULTS: Full-length ORF (1448 bp) of Δ6Des-Iso was isolated from Isochrysis sp. and characterized using multiple sequence alignment, phylogenetic analysis, transmembrane domain, and protein tertiary structure. Δ6Des-Iso is a front-end desaturase consisting of three conserved histidine domains and a cytochrome b5 domain. Δ6Des-Iso was cloned and expressed in E. coli with the production of GLA and SDA. Recombinant E. coli utilized 27 and 8% of exogenously supplied alpha-linolenic acid (ALA) and linoleic acid (LA) to produce 6.3% of SDA and 2.3% of GLA, respectively, suggesting that isolated Δ6Des-Iso is specific to the omega-3 pathway. CONCLUSION: For the first time production of GLA and SDA in a prokaryotic system was achieved.


Assuntos
Ácidos Graxos Insaturados/metabolismo , Haptófitas/enzimologia , Linoleoil-CoA Desaturase/metabolismo , Microalgas/enzimologia , Proteínas Recombinantes/metabolismo , Escherichia coli/genética , Ácidos Graxos Insaturados/química , Haptófitas/genética , Linoleoil-CoA Desaturase/química , Linoleoil-CoA Desaturase/genética , Microalgas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade por Substrato
7.
J Exp Bot ; 68(14): 3959-3969, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28582571

RESUMO

The haptophyte algae are a cosmopolitan group of primary producers that contribute significantly to the marine carbon cycle and play a major role in paleo-climate studies. Despite their global importance, little is known about carbon assimilation in haptophytes, in particular the kinetics of their Form 1D CO2-fixing enzyme, Rubisco. Here we examine Rubisco properties of three haptophytes with a range of pyrenoid morphologies (Pleurochrysis carterae, Tisochrysis lutea, and Pavlova lutheri) and the diatom Phaeodactylum tricornutum that exhibit contrasting sensitivities to the trade-offs between substrate affinity (Km) and turnover rate (kcat) for both CO2 and O2. The pyrenoid-containing T. lutea and P. carterae showed lower Rubisco content and carboxylation properties (KC and kCcat) comparable with those of Form 1D-containing non-green algae. In contrast, the pyrenoid-lacking P. lutheri produced Rubisco in 3-fold higher amounts, and displayed a Form 1B Rubisco kCcat-KC relationship and increased CO2/O2 specificity that, when modeled in the context of a C3 leaf, supported equivalent rates of photosynthesis to higher plant Rubisco. Correlation between the differing Rubisco properties and the occurrence and localization of pyrenoids with differing intracellular CO2:O2 microenvironments has probably influenced the divergent evolution of Form 1B and 1D Rubisco kinetics.


Assuntos
Cloroplastos/metabolismo , Haptófitas/metabolismo , Microalgas/metabolismo , Fotossíntese , Ribulose-Bifosfato Carboxilase/metabolismo , Haptófitas/enzimologia , Cinética , Microalgas/enzimologia , Especificidade da Espécie
8.
ACS Chem Biol ; 12(1): 41-46, 2017 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-28103686

RESUMO

Atmospheric dimethylsulfide (DMS) is massively produced in the oceans by bacteria, algae, and corals. To enable identification of DMS sources, we developed a potent mechanism-based inhibitor of the algal Alma dimethylsulfoniopropionate lyase family that does not inhibit known bacterial lyases. Its application to coral holobiont indicates that DMS originates from Alma lyase(s). This biochemical profiling may complement meta-genomics and transcriptomics to provide better understanding of the marine sulfur cycle.


