RESUMO
Recent global marine lipidomic analysis reveals a strong relationship between ocean temperature and phytoplanktonic abundance of omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are essential for human nutrition and primarily sourced from phytoplankton in marine food webs. In phytoplanktonic organisms, EPA may play a major role in regulating the phase transition temperature of membranes, while the function of DHA remains unexplored. In the oleaginous diatom Phaeodactylum tricornutum, DHA is distributed mainly on extraplastidial phospholipids, which is very different from the EPA enriched in thylakoid lipids. Here, CRISPR/Cas9-mediated knockout of delta-5 elongase (ptELO5a), which encodes a delta-5 elongase (ELO5) catalyzing the elongation of EPA to synthesize DHA, led to a substantial interruption of DHA synthesis in P. tricornutum. The ptELO5a mutants showed some alterations in transcriptome and glycerolipidomes, including membrane lipids and triacylglycerols under normal temperature (22°C), and were more sensitive to elevated temperature (28°C) than wild type. We conclude that PtELO5a-mediated synthesis of small amounts of DHA has indispensable functions in regulating membrane lipids, indirectly contributing to storage lipid accumulation, and maintaining thermomorphogenesis in P. tricornutum. This study also highlights the significance of DHA synthesis and lipid composition for environmental adaptation of P. tricornutum.
RESUMO
BACKGROUND: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), belonging to ω-3 long-chain polyunsaturated fatty acids (ω3-LC-PUFAs), are essential components of human diet. They are mainly supplemented by marine fish consumption, although their native producers are oleaginous microalgae. Currently, increasing demand for fish oils is insufficient to meet the entire global needs, which puts pressure on searching for the alternative solutions. One possibility may be metabolic engineering of plants with an introduced enzymatic pathway producing ω3-LC-PUFAs. RESULT: In this study we focused on the acyl-CoA:diacylglycerol acyltransferase2b (PtDGAT2b) from the diatom Phaeodactylum tricornutum, an enzyme responsible for triacylglycerol (TAG) biosynthesis via acyl-CoA-dependent pathway. Gene encoding PtDGAT2b, incorporated into TAG-deficient yeast strain H1246, was used to confirm its activity and conduct biochemical characterization. PtDGAT2b exhibited a broad acyl-CoA preference with both di-16:0-DAG and di-18:1-DAG, whereas di-18:1-DAG was favored. The highest preference for acyl donors was observed for 16:1-, 10:0- and 12:0-CoA. PtDGAT2b also very efficiently utilized CoA-conjugated ω-3 LC-PUFAs (stearidonic acid, eicosatetraenoic acid and EPA). Additionally, verification of the potential role of PtDGAT2b in planta, through its transient expression in tobacco leaves, indicated increased TAG production with its relative amount increasing to 8%. Its co-expression with the gene combinations aimed at EPA biosynthesis led to, beside elevated TAG accumulation, efficient accumulation of EPA which constituted even 25.1% of synthesized non-native fatty acids (9.2% of all fatty acids in TAG pool). CONCLUSIONS: This set of experiments provides a comprehensive biochemical characterization of DGAT enzyme from marine microalgae. Additionally, this study elucidates that PtDGAT2b can be used successfully in metabolic engineering of plants designed to obtain a boosted TAG level, enriched not only in ω-3 LC-PUFAs but also in medium-chain and ω-7 fatty acids.
