enoyl-CoA Reductase Research Articles

Application of deep brain stimulation for the treatment of childhood-onset dystonia in patients with MEPAN syndrome.

Mitochondrial Enoyl CoA Reductase Protein-Associated Neurodegeneration (MEPAN) syndrome is a rare inherited metabolic condition caused by MECR gene mutations. This gene encodes a protein essential for fatty acid synthesis, and defects cause progressively worsening childhood-onset dystonia, optic atrophy, and basal ganglia abnormalities. Deep brain stimulation (DBS) has shown mixed improvement in other childhood-onset dystonia conditions. To the best of our knowledge, DBS has not been investigated as a treatment for dystonia in patients with MEPAN syndrome. Two children with MEPAN were identified as possible DBS candidates due to severe generalized dystonia unresponsive to pharmacotherapy. Temporary depth electrodes were placed in six locations bilaterally and tested during a 6-day hospitalization to determine the best locations for permanent electrode placement. The Burke-Fahn-Marsden Dystonia Rating Scale (BFMDRS) and Barry-Albright Dystonia Scale (BADS) were used for preoperative and postoperative testing to quantitatively assess dystonia severity changes. Patient 1 had permanent electrodes placed at the globus pallidus internus (GPi) and pedunculopontine nucleus (PPN). Patient 2 had permanent electrodes placed at the GPi and ventralis intermedius nucleus of the thalamus (VIM). Both patients successfully underwent DBS placement with no perioperative complications and significant improvement in their BFMDRS score. Patient 2 also demonstrated improvement in the BADS. We demonstrated a novel application of DBS in MEPAN syndrome patients with childhood-onset dystonia. These patients showed clinically significant improvements in dystonia following DBS, indicating that DBS can be considered for dystonia in patients with rare metabolic disorders that currently have no other proven treatment options.

Novel utilization of deep brain stimulation in the pedunculopontine nucleus with globus pallidus internus for treatment of childhood-onset dystonia

IntroductionDeep brain stimulation (DBS) is a well-documented therapy for dystonia utilized in many adult and pediatric movement disorders. Pedunculopontine nucleus (PPN) has been investigated as a DBS target primarily in adult patients with dystonia or dyskinesias from Parkinson’s disease, showing improvement in postural instability and gait dysfunction. Due to the difficulty in targeting PPN using standard techniques, it is not commonly chosen as a target for adult or pediatric pathology. There is no current literature describing the targeting of PPN in DBS for childhood-onset dystonia.MethodsTwo pediatric and one young adult patient with childhood-onset dystonia who underwent DBS implantation at our institution were identified. Patient 1 has Mitochondrial Enoyl CoA Reductase Protein-Associated Neurodegeneration (MEPAN) syndrome. Patient 2 has Glutaric Aciduria Type 1 (GA1). Patient 3 has atypical pantothenate kinase-associated neurodegeneration (PKAN). PPN was identified as a potential target for these patients due to axial or orofacial dystonia. Pre- and post-operative videos taken as part of routine clinical assessments were evaluated and scored on the Burke-Fahn-Marsden Dystonia Rating Scale (BFMDRS) and Barry-Albright Dystonia Scale (BADS). All patients had permanent electrodes placed bilaterally in PPN and globus pallidus internus (GPi). A Likert scale on quality of life was also obtained from the patient/parents as applicable.ResultsSignificant programming was necessary over the first 3–12 months to optimize patients’ response to stimulation. All patients experienced at least a 34% improvement in the BFMDRS score. Patients 2 and 3 also experienced an over 30% improvement in BADS score. All patients/parents appreciated improvement in quality of life postoperatively.DiscussionDeep brain stimulation in PPN was safely and successfully used in two pediatric patients and one young adult patient with childhood-onset dystonia. These patients showed clinically significant improvements in BFMDRS scoring post operatively. This represents the first reported DBS targeting of PPN in pediatric patients, and suggests that PPN is a possible target for pediatric-onset dystonia with axial and orofacial symptoms that may be refractory to traditional pallidal stimulation alone.