Assuntos
Antozoários/metabolismo , Haptófitas/metabolismo , Sulfetos/metabolismo , Animais , Antozoários/efeitos dos fármacos , Antozoários/enzimologia , Bactérias/efeitos dos fármacos , Bactérias/enzimologia , Bactérias/metabolismo , Liases de Carbono-Enxofre/antagonistas & inibidores , Liases de Carbono-Enxofre/metabolismo , Inibidores Enzimáticos/metabolismo , Haptófitas/efeitos dos fármacos , Haptófitas/enzimologia , Oceanos e Mares
9.
PLoS One ; 11(7): e0158103, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27433934

RESUMO

Eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17) and Docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19) are nutritionally beneficial to human health. Transgenic production of EPA and DHA in oilseed crops by transferring genes originating from lower eukaryotes, such as microalgae and fungi, has been attempted in recent years. However, the low yield of EPA and DHA produced in these transgenic crops is a major hurdle for the commercialization of these transgenics. Many factors can negatively affect transgene expression, leading to a low level of converted fatty acid products. Among these the codon bias between the transgene donor and the host crop is one of the major contributing factors. Therefore, we carried out codon optimization of a fatty acid delta-6 desaturase gene PinD6 from the fungus Phytophthora infestans, and a delta-9 elongase gene, IgASE1 from the microalga Isochrysis galbana for expression in Saccharomyces cerevisiae and Arabidopsis respectively. These are the two key genes encoding enzymes for driving the first catalytic steps in the Δ6 desaturation/Δ6 elongation and the Δ9 elongation/Δ8 desaturation pathways for EPA/DHA biosynthesis. Hence expression levels of these two genes are important in determining the final yield of EPA/DHA. Via PCR-based mutagenesis we optimized the least preferred codons within the first 16 codons at their N-termini, as well as the most biased CGC codons (coding for arginine) within the entire sequences of both genes. An expression study showed that transgenic Arabidopsis plants harbouring the codon-optimized IgASE1 contained 64% more elongated fatty acid products than plants expressing the native IgASE1 sequence, whilst Saccharomyces cerevisiae expressing the codon optimized PinD6 yielded 20 times more desaturated products than yeast expressing wild-type (WT) PinD6. Thus the codon optimization strategy we developed here offers a simple, effective and low-cost alternative to whole gene synthesis for high expression of foreign genes in yeast and Arabidopsis.


Assuntos
Acetiltransferases/genética , Arabidopsis/genética , Ácidos Docosa-Hexaenoicos/biossíntese , Ácido Eicosapentaenoico/biossíntese , Linoleoil-CoA Desaturase/genética , Transgenes , Acetiltransferases/metabolismo , Arabidopsis/enzimologia , Sequência de Bases , Códon , Ácidos Docosa-Hexaenoicos/genética , Ácido Eicosapentaenoico/genética , Regulação da Expressão Gênica , Engenharia Genética , Haptófitas/enzimologia , Haptófitas/genética , Linoleoil-CoA Desaturase/metabolismo , Phytophthora infestans/enzimologia , Phytophthora infestans/genética , Plantas Geneticamente Modificadas , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética
10.
PLoS One ; 11(1): e0146158, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26727469

RESUMO

Microalgae have attracted wide attention as one of the most versatile renewable feedstocks for production of biofuel. To develop genetically engineered high lipid yielding algal strains, a thorough understanding of the lipid biosynthetic pathway and the underpinning enzymes is essential. In this work, we have systematically mined the genomes of fifteen diverse algal species belonging to Chlorophyta, Heterokontophyta, Rhodophyta, and Haptophyta, to identify and annotate the putative enzymes of lipid metabolic pathway. Consequently, we have also developed a database, dEMBF (Database of Enzymes of Microalgal Biofuel Feedstock), which catalogues the complete list of identified enzymes along with their computed annotation details including length, hydrophobicity, amino acid composition, subcellular location, gene ontology, KEGG pathway, orthologous group, Pfam domain, intron-exon organization, transmembrane topology, and secondary/tertiary structural data. Furthermore, to facilitate functional and evolutionary study of these enzymes, a collection of built-in applications for BLAST search, motif identification, sequence and phylogenetic analysis have been seamlessly integrated into the database. dEMBF is the first database that brings together all enzymes responsible for lipid synthesis from available algal genomes, and provides an integrative platform for enzyme inquiry and analysis. This database will be extremely useful for algal biofuel research. It can be accessed at http://bbprof.immt.res.in/embf.