Assuntos
Diacilglicerol O-Aciltransferase , Diatomáceas , Nicotiana , Diatomáceas/genética , Diatomáceas/enzimologia , Diatomáceas/metabolismo , Diacilglicerol O-Aciltransferase/genética , Diacilglicerol O-Aciltransferase/metabolismo , Nicotiana/genética , Nicotiana/enzimologia , Nicotiana/metabolismo , Acil Coenzima A/metabolismo , Plantas Geneticamente Modificadas , Triglicerídeos/biossíntese , Triglicerídeos/metabolismo , Ácido Eicosapentaenoico/biossíntese , Ácido Eicosapentaenoico/metabolismo , Ácidos Graxos Ômega-3/biossíntese , Ácidos Graxos Ômega-3/metabolismo , Engenharia MetabólicaRESUMO
Continuous research on obtaining an even more efficient production of very long-chain polyunsaturated fatty acids (VLC-PUFAs) in plants remains one of the main challenges of scientists working on plant lipids. Since crops are not able to produce these fatty acids due to the lack of necessary enzymes, genes encoding them must be introduced exogenously from native organisms producing VLC-PUFAs. In this study we reported, in tobacco leaves, the characterization of three distinct ∆6-desaturases from diatom Phaeodactylum tricornutum, fungi Rhizopus stolonifer and microalge Osterococcus tauri and two different ∆5-desaturases from P. tricornutum and single-celled saprotrophic eukaryotes Thraustochytrium sp. The in planta agroinfiltration of essential ∆6-desaturases, ∆6-elongases and ∆5-desaturases allowed for successful introduction of eicosapentaenoic acid (20:5∆5,8,11,14,17) biosynthesis pathway. However, despite the desired, targeted production of ω3-fatty acids we detected the presence of ω6-fatty acids, indicating and confirming previous results that all tested desaturases are not specifically restricted to neither ω3- nor ω6-pathway. Nevertheless, the additional co-expression of acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) from Phaeodactylum tricornutum boosted the proportion of ω3-fatty acids in newly synthesized fatty acid pools. For the most promising genes combinations the EPA content reached at maximum 1.4% of total lipid content and 4.5% of all fatty acids accumulated in the TAG pool. Our results for the first time describe the role of LPCAT enzyme and its effectiveness in alleviating a bottleneck called 'substrate dichotomy' for improving the transgenic production of VLC-PUFAs in plants.
Assuntos
Diatomáceas , Ácidos Graxos Dessaturases , Ácidos Graxos Ômega-3 , Engenharia Metabólica , Nicotiana , Plantas Geneticamente Modificadas , Diatomáceas/genética , Diatomáceas/metabolismo , Diatomáceas/enzimologia , Engenharia Metabólica/métodos , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Ômega-3/biossíntese , Plantas Geneticamente Modificadas/genética , Nicotiana/genética , Nicotiana/metabolismoRESUMO
Diatoms such as Phaeodactylum tricornutum arose through a process termed secondary endosymbiosis, in which red alga-derived plastids are surrounded by a complicated membrane system. Subcellular marker proteins provide defined localizations on the compartmental and even sub-compartmental levels in the complex plastids of diatoms. Here we introduce how to use subcellular marker proteins and in vivo co-localization in the diatom P. tricornutum by presenting a step-by-step method allowing the determination of subcellular localization of proteins in different membranes of the secondary plastid. This chapter describes the materials required and the procedures of transformation and microscopic observation.
Assuntos
Diatomáceas , Diatomáceas/metabolismo , Proteínas/metabolismo , Membranas , Simbiose , Plastídeos/metabolismoRESUMO
Lysophosphatidic acid acyltransferases (LPAATs) catalyze the formation of phosphatidic acid (PA), a central metabolite in both prokaryotic and eukaryotic organisms for glycerolipid biosynthesis. Phaeodactylum tricornutum contains at least two plastid-localized LPAATs (ptATS2a and ptATS2b), but their roles in lipid synthesis remain unknown. Both ptATS2a and ptATS2b could complement the high temperature sensitivity of the bacterial plsC mutant deficient in LPAAT. In vitro enzyme assays showed that they prefer lysophosphatidic acid over other lysophospholipids. ptATS2a is localized in the plastid inner envelope membrane and CRISPR/Cas9-generated ptATS2a mutants showed compromised cell growth, significantly changed plastid and extra-plastidial membrane lipids at nitrogen-replete condition and reduced triacylglycerols (TAGs) under nitrogen-depleted condition. ptATS2b is localized in thylakoid membranes and its knockout led to reduced growth rate and TAG content but slightly altered molecular composition of membrane lipids. The changes in glycerolipid profiles are consistent with the role of both LPAATs in the sn-2 acylation of sn-1-acyl-glycerol-3-phosphate substrates harboring 20:5 at the sn-1 position. Our findings suggest that both LPAATs are important for membrane lipids and TAG biosynthesis in P. tricornutum and further highlight that 20:5-Lyso-PA is likely involved in the massive import of 20:5 back to the plastid to feed plastid glycerolipid syntheses.