Formation of plant resistance to pathogens involving epigenetic factors and phytohormones

The development of resistance to plant pathogens is determined by the presence of resistance genes and regulation of their activity due to phytohormones, the activity of the nuclear pore complex, and epigenetic modifications at the post-transcriptional and post-translational levels of DNA and histones, respectively. Changes in gene expression due to such modifications can be inherited in generations and contribute to the selection of resistant plants in populations.The nuclear pore complex is composed of nucleoporins, a nuclear pore basket, cytoplasmic filaments and is able to selectively transport transcription factors to the nucleus from the cytoplasm and mRNA in the opposite direction, affecting gene expression. Methylation is the most common and well-studied among the epigenetic modifications. It is described for DNA and histones and ensures genome stability and the availability of transcription factors. Incorporation of methyl groups to amino acid residues is not always a factor in gene silencing for histones. The number of the incorporated groups as well as an amino acid they are attached to are crucial. Histone acetylation is associated with the transition to transcriptional activity.The general control histone acetyltransferase TaGCN5 is able to promote expression of the enoyl-CoA reductase gene in allohexaploid wheat, which is involved in the biosynthesis of cuticular wax as one of the resistance factors. The reverse process of deacetylation acts both as a positive regulator, through ethylene response factors, and a negative one, as it interferes with acetylation and methylation of histone amino acids. Jasmonic and salicylic acids, along with combinations of the mentioned phytohormones, mediate formation of systemic acquired resistance in addition to ethylene. Auxin is a positive phytohormone for pathogens due to its ability to influence the structure of the cell wall. At the same time, pathogens, due to effectors, are able to inhibit the plant’s immune responses, so there is a constant “arms race” resulting in the selection of more effective means of penetration and development in the plant as well as its protective reactions. Investigation of the mechanisms of resistance formation and identifying the main factors of resistance, such as either presence of a gene sequence and/or factors regulating its expression, are of great importance.

Integrated proteomics and metabolomics analysis revealed the mechanisms underlying the effect of irradiation on the fat quality of Chinese bacon

Irradiation increases the security and storage period of preserved Chinese bacon; nevertheless, the biological mechanisms underlying the changes in fat quality caused by irradiation are unknown. We investigated the influence of irradiation on Chinese bacon by proteomic and metabolomic. We identified 24 proteins that participated in metabolism and 40 common differential metabolites enriched in 16 signalling pathways. Correlation analysis revealed that irradiation altered 11 pathways shared between the proteome and metabolome, including two lipid metabolism pathways. Acetyl-CoA carboxylase, ACSL, octanoic acid, decanoic acid, palmitic acid, and oleic acid participated in fatty acid biosynthesis. Acyl-CoA thioesterase 1/2/4, enoyl-CoA reductase, acetyl-CoA acyltransferase 1, enoyl-CoA hydratase 2, palmitic acid, and oleic acid participated in unsaturated fatty acid biosynthesis. These findings lay the groundwork for multi-omics research on the effects of irradiation on Chinese bacon quality, assisting in assessing irradiated Chinese bacon quality, and developing effective strategies to standardise quality parameters.

Alternative splicing liberates a cryptic cytoplasmic isoform of mitochondrial MECR that antagonizes influenza virus.

Viruses must balance their reliance on host cell machinery for replication while avoiding host defense. Influenza A viruses are zoonotic agents that frequently switch hosts, causing localized outbreaks with the potential for larger pandemics. The host range of influenza virus is limited by the need for successful interactions between the virus and cellular partners. Here we used immunocompetitive capture-mass spectrometry to identify cellular proteins that interact with human- and avian-style viral polymerases. We focused on the proviral activity of heterogenous nuclear ribonuclear protein U-like 1 (hnRNP UL1) and the antiviral activity of mitochondrial enoyl CoA-reductase (MECR). MECR is localized to mitochondria where it functions in mitochondrial fatty acid synthesis (mtFAS). While a small fraction of the polymerase subunit PB2 localizes to the mitochondria, PB2 did not interact with full-length MECR. By contrast, a minor splice variant produces cytoplasmic MECR (cMECR). Ectopic expression of cMECR shows that it binds the viral polymerase and suppresses viral replication by blocking assembly of viral ribonucleoprotein complexes (RNPs). MECR ablation through genome editing or drug treatment is detrimental for cell health, creating a generic block to virus replication. Using the yeast homolog Etr1 to supply the metabolic functions of MECR in MECR-null cells, we showed that specific antiviral activity is independent of mtFAS and is reconstituted by expressing cMECR. Thus, we propose a strategy where alternative splicing produces a cryptic antiviral protein that is embedded within a key metabolic enzyme.

Ectopic Overexpression of CsECR From Navel Orange Increases Cuticular Wax Accumulation in Tomato and Enhances Its Tolerance to Drought Stress.