Assuntos
Proteínas de Algas , Biocombustíveis , Bases de Dados de Proteínas , Enzimas , Microalgas/enzimologia , Proteínas de Algas/química , Proteínas de Algas/genética , Motivos de Aminoácidos , Aminoácidos/análise , Clorófitas/enzimologia , Clorófitas/genética , Bases de Dados como Assunto , Ontologia Genética , Haptófitas/enzimologia , Haptófitas/genética , Interações Hidrofóbicas e Hidrofílicas , Metabolismo dos Lipídeos/genética , Microalgas/genética , Filogenia , Conformação Proteica , Energia Renovável , Rodófitas/enzimologia , Rodófitas/genética , Software , Estramenópilas/enzimologia , Estramenópilas/genética
12.
Science ; 348(6242): 1466-9, 2015 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-26113722

RESUMO

Algal blooms produce large amounts of dimethyl sulfide (DMS), a volatile with a diverse signaling role in marine food webs that is emitted to the atmosphere, where it can affect cloud formation. The algal enzymes responsible for forming DMS from dimethylsulfoniopropionate (DMSP) remain unidentified despite their critical role in the global sulfur cycle. We identified and characterized Alma1, a DMSP lyase from the bloom-forming algae Emiliania huxleyi. Alma1 is a tetrameric, redox-sensitive enzyme of the aspartate racemase superfamily. Recombinant Alma1 exhibits biochemical features identical to the DMSP lyase in E. huxleyi, and DMS released by various E. huxleyi isolates correlates with their Alma1 levels. Sequence homology searches suggest that Alma1 represents a gene family present in major, globally distributed phytoplankton taxa and in other marine organisms.


Assuntos
Proteínas de Algas/química , Liases de Carbono-Enxofre/química , Haptófitas/enzimologia , Sulfetos/metabolismo , Proteínas de Algas/classificação , Proteínas de Algas/genética , Sequência de Aminoácidos , Bactérias/enzimologia , Bactérias/genética , Liases de Carbono-Enxofre/classificação , Liases de Carbono-Enxofre/genética , Haptófitas/genética , Dados de Sequência Molecular , Filogenia , Fitoplâncton/enzimologia , RNA Mensageiro/biossíntese , Proteínas Recombinantes/química
13.
Mol Biol Rep ; 41(11): 7235-40, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25055977

RESUMO

Thiolases are functionally divided into two groups: 3-ketoacyl-CoA thiolase and acetoacetyl-CoA thiolase (ACAT). Acetoacetyl-CoA thiolase plays a key role in the mevalonate pathway. In this study, a novel gene, IgACAT, which encodes ACAT was cloned from Isochrysis galbana and characterized. The cDNA of IgACAT was 1551 bp in length, consisting of an open reading frame of 1173 bp, a 5' untranslated region of 69 bp and a 3' untranslated region of 309 bp. The deduced amino acid sequence of IgACAT was 390 amino acid residues in length with a predicted molecular weight of 53.59 kDa and an isoelectric point of pH 9.04. The triterpenes content and the expression of IgACAT under nitrogen and temperature stress were analyzed. When I. galbana was treated with excessive nitrogen and at 35 °C, respectively, both the triterpenes content and the abundance of IgACAT gene transcript increased. Our findings will facilitate the regulation of gene expression and genetic modification of the triterpenes synthesis pathway of I. galbana.