Assuntos
Aciltransferases , Lipídeos de Membrana , Triglicerídeos , Aciltransferases/metabolismo , Plastídeos/metabolismo , Ácidos Fosfatídicos , NitrogênioRESUMO
Phenolic recovery from agricultural byproducts has been highlighted due to their health-promoting bioactivities. However, uncontrolled discard of residues after extraction process would induce environmental pollution and bioresource waste. In this study, biorefining of phenolic-rich rapeseed meal (RSM) and its defatted sample (dRSM) was attempted by holistic utilization of phenolic extract and residue separately. Phenolic removal could significantly improve residues' Cr(VI) adsorption capacities by about 21%, which presented extended physical surface and more released functional groups. Moreover, simulating raw material by remixing 3% separated phenolic extracts or main component sinapic acid therein with corresponding residues further improved about 12% adsorption efficiencies. These indicated that the different present forms of phenolics had opposite effects on Cr(VI) removal. While natural conjugational form inhibited hosts' biosorption, free form had enhanced functions for either extract or residue. Four optimal adsorption parameters (pH, adsorbent dosage, contact time and initial Cr(VI) concentration), three kinetic (pseudo-first order, pseudo-second order and intra-particle diffusion) models and two isotherms (Langmuir and Freundlich) were used to reveal the adsorption process. The maximum Cr(VI) adsorption capacity on residues could reach about 100 mg/g, which was superior to that of most biosorbents derived from agricultural byproducts, even some biochar. Together with the residues' advantages with everlasting capacity after 3 adsorption-desorption cycles and excellent abilities for adsorbing multiple co-existed metal ions (Cr(VI), Cd(II), Cu(II), Pb(II), Ni(II) and Zn(II)), phenolic recovery was first proved to be a new and sustainable strategy for modifying biosorbents from agricultural byproducts with zero waste.
Assuntos
Brassica napus , Poluentes Químicos da Água , Concentração de Íons de Hidrogênio , Cromo/química , Adsorção , Cinética , Extratos Vegetais , Poluentes Químicos da Água/análiseRESUMO
Phaeodactylum tricornutum (Pt) is a critical microbial cell factory to produce a wide spectrum of marketable products including recombinant biopharmaceutical N-glycoproteins. N-glycosylation modification of proteins is important for their activity, stability, and half-life, especially some special modifications, such as fucose-modification by fucosyltransferase (FucT). Three PtFucTs were annotated in the genome of P. tricornutum, PtFucT1 was located on the medial/trans-Golgi apparatus and PtFucT2-3 in the plastid stroma. Algal growth, biomass and photosynthesis efficiency were significantly inhibited in a knockout mutant of PtFucT1 (PtFucT1-KO). PtFucT1 played a role in non-core fucose modification of N-glycans. The knockout of PtFucT1 might affect the activity of PtGnTI in the complex and change the complex N-glycan to mannose type N-glycan. The study provided critical information for understanding the mechanism of protein N-glycosylation modification and using microalgae as an alternative ecofriendly cell factory to produce biopharmaceuticals.