Drought stress often occurred in citrus to limit its growth, distribution, and fruit quality. Cuticular waxes play an important role in regulating plant tolerance to drought stress. Plant enoyl-CoA reductase (ECR) is involved in the biosynthesis of cuticular waxes and catalyzes the last step of very long-chain fatty acids (VLCFAs) elongation. In this study, a putative ECR gene, named CsECR, was cloned from “Newhall” navel orange. CsECR protein has high identities with other plant ECR proteins and contained a conserved NADP/NAD-binding motif and three conserved functional sites. The highest expression of CsECR was observed in leaves, followed by stems, flavedos, ovaries, juice sacs, stigmas, stamens, albedos, and petals. Besides, the expression of CsECR was significantly induced by PEG6000 and ABA treatments. Ectopic overexpression of CsECR increased the contents of total waxes and aliphatic wax fractions (n-fatty acids, unsaturated fatty acids, n-alkanes, alkenes, iso-, and anteiso-alkanes) in the leaves and fruits of the transgenic tomato. Furthermore, ectopic overexpression of CsECR reduced the cuticle permeability in the leaves and fruits of the transgenic tomato and increased its tolerance to drought stress. Taken together, our results revealed that CsECR plays an important role in plant response to drought stresses by regulating cuticular wax biosynthesis.

ECERIFERUM 10 Encoding an Enoyl-CoA Reductase Plays a Crucial Role in Osmotolerance and Cuticular Wax Loading in Arabidopsis

Acquired osmotolerance induced after salt stress is widespread across Arabidopsis thaliana (Arabidopsis) accessions (e.g., Bu-5). However, it remains unclear how this osmotolerance is established. Here, we isolated a mutant showing an acquired osmotolerance-defective phenotype (aod2) from an ion-beam-mutagenized M2 population of Bu-5. aod2 was impaired not only in acquired osmotolerance but also in osmo-shock, salt-shock, and long-term heat tolerances compared with Bu-5, and it displayed abnormal morphology, including small, wrinkled leaves, and zigzag-shaped stems. Genetic analyses of aod2 revealed that a 439-kbp region of chromosome 4 was translocated to chromosome 3 at the causal locus for the osmosensitive phenotype. The causal gene of the aod2 phenotype was identical to ECERIFERUM 10 (CER10), which encodes an enoyl-coenzyme A reductase that is involved in the elongation reactions of very-long-chain fatty acids (VLCFAs) for subsequent derivatization into cuticular waxes, storage lipids, and sphingolipids. The major components of the cuticular wax were accumulated in response to osmotic stress in both Bu-5 WT and aod2. However, less fatty acids, primary alcohols, and aldehydes with chain length ≥ C30 were accumulated in aod2. In addition, aod2 exhibited a dramatic reduction in the number of epicuticular wax crystals on its stems. Endoplasmic reticulum stress mediated by bZIP60 was increased in aod2 under osmotic stress. The only cer10 showed the most pronounced loss of epidermal cuticular wax and most osmosensitive phenotype among four Col-0-background cuticular wax-related mutants. Together, the present findings suggest that CER10/AOD2 plays a crucial role in Arabidopsis osmotolerance through VLCFA metabolism involved in cuticular wax formation and endocytic membrane trafficking.

In silico Design, Synthesis and Antitubercular Activity of Some Metal Complexes Derived from Salicylaldehyde and Amino Acid

Background: A new series of copper (II), cobalt (II), zinc (II), manganese (II), and iron (II) metal complexes were synthesized by the condensation of a novel Schiff base with various metal chlorides in ethanol. Schiff base was synthesized by reacting salicylaldehyde with L-glutamic acid and L-tyrosine dissolved in ethanol, respectively Methods: The structures of all the synthesized metal complexes (4a-e, 7a-e) were investigated using elemental analysis, FT-IR,1H NMR,13C NMR and MS spectral data. The metal complexes were also screened for their anti-bacterial, anti-fungal, and anti-tubercular activities against various tested strains. Results: Assessment of in silico ADMET properties of all metal complexes showed to be in accordance with Lipinski’s rule of five. Further enzymatic assay was aided by a molecular docking study of Enoyl CoA reductase (INHA) using Autodock Vina and evaluated by Autodock 4.0. Conclusion: The metal complexes, 4b,4c, 4d,7b and 7d, containing metals, like Zn, Co, and Fe, exhibited good anti-bacterial, anti-fungal and anti-tubercular activities against the tested strains.