Assuntos
Acetil-CoA C-Acetiltransferase/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Haptófitas/enzimologia , Nitrogênio/farmacologia , Temperatura , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Análise por Conglomerados , Primers do DNA/genética , DNA Complementar/genética , Haptófitas/genética , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNA , Triterpenos/metabolismo
14.
J Exp Bot ; 65(6): 1637-49, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24609499

RESUMO

IgASE1, a C18 Δ(9)-specific polyunsaturated fatty acid elongase from the marine microalga Isochrysis galbana, is able to convert linoleic acid and α-linolenic acid to eicosadienoic acid and eicosatrienoic acid in Arabidopsis. Eicosadienoic acid and eicosatrienoic acid are precursors of arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid, which are synthesized via the Δ(8) desaturation biosynthetic pathways. This study shows that the IgASE1-expressing transgenic Arabidopsis exhibited altered morphology (decreased leaf area and biomass) and enhanced drought resistance compared to wild-type plants. The transgenic Arabidopsis were hypersensitive to abscisic acid (ABA) during seed germination, post-germination growth, and seedling development. They had elevated leaf ABA levels under well-watered and dehydrated conditions and their stomata were more sensitive to ABA. Exogenous application of eicosadienoic acid and eicosatrienoic acid can mimic ABA and drought responses in the wild type plants, similar to that found in the transgenic ones. The transcript levels of genes involved in the biosynthesis of ABA (NCED3, ABA1, AAO3) as well as other stress-related genes were upregulated in this transgenic line upon osmotic stress (300 mM mannitol). Taken together, these results indicate that these two eicosapolyenoic acids or their derived metabolites can mitigate the effects of drought in transgenic Arabidopsis, at least in part, through the action of ABA.


Assuntos
Ácido Abscísico/metabolismo , Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas , Haptófitas/enzimologia , Reguladores de Crescimento de Planta/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Biomassa , Secas , Ácidos Graxos Insaturados/metabolismo , Regulação Enzimológica da Expressão Gênica , Germinação , Haptófitas/genética , Manitol/metabolismo , Pressão Osmótica , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/fisiologia , Plantas Geneticamente Modificadas , Transgenes , Ácido alfa-Linoleico/metabolismo
15.
J Microbiol Biotechnol ; 23(10): 1413-21, 2013 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-23851273

RESUMO

The marine microalga Isochrysis sphaerica is rich in the very-long-chain polyunsaturated fatty acids, including eicosapentaenoic acid (EPA, C20:5ω-3) and docosahexaenoic acid (DHA, C22:6ω-3) that are important to human health. Here, we report a functional characterization of a Δ4-fatty acid desaturase gene (FAD4) from I. sphaerica. IsFAD4 contains a 1,284 bp open reading frame encoding a 427 amino acid polypeptide. The deduced amino sequence comprises three conserved histidine motifs and a cytochrome b5 domain at its N-terminus. Phylogenetic analysis indicated that IsFad4 formed a unique Isochrysis clade distinct from the counterparts of other eukaryotes. Heterologous expression of IsFAD4 in Pichia pastoris showed that IsFad4 was able to desaturate docosapentaenoic acid (DPA) to form DHA, and the rate of converting DPA to DHA was 79.8%. These results throw light on the potential industrial production of specific polyunsaturated fatty acids through IsFAD4 transgenic yeast or oil crops.


Assuntos
Ácidos Docosa-Hexaenoicos/metabolismo , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos Insaturados/metabolismo , Haptófitas/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Análise por Conglomerados , DNA de Algas/química , DNA de Algas/genética , Ácidos Graxos Dessaturases/genética , Expressão Gênica , Haptófitas/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Pichia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos
16.
Biotechnol Lett ; 35(8): 1271-82, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23546943

RESUMO

The marine microalga, Pavlova viridis, contains long-chain polyunsatured fatty acids including eicosapentaenoic acid (EPA, 20:5n-3) and docosapentaenoic acid (DPA, 22:5n-3). A full-length cDNA sequence, pvelo5, was isolated from P. viridis. From sequence alignment, the gene was homologous to fatty acyl elongases from other organisms. Heterologous expression of pvelo5 in Saccharomyces cerevisiae confirmed that it encoded a specific C20-elongase within the n-3 and n-6 pathways. Elongation activity was confined exclusively to EPA and arachidonic acid (20:4n-6). GC analysis indicated that pvelo5 could co-express with other genes for biosynthesis to reconstitute the Δ8 and Δ6 pathways. Real-time PCR results and fatty acid analysis demonstrated that long-chain polyunsatured fatty acids production by the Δ8 pathway might be more effective than that by the Δ6 pathway.