Assuntos
Diatomáceas , Fucosiltransferases , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Diatomáceas/genética , Diatomáceas/metabolismo , Fucose/metabolismo , Sistemas CRISPR-Cas , Proteínas Recombinantes/metabolismo , Polissacarídeos/metabolismo , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Galactosídeo 2-alfa-L-FucosiltransferaseRESUMO
Triacylglycerols (TAGs) are the main storage lipids in photosynthetic organisms under stress. In the oleaginous alga Nannochloropsis oceanica, while multiple acyl CoA:diacylglycerol (DAG) acyltransferases (NoDGATs) are involved in TAG production, the role of the unique phospholipid:DAG acyltransferase (NoPDAT) remains unknown. Here, we performed a functional complementation assay in TAG-deficient yeast (Saccharomyces cerevisiae) and an in vitro assay to probe the acyltransferase activity of NoPDAT. Subcellular localization, overexpression, and knockdown (KD) experiments were also conducted to elucidate the role of NoPDAT in N. oceanica. NoPDAT, residing at the outermost plastid membrane, does not phylogenetically fall into the clades of algae or plants and uses phosphatidylethanolamine (PE) and phosphatidylglycerol with 16:0, 16:1, and 18:1 at position sn-2 as acyl-donors in vivo. NoPDAT KD, not triggering any compensatory mechanism via DGATs, led to an â¼30% decrease of TAG content, accompanied by a vast accumulation of PEs rich in 16:0, 16:1, and 18:1 fatty acids (referred to as "LU-PE") that was positively associated with CO2 availability. We conclude that the NoPDAT pathway is parallel to and independent of the NoDGAT pathway for oil production. LU-PE can serve as an alternative carbon sink for photosynthetically assimilated carbon in N. oceanica when PDAT-mediated TAG biosynthesis is compromised or under stress in the presence of high CO2 levels.
Assuntos
Aciltransferases , Microalgas , Fosfatidiletanolaminas , Aciltransferases/genética , Aciltransferases/metabolismo , Dióxido de Carbono/metabolismo , Sequestro de Carbono/genética , Sequestro de Carbono/fisiologia , Diacilglicerol O-Aciltransferase/metabolismo , Microalgas/genética , Microalgas/metabolismo , Fosfatidiletanolaminas/genética , Fosfatidiletanolaminas/metabolismo , Plantas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Triglicerídeos/genética , Triglicerídeos/metabolismoRESUMO
Long-chain acyl-CoA synthetases (LACS) play diverse and fundamentally important roles in lipid metabolism. While their functions have been well established in bacteria, yeast and plants, the mechanisms by which LACS isozymes regulate lipid metabolism in unicellular oil-producing microalgae, including the diatom Phaeodactylum tricornutum, remain largely unknown. In P. tricornutum, a family of five genes (ptACSL1-ptACSL5) encodes LACS activities. We generated single lacs knockout/knockdown mutants using multiplexed CRISPR/Cas9 method, and determined their substrate specificities towards different fatty acids (FAs) and subcellular localisations. ptACSL3 is localised in the mitochondria and its disruption led to compromised growth and reduced triacylglycerol (TAG) content when cells were bubbled with air. The ptACSL3 mutants showed altered FA profiles in two galactoglycerolipids and phosphatidylcholine (PC) with significantly reduced distribution of 16:0 and 16:1. ptACSL5 is localised in the peroxisome and its knockdown resulted in reduced growth rate and altered molecular species of PC and TAG, indicating a role in controlling the composition of acyl-CoAs for lipid synthesis. Our work demonstrates the potential of generating gene knockout mutants with the mutation of large fragment deletion using multiplexed CRISPR/Cas9 and provides insight into the functions of LACS isozymes in lipid metabolism in the oleaginous microalgae.
Assuntos
Diatomáceas , Sistemas CRISPR-Cas/genética , Coenzima A/genética , Coenzima A/metabolismo , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Diatomáceas/genética , Diatomáceas/metabolismo , Ácidos Graxos/metabolismo , Mitocôndrias/metabolismoRESUMO
Rapeseed meal (RSM) is a major by-product of oil extraction from rapeseed, consists mainly of proteins and phenolic compounds. The use of RSM as protein feedstock for microbial fermentation is always hampered by phenolic compounds, which have antioxidant property with health-promoting benefits but inhibit bacterial growth. However, there is still not any good process that simultaneously improve extraction efficiency of phenolic compounds with conversion efficiency of protein residue into microbial production. Here we established a two-step strategy including fungal pretreatment followed by extraction of phenolic compounds. This could not only increase extraction efficiency and antioxidant property of phenolic compounds by about 2-fold, but also improve conversion efficiency of protein residue into iturin A production by Bacillus amyloliquefaciens CX-20 by about 33%. The antioxidant and antibacterial activities of phenolic extracts were influenced by both total phenolic content and profile, while microbial feedstock value of residue was greatly improved because protein content was increased by â¼5% and phenolic content was decreased by â¼60%. Moreover, this two-step process resulted in isolating more proteins from RSM, bringing iturin A production to 1.95 g/L. In conclusion, high-value-added and graded utilization of phenolic extract and protein residue from RSM with zero waste is realized by a two-step strategy, which combines both benefits of fungal pretreatment and phenolic extraction procedures.