High resolution mass spectrometric analysis reveals that Giardia cysts are metabolically active and generates membrane lipids with long chain fatty acyl moieties

Giardia lamblia, the causative agent of “giardiasis” affects people throughout the world. Metronidazole (Mtz, Flagyl®), a nitroimidazole group of compounds is the commonly prescribed treatment for giardiasis. However, Mtz is a genotoxic agent and a potential carcinogen. Therefore, it is imperative to identify new and specific targets in Giardia that could be exploited to develop non-Mtz classes of drugs to treat giardiasis. Because of its limited lipid synthetic ability, Giardia was proposed to scavenge phospholipids, sterols and free fatty acids from the hosts or its environment for membrane and organelle biogenesis. In the current study, we utilized a quantitative lipidomic and proteomic approach to further elucidate the lipid metabolic pathways in Giardia. Vegetative and encysting trophozoites as well as osmotically resistant cysts were cultured in the laboratory, collected, and subjected to lipid extraction or tryptic digestion followed by the high-resolution mass spectrometric analysis (Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer equipped with a heated electrospray (HESI-II) or a Nanospray Flex Ion source, Thermo Fisher). Lipid identification and confirmation was performed on open-source software MS-DIAL and LipidMatch flow. Proteomic analysis was performed on a licensed version of Proteome Discoverer. Lipidomic analysis revealed the dynamic changes of overall lipid and fatty acid (mostly C16, C18 moieties) profiles throughout the life stages of Giardia. Lipid classes that were found in cysts included LPC, P- and O-LPC, LPE, DG, TG, CE, PC, P-PC (Plasmalogen), and ceramides. Lipids, such as SM, PG, BMP were present mostly in encysting trophozoites rather than cysts. Ether linked PC species revealed to be most abundant in trophozoites. Proteomic analysis identified increased levels of several enzymes from lipid elongation pathway i.e., acyl-CoA ligases, long-chain fatty acid elongase, oxidoreductase and enoyl-CoA reductase in cysts.These studies suggest that although limited, Giardia maintains an active and robust lipid metabolism in all stages including the water-resistant non-motile cysts. It is possible that active lipid metabolism in cysts is required for the successful excystation to produce infective membranous trophozoites.

AP2/ERF transcription factors orchestrate very long chain fatty acid biosynthesis during Arabidopsis lateral root development

AP2/ERF transcription factors orchestrate very long chain fatty acid biosynthesis during Arabidopsis lateral root development

Epigenetic Activation of Enoyl-CoA Reductase By An Acetyltransferase Complex Triggers Wheat Wax Biosynthesis.

The epidermal surface of bread wheat (Triticum aestivum) is coated with a hydrophobic cuticular wax layer that protects plant tissues against environmental stresses. However, the regulatory mechanism of cuticular wax biosynthesis remains to be uncovered in bread wheat. Here, we identified wheat Enoyl-CoA Reductase (TaECR) as a core component responsible for biosynthesis of wheat cuticular wax. Silencing of TaECR in bread wheat resulted in a reduced cuticular wax load and attenuated conidia germination of the adapted fungal pathogen powdery mildew (Blumeria graminis f.sp. tritici). Furthermore, we established that TaECR genes are direct targets of TaECR promoter-binding MYB transcription factor1 (TaEPBM1), which could interact with the adapter protein Alteration/Deficiency in Activation2 (TaADA2) and recruit the histone acetyltransferase General Control Nonderepressible5 (TaGCN5) to TaECR promoters. Most importantly, we demonstrated that the TaEPBM1-TaADA2-TaGCN5 ternary protein complex activates TaECR transcription by potentiating histone acetylation and enhancing RNA polymerase II enrichment at TaECR genes, thereby contributing to the wheat cuticular wax biosynthesis. Finally, we identified very-long-chain aldehydes as the wax signals provided by the TaECR-TaEPBM1-TaADA2-TaGCN5 circuit for triggering B graminis f.sp. tritici conidia germination. These results demonstrate that specific transcription factors recruit the TaADA2-TaGCN5 histone acetyltransferase complex to epigenetically regulate biosynthesis of wheat cuticular wax, which is required for triggering germination of the adapted powdery mildew pathogen.

Ternary complex formation of AFN-1252 with Acinetobacter baumannii FabI and NADH: Crystallographic and biochemical studies.