Assuntos
Acetiltransferases/genética , Acetiltransferases/metabolismo , Vias Biossintéticas/genética , Ácidos Graxos/biossíntese , Haptófitas/enzimologia , Saccharomyces cerevisiae/metabolismo , DNA de Algas/química , DNA de Algas/genética , Ácidos Graxos/química , Cromatografia Gasosa-Espectrometria de Massas , Expressão Gênica , Haptófitas/genética , Engenharia Metabólica/métodos , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA
17.
Proc Natl Acad Sci U S A ; 109(47): 19327-32, 2012 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-23134731

RESUMO

Marine viruses are major evolutionary and biogeochemical drivers in marine microbial foodwebs. However, an in-depth understanding of the cellular mechanisms and the signal transduction pathways mediating host-virus interactions during natural bloom dynamics has remained elusive. We used field-based mesocosms to examine the "arms race" between natural populations of the coccolithophore Emiliania huxleyi and its double-stranded DNA-containing coccolithoviruses (EhVs). Specifically, we examined the dynamics of EhV infection and its regulation of cell fate over the course of bloom development and demise using a diverse suite of molecular tools and in situ fluorescent staining to target different levels of subcellular resolution. We demonstrate the concomitant induction of reactive oxygen species, caspase-specific activity, metacaspase expression, and programmed cell death in response to the accumulation of virus-derived glycosphingolipids upon infection of natural E. huxleyi populations. These subcellular responses to viral infection simultaneously resulted in the enhanced production of transparent exopolymer particles, which can facilitate aggregation and stimulate carbon flux. Our results not only corroborate the critical role for glycosphingolipids and programmed cell death in regulating E. huxleyi-EhV interactions, but also elucidate promising molecular biomarkers and lipid-based proxies for phytoplankton host-virus interactions in natural systems.


Assuntos
Linhagem da Célula , Haptófitas/citologia , Haptófitas/virologia , Interações Hospedeiro-Patógeno/fisiologia , Phycodnaviridae/fisiologia , Biopolímeros/biossíntese , Caspases/metabolismo , Ativação Enzimática , Eutrofização , Haptófitas/enzimologia , Noruega , Frações Subcelulares/virologia , Fatores de Tempo
18.
Biotechnol Lett ; 34(12): 2265-74, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22941368

RESUMO

Isochrysis galbana, produces long chain polyunsaturated fatty acids including docosahexaenoic acid (DHA, 22:6n-3). A novel gene (IgFAD4-2), encoding a C22-∆4 polyunsaturated fatty acid specific desaturase, has been isolated and characterized from I. galbana. A full-length cDNA of 1,302 bp was cloned by LA-PCR technique. The IgFAD4-2 encoded a protein of 433 amino acids that shares 78 % identity with a previously reported ∆4-desaturase (IgFAD4-1) from I. galbana. The function of IgFAD4-2 was deduced by its heterologous expression in Saccharomyces cerevisiae, which then desaturated docosapentaenoic acid (DPA, 22:5n-3) to DHA. The conversion ratio of DPA to DHA was 34 %, which is higher than other ∆4-desaturases cloned from algae. However, IgFAD4-2 did not catalyze the desaturation or elongation reactions with other fatty acids. These results confirm that IgFAD4-2 has C22-∆4-PUFAs-specific desaturase activity.