RESUMO
The search of the Phaeodactylum tricornutum genome database revealed the existence of six genes potentially encoding lysophospholipid acyltransferases. One of these genes, Phatr3_J20460, after introduction to yeast ale1 mutant disrupted in the LPCAT gene, produced a very active acyl-CoA:lysophosphatidylcholine (LPCAT) enzyme. Using in vitro assays applying different radioactive and non-radioactive substrates and microsomal fractions from such yeast, we have characterized the biochemical properties and substrate specificities of this PtLPCAT1. We have found that the substrate specificity of this enzyme indicates that it can completely supply phosphatidylcholine (PC) with all fatty acids connected with a biosynthetic pathway of very long-chain polyunsaturated fatty acids (VLC-PUFAs) used further for the desaturation process. Additionally, we have shown that biochemical properties of the PtLPCAT1 in comparison to plant LPCATs are in some cases similar (such as the dependency of its activity on pH value), differ moderately (such as in response to temperature changes), or express completely different properties (such as in reaction to calcium and magnesium ions or toward some acyl-CoA with 20C polyunsaturated fatty acids). Moreover, the obtained results suggest that cloned "Phatr3_J20460" gene can be useful in oilseeds plant engineering toward efficient production of VLC-PUFA as LPCAT it encodes can (contrary to plant LPCATs) introduce 20:4-CoA (n-3) to PC for further desaturation to 20:5 (EPA, eicosapentaenoic acid).
Assuntos
1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Diatomáceas/enzimologia , 1-Acilglicerofosfocolina O-Aciltransferase/genética , Animais , Brassicaceae , Diatomáceas/genética , Humanos , Proteínas de Plantas/metabolismo , Especificidade por SubstratoRESUMO
The International Symposium on Lipid Science and Health (ISLSH) has been organized annually by the Oil Crops Research Institute of Chinese Academy of Agricultural Sciences (OCRI-CAAS) since 2016. The purpose of the symposium was to bring together the leading lipid science and health researchers throughout the world to discuss the current state of knowledge as well as research needs with respect to chemistry and beneficial health properties of lipids. The Fifth International Symposium on Lipid Science and Health was held on October 2020 in Wuhan, Hubei, China. Speakers from China, the United States, Australia, Finland, and other countries delivered wonderful presentations. The presentations covered such diverse topics as lipid profiling and characterization, lipid preparation and modification, lipid improvement and regulation, and lipid nutrition and health. As a record of the symposium proceedings, this special issue comprises a selection of 27 papers from oral presentations and poster contributions and is prefaced by this introduction.
Assuntos
Produtos Agrícolas , Lipídeos , Austrália , China , Finlândia , HumanosRESUMO
Fungal pretreatment is the most common strategy for improving the conversion of rapeseed meal (RSM) into value-added microbial products. It was demonstrated that Bacillus amyloliquefaciens CX-20 could directly use RSM as the sole source of all nutrients except the carbon source for iturin A fermentation with high productivity. However, whether fungal pretreatment has an impact on iturin A production is still unknown. In this study, the effects of fungal pretreatment and direct bio-utilization of RSM for iturin A fermentation were comparatively analysed through screening suitable fungal species, and evaluating the relationships between iturin A production and the composition of solid fermented RSM and liquid hydrolysates. Three main unconventional adverse effects were identified. (1) Solid-state fermentation by fungi resulted in a decrease of the total nitrogen for B. amyloliquefaciens CX-20 growth and metabolism, which caused nitrogen waste from RSM. (2) The released free ammonium nitrogen in liquid hydrolysates by fungal pretreatment led to the reduction of iturin A. (3) The insoluble precipitates of hydrolysates, which were mostly ignored and wasted in previous studies, were found to have beneficial effects on producing iturin A. In conclusion, our study verifies the unconventional adverse effects of fungal pretreatment on iturin A production by B. amyloliquefaciens CX-20 compared with direct bio-utilization of RSM.