Acinetobacter baumannii is an opportunistic Gram-negative bacterial pathogen, associated mostly with hospital-acquired infections. The emergence of drug resistance strains made it necessary to explore new pathways for the development of more effective antibiotics. Enoyl CoA reductase (FabI), a key enzyme in the fatty acid biosynthesis (FAS) pathway, has emerged as a potential target for antibacterial drug development. Earlier reports show that the lead SaFabI inhibitor AFN-1252 can inhibit FabI from other organisms including Escherichia coli and Burkholderia pseudomallei, but with differential potency. In the present work, we show that AFN-1252 is a moderate inhibitor of AbFabI with an IC50 of 216nM. AFN-1252 stabilized AbFabI with a 4.2°C increase in the melting temperature (Tm ) and, interestingly, the stabilization effect was significantly increased in presence of the cofactor NADH (∆Tm =17°C), suggesting the formation of a ternary complex AbFabI: AFN-1252: NADH. X-ray crystallography studies of AbFabI co-crystalized with AFN-1252 and NADH confirmed the ternary complex formation. The critical interactions of AFN-1252 with AbFabI and NADH identified from the co-crystal structure may facilitate the design and development of new drugs against A. baumannii infections by targeting the FAS pathway.

Development of camelina enhanced with drought stress resistance and seed oil production by co-overexpression of MYB96A and DGAT1C

Camelina (Camelina sativa, Cs) is an emerging crop for the production of biodiesel and biofeedstock. This study aims to develop transgenic plants that have the advantage of two traits: improved drought resistance and oil content. Three genes, CsMYB96A, CsMYB96B, and CsMYB96C, were isolated from camelina stem. The deduced amino acid sequence of the three CsMYB96s showed at least 93% identity with Arabidopsis MYB96. CsMYB96A, B, and C transcripts were detected in various camelina tissues. Fluorescence signal from the fusion of CsMYB96A: enhanced yellow fluorescent protein was confined to the nucleus of tobacco epidermal cells. Transactivation analysis of tobacco protoplasts revealed that CsMYB96A was a transcription activator. Wax biosynthesis genes such as camelina β-ketoacyl-CoA synthase 2, β-ketoacyl-CoA synthase 6, β-ketoacyl-CoA reductase 1-1, β-ketoacyl-CoA reductase 1-2, enoyl-CoA reductase, ECERIFERUM 1 and ECERIFERUM 3 were upregulated approximately 2 to 120 times by CsMYB96A, indicating that CsMYB96A was involved in the activating of cuticular wax synthesis on plant epidemics. Camelina diacylglycerol acyltransferase 1C (CsDGAT1C) has been shown to increased oil synthesis genes in Arabidopsis. When CsDGAT1C and CsMYB96A were co-overexpressed in camelina, total fatty acid levels in transgenic seeds increased by approximately 21%. In addition, the transgenic camelina plants showed improved resistance to drought stress. This result suggests that the transgenic camelina can be grown to produce biodiesel and biofeedstock in arid or semi-arid lands.

Genome-wide association analysis of the lipid and fatty acid metabolism regulatory network in the mesocarp of oil palm (Elaeis guineensis Jacq.) based on small noncoding RNA sequencing.

Oil palm (Elaeis guineensis Jacq.) is the highest oil-yielding crop in the plant kingdom and accumulates 90% of palm oil in the mesocarp. However, the regulatory mechanisms of lipid and fatty acid (FA) metabolism in oil palm are just beginning to be understood, and more studies are needed, especially in the understanding of small noncoding RNA (ncRNA) and mRNA. Based on the deep sequencing of small noncoding RNAs and the degradome in five developmental mesocarp stages, 452 microRNAs (miRNAs), including 170 conserved known-miRNAs (kn-miRNAs) and 282 novel-miRNA (nov-miRNAs), were identified. After predicting the targets of those miRNAs to 37 FA synthesis-related genes, we found that 22 kn-miRNAs and 14 nov-miRNAs might be involved in FA metabolism pathways. Among them, eg-miR156c, eg-miR397, eg-miR444b and nov-miR129 regulated FA synthesis in plastids and the transport of FA-ACP from plastids to the endoplasmic reticulum by targeting acetyl-CoA carboxylase 1 (ACC1), long-chain acyl-CoA synthetase 9 (LACS9), LACS4 and enoyl-ACP reductase (ENR), respectively. Nov-miR138 and nov-miR59 targeted glycerol-3-phosphate acyltransferase (GPAT), and nov-miR274 targeted phosphatidate phosphatase 1 (PAP1). Both target genes are involved in triacylglycerol synthesis in the endoplasmic reticulum. Eg-miR156e and eg-miR156j played pivotal roles by targeting β-ketoacyl-CoA synthase 12 (KCS12), and nov-miR201 targets very-long-chain enoyl-CoA reductase (ECR). Several miRNAs were also predicted to indirectly regulate FA synthesis and lipid metabolism through the squamosa promoter-binding protein-like gene (SPL), NAC and MYB transcription factors. As a whole, indications of a complex and extensive miRNA-mRNA regulatory network associated with FA metabolism in the mesocarp of the oil palm is presented. The results help to broaden the knowledge of potential mechanisms that might be regulated by miRNAs through modulation of the expression of FA-related target gene metabolism in the oil palm.