Assuntos
Ácidos Docosa-Hexaenoicos/metabolismo , Ácidos Graxos Dessaturases/metabolismo , Expressão Gênica , Haptófitas/enzimologia , Sequência de Aminoácidos , Clonagem Molecular , Análise por Conglomerados , Ácidos Graxos Dessaturases/genética , Haptófitas/genética , Dados de Sequência Molecular , Filogenia , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos
19.
Metallomics ; 4(11): 1160-6, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23011578

RESUMO

Iron is an essential element for all living organisms due to its ubiquitous role in redox and other enzymes, especially in the context of respiration and photosynthesis. The iron uptake and storage systems of terrestrial/higher plants are now reasonably well understood with two basic strategies for iron uptake being distinguished: strategy I plants use a mechanism involving soil acidification and induction of Fe(III)-chelate reductase (ferrireductase) and Fe(II) transporter proteins while strategy II plants have evolved sophisticated systems based on high-affinity, iron specific, binding compounds called phytosiderophores. In contrast, there is little knowledge about the corresponding systems in marine plant-like lineages. Herein we report a study of the iron uptake and storage mechanisms in the coccolithophore Emiliania huxleyi. Short term radio-iron uptake studies indicate that iron is taken up by Emiliania in a time and concentration dependent manner consistent with an active transport process. Based on inhibitor studies it appears that iron is taken up directly as Fe(iii). However if a reductive step is involved the Fe(II) must not be accessible to the external environment. Upon long term exposure to (57)Fe we have been able, using a combination of Mössbauer and XAS spectroscopies, to identify a single metabolite which displays spectral features similar to the phosphorus-rich mineral core of bacterial and plant ferritins.


Assuntos
Haptófitas/química , Haptófitas/metabolismo , Ferro/química , Ferro/metabolismo , Transporte Biológico , Haptófitas/enzimologia , Isótopos de Ferro/química , Isótopos de Ferro/metabolismo , Proteínas de Ligação ao Ferro , Proteínas de Membrana , Oxirredução , Oxirredutases/química , Oxirredutases/metabolismo , Análise Espectral
20.
Plant Cell Physiol ; 53(6): 1043-52, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22492231

RESUMO

Pyruvate carboxylase (PYC) catalyzes the ß-carboxylation of pyruvate to yield oxaloacetate (OAA). We previously isolated a cDNA encoding a putative PYC (EhPYC1) from the haptophyte alga Emiliania huxleyi and then proposed that EhPYC1 contributes to active anaplerotic ß-carboxylation during photosynthesis although PYC activity was not detected in the cell extracts. Involvement of PYC in photosynthetic carbon metabolism is unique, since PYC generally functions in non-photosynthetic organisms. In the present study, we demonstrate that EhPYC1 is highly sensitive to endogenous proteases and therefore is easily degraded in cell extracts. By avoiding proteolytic degradation, PYC activity can be detected in the cell extracts of E. huxleyi. The activity of a recombinant His-tagged EhPYC1 expressed in Streptomyces lividans was inhibited by l-malate in a mixed non-competitive manner. Immunofluorescence labeling showed that EhPYC1 is located in the plastid. This result agrees with the prediction that a bipartite plastid-targeting signal is present that functions to deliver proteins into the four-membrane plastid of haptophyte algae. This is the first finding of a plastid-located PYC. These results indicate that E. huxleyi possesses a unique pathway to produce OAA catalyzed by PYC, and the pathway may provide carbon skeletons for amino acid biosynthesis in the plastid. A database search indicates that PYC genes are widespread in green algae, diatoms and brown algae, suggesting the crucial role of PYC in various aquatic phototrophs.


Assuntos
Proteínas de Algas/metabolismo , Haptófitas/enzimologia , Plastídeos/enzimologia , Piruvato Carboxilase/metabolismo , Proteínas de Algas/genética , Sequência de Aminoácidos , Ácido Aspártico/farmacologia , Avidina , Carbono/metabolismo , Proteínas de Cloroplastos/genética , Proteínas de Cloroplastos/metabolismo , Ativação Enzimática , Genes de Plantas , Haptófitas/genética , Membranas Intracelulares/metabolismo , Luz , Malatos/farmacologia , Mitocôndrias/genética , Mitocôndrias/metabolismo , Ácido Oxaloacético/metabolismo , Fotossíntese , Plastídeos/genética , Transporte Proteico , Proteólise , Piruvato Carboxilase/antagonistas & inibidores , Piruvato Carboxilase/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Streptomyces lividans/genética , Streptomyces lividans/metabolismo
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