Assuntos
Bacillus amyloliquefaciens , Bacillus amyloliquefaciens/metabolismo , Fermentação , Fungos/metabolismo , Peptídeos Cíclicos/metabolismoRESUMO
There is an increasing demand for astaxanthin in food, feed, cosmetics and pharmaceutical applications because of its superior anti-oxidative and coloring properties. However, naturally produced astaxanthin is expensive, mainly due to low productivity and limited sources. Reprogramming of microorganisms for astaxanthin production via metabolic engineering is a promising strategy. We primarily focus on the application of synthetic biology, enzyme engineering and metabolic engineering in enhancing the synthesis and accumulation of astaxanthin in microorganisms in this review. We also discuss the biosynthetic pathways of astaxanthin within natural producers, and summarize the achievements and challenges in reprogramming microorganisms for enhancing astaxanthin production. This review illuminates recent biotechnological advances in microbial production of astaxanthin. Future perspectives on utilization of new technologies for boosting microbial astaxanthin production are also discussed.
Assuntos
Engenharia Metabólica , Xantofilas , Vias Biossintéticas , Biotecnologia , Xantofilas/metabolismoRESUMO
BACKGROUND: In photosynthetic oleaginous microalgae, acyl-CoA molecules are used as substrates for the biosynthesis of membrane glycerolipids, triacylglycerol (TAG) and other acylated molecules. Acyl-CoA can also be directed to beta-oxidative catabolism. They can be utilized by a number of lipid metabolic enzymes including endogenous thioesterases, which catalyze their hydrolysis to release free fatty acids. Acyl-CoA availability thus plays fundamental roles in determining the quantity and composition of membrane lipids and storage lipids. RESULTS: Here, we have engineered the model diatom Phaeodactylum tricornutum to produce significantly increased TAGs by disruption of the gene encoding a Hotdog-fold thioesterase involved in acyl-CoA hydrolysis (ptTES1). This plastidial thioesterase can hydrolyze both medium- and long-chain fatty acyl-CoAs, but has the highest activity toward long-chain saturated and monounsaturated fatty acyl-CoAs. The maximum rate was found with oleoyl-CoA, which is hydrolyzed at 50 nmol/min/mg protein. The stable and targeted interruption of acyl-CoA thioesterase gene was achieved using a genome editing technique, transcription activator-like effector nucleases (TALENs). Disruption of native ptTES1 gene resulted in a 1.7-fold increase in TAG content when algal strains were grown in nitrogen-replete media for 8 days, whereas the content of other lipid classes, including phosphoglycerolipids and galactoglycerolipids, remained almost unchanged. The engineered algal strain also exhibited a marked change in fatty acid profile, including a remarkable increase in 16:0 and 16:1 and a decrease in 20:5. Nitrogen deprivation for 72 h further increased TAG content and titer of the engineered strain, reaching 478 µg/109 cells and 4.8 mg/L, respectively. Quantitative determination of in vivo acyl-CoAs showed that the total acyl-CoA pool size was significantly higher in the engineered algal strain than that in the wild type. CONCLUSIONS: This study supports the role of ptTES1 in free fatty acid homeostasis in the plastid of Phaeodactylum and demonstrates the potential of TALEN-based genome editing technique to generate an enhanced lipid-producing algal strain through blocking acyl-CoA catabolism.
RESUMO
Nε-lysine acetylation represents a highly dynamic and reversibly regulated post-translational modification widespread in almost all organisms, and plays important roles for regulation of protein function in diverse metabolic pathways. However, little is known about the role of lysine acetylation in photosynthetic eukaryotic microalgae. We integrated proteomic approaches to comprehensively characterize the lysine acetylome in the model diatom Phaeodactylum tricornutum In total, 2324 acetylation sites from 1220 acetylated proteins were identified, representing the largest data set of the lysine acetylome in plants to date. Almost all enzymes involved in fatty acid synthesis were found to be lysine acetylated. Six putative lysine acetylation sites were identified in a plastid-localized long-chain acyl-CoA synthetase. Site-directed mutagenesis and site-specific incorporation of N-acetyllysine in acyl-CoA synthetase show that acetylation at K407 and K425 increases its enzyme activity. Moreover, the nonenzymatically catalyzed overall hyperacetylation of acyl-CoA synthetase by acetyl-phosphate can be effectively deacetylated and reversed by a sirtuin-type NAD+-dependent deacetylase with subcellular localization of both the plastid and nucleus in Phaeodactylum This work indicates the regulation of acyl-CoA synthetase activity by site-specific lysine acetylation and highlights the potential regulation of fatty acid metabolism by lysine actetylation in the plastid of the diatom Phaeodactylum.
Assuntos
Diatomáceas/metabolismo , Ácidos Graxos/metabolismo , Lisina/metabolismo , Acetilação , Processamento de Proteína Pós-Traducional , Proteoma , ProteômicaRESUMO
BACKGROUND: The biological control agent Aspergillus aculeatus Asp-4 colonizes and degrades sclerotia of Sclerotinia sclerotiorum resulting in reduced germination and disease caused by this important plant pathogen. Molecular mechanisms of mycoparasites underlying colonization, degradation, and reduction of germination of sclerotia of this and other important plant pathogens remain poorly understood. RESULTS: An RNA-Seq screen of Asp-4 growing on autoclaved, ground sclerotia of S. sclerotiorum for 48 h identified 997 up-regulated and 777 down-regulated genes relative to this mycoparasite growing on potato dextrose agar (PDA) for 48 h. qRT-PCR time course experiments characterized expression dynamics of select genes encoding enzymes functioning in degradation of sclerotial components and management of environmental conditions, including environmental stress. This analysis suggested co-temporal up-regulation of genes functioning in these two processes. Proteomic analysis of Asp-4 growing on this sclerotial material for 48 h identified 26 up-regulated and 6 down-regulated proteins relative to the PDA control. Certain proteins with increased abundance had putative functions in degradation of polymeric components of sclerotia and the mitigation of environmental stress. CONCLUSIONS: Our results suggest co-temporal up-regulation of genes involved in degradation of sclerotial compounds and mitigation of environmental stress. This study furthers the analysis of mycoparasitism of sclerotial pathogens by providing the basis for molecular characterization of a previously uncharacterized mycoparasite-sclerotial interaction.
Assuntos
Ascomicetos/genética , Ascomicetos/metabolismo , Aspergillus/metabolismo , Micélio/metabolismo , Proteômica , Ascomicetos/crescimento & desenvolvimento , Biomassa , Perfilação da Expressão Gênica , Anotação de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
Since methylmalonyl-CoA epimerase appears to be absent in the majority of photosynthetic organisms, including diatoms, (S)-methylmalonyl-CoA, the intermediate of isoleucine (Ile) catabolism, cannot be metabolized to (R)-methylmalonyl-CoA then to succinyl-CoA. In this study, propionyl-CoA carboxylase (PCC) RNAi silenced strains and 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) overexpression strains were constructed to elucidate the Ile degradation pathway and its influence on lipid accumulation in Phaeodactylum tricornutum based on growth, neutral lipid content and metabolite profile analysis. Knockdown of PCC disturbed the metabolism of Ile through propionyl-CoA to methylmalonyl-CoA, as illustrated by much higher Ile content at day 6. However, Ile decreased to comparable levels to the wild-type at day 10. PCC silencing redirected propionyl-CoA to acetyl-CoA via a modified ß-oxidation pathway, and transcript levels for some branched-chain amino acid (BCAA) degradation-related genes, especially HIBCH, significantly upregulated in the PCC mutant, which enhanced the BCAA degradations and thus resulted in higher triacylglycerol (TAG) content. Overexpression of HIBCH accelerates Ile degradation and results in a lowered Ile content in the overexpression strains, thus enhancing carbon skeletons to the tricarboxylic acid cycle and giving rise to increasing TAG accumulation. Our study provides a good strategy to obtain high-lipid-yield transgenic diatoms by modifying the propionyl-CoA metabolism.This article is part of the themed issue 'The peculiar carbon metabolism in diatoms'.
Assuntos
Proteínas de Algas/metabolismo , Diatomáceas/metabolismo , Isoleucina/metabolismo , Tioléster Hidrolases/metabolismo , Triglicerídeos/metabolismo , Diatomáceas/enzimologia , MetabolismoRESUMO
BACKGROUND: Triacylglycerols (TAGs) and wax esters (WEs) are important neutral lipids which serve as energy reservoir in some plants and microorganisms. In recent years, these biologically produced neutral lipids have been regarded as potential alternative energy sources for biofuel production because of the increased interest on developing renewable and environmentally benign alternatives for fossil fuels. In bacteria, the final step in TAG and WE biosynthetic pathway is catalyzed by wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT). This bifunctional WS/DGAT enzyme is also a key enzyme in biotechnological production of liquid WE via engineering of plants and microorganisms. To date, knowledge about this class of biologically and biotechnologically important enzymes is mainly from biochemical characterization of WS/DGATs from Arabidopsis, jojoba and some bacteria that can synthesize both TAGs and WEs intracellularly, whereas little is known about WS/DGATs from eukaryotic microorganisms. RESULTS: Here, we report the identification and characterization of two bifunctional WS/DGAT enzymes (designated TrWSD4 and TrWSD5) from the marine protist Thraustochytrium roseum. Both TrWSD4 and TrWSD5 comprise a WS-like acyl-CoA acyltransferase domain and the recombinant proteins purified from Escherichia coli Rosetta (DE3) have substantial WS and lower DGAT activity. They exhibit WS activity towards various-chain-length saturated and polyunsaturated acyl-CoAs and fatty alcohols ranging from C10 to C18. TrWSD4 displays WS activity with the lowest Km value of 0.14 µM and the highest kcat/Km value of 1.46 × 105 M-1 s-1 for lauroyl-CoA (C12:0) in the presence of 100 µM hexadecanol, while TrWSD5 exhibits WS activity with the lowest Km value of 0.96 µM and the highest kcat/Km value of 9.83 × 104 M-1 s-1 for decanoyl-CoA (C10:0) under the same reaction condition. Both WS/DGAT enzymes have the highest WS activity at 37 and 47 °C, and WS activity was greatly decreased when temperature exceeds 47 °C. TrWSD4 and TrWSD5 are insensitive to ionic strength and reduced WS activity was observed when salt concentration exceeded 800 mM. The potential of T. roseum WS/DGATs to establish novel process for biotechnological production of WEs was demonstrated by heterologous expression in recombinant yeast. Expression of either TrWSD4 or TrWSD5 in Saccharomyces cerevisiae quadruple mutant H1246, which is devoid of storage lipids, resulted in the accumulation of WEs, but not any detectable TAGs, indicating a predominant WS activity in yeast. CONCLUSIONS: This study demonstrates both in vitro WS and DGAT activity of two T. roseum WS/DGATs, which were characterized as unspecific acyltransferases accepting a broad range of acyl-CoAs and fatty alcohols as substrates for WS activity but displaying substrate preference for medium-chain acyl-CoAs. In vivo characterization shows that these two WS/DGATs predominantly function as wax synthase and presents the feasibility for production of WEs by heterologous hosts.