Identification and characterization of candidates involved in production of OMEGAs in microalgae: a gene mining and phylogenomic approach

Optimizing the production of the high-value renewables such as OMEGAs through pathway engineering requires an in-depth understanding of the structure–function relationship of genes involved in the OMEGA biosynthetic pathways. In this preliminary study, our rationale is to identify and characterize the ∼221 putative genes involved in production of OMEGAs using bioinformatic analysis from the Streptophyte (plants), Chlorophyte (green algae), Rhodophyta (red algae), and Bacillariophyta (diatoms) lineages based on their phylogenomic profiling, conserved motif/domain organization and physico-chemical properties. The MEME suite predicted 12 distinct protein domains, which are conserved among these putative genes. The phylogenomic analysis of the putative candidate genes [such as FAD2 (delta-12 desaturase); ECR (enoyl-CoA reductase); FAD2 (delta-12 desaturase); ACOT (acyl CoA thioesterase); ECH (enoyl-CoA hydratase); and ACAT (acetyl-CoA acyltransferase)] with similar domains and motif patterns were remarkably well conserved. Furthermore, the subcellular network prediction of OMEGA biosynthetic pathway genes revealed a unique interaction between the light-dependent chlorophyll biosynthesis and glycerol-3-phosphate dehydrogenase, which predicts a major cross-talk between the key essential pathways. Such bioinformatic analysis will provide insights in finding the key regulatory genes to optimize the productivity of OMEGAs in microalgal cell factories.

Selective production of decanoic acid from iterative reversal of β-oxidation pathway.

Decanoic acid is a valuable compound used as precursor for industrial chemicals, pharmaceuticals, and biofuels. Despite efforts to produce it from renewables, only limited achievements have been reported. Here, we report an engineered cell factory able to produce decanoic acid as a major product from glycerol, and abundant and renewable feedstock. We exploit the overlapping chain-length specificity of β-oxidation reversal (r-BOX) and thioesterase enzymes to selectively generate decanoic acid. This was achieved by selecting r-BOX enzymes that support the synthesis of acyl-CoA of up to 10 carbons (thiolase BktB and enoyl-CoA reductase EgTER) and a thioesterase that exhibited high activity toward decanoyl-CoA and longer-chain acyl-CoAs (FadM). Combined chromosomal and episomal expression of r-BOX core enzymes such as enoyl-CoA reductase and thiolase (in the presence of E. coli thioesterase FadM) increased titer and yield of decanoic acid, respectively. The carbon flux toward decanoic acid was substantially increased by the use of an organic overlay, which decreased its intracellular accumulation and presumably increased its concentration gradient across cell membrane, suggesting that decanoic acid transport to the extracellular medium might be a major bottleneck. When cultivated in the presence of a n-dodecane overlay, the final engineered strain produced 2.1 g/L of decanoic acid with a yield of 0.1 g/g glycerol. Collectively, our data suggests that r-BOX can be used as a platform to selectively produce decanoic acid and its derivatives at high yield, titer and productivity.

A novel synthetic pathway for glutarate production in recombinant Escherichia coli

Abstract Glutarate is an important C5 linear chain dicarboxylic acid having wide applications in chemical industry. In this work we report glutarate production by Escherichia coli with a newly constructed biosynthetic pathway containing part of known glutaconate biosynthetic pathway and a gap-filling module employing trans -enoyl-CoA reductase (Ter). Overall the artificial pathway comprises reduction of the central carbon metabolite α-ketoglutarate to 2-hydroxyglutarate, activation to 2-hydroxyglutaryl-CoA, dehydration to trans -glutaconyl-CoA, hydrogenation to glutaryl-CoA by Ter and thioester hydrolysis to finally yield glutarate. The pathway introduced into E. coli resulted in a recombinant strain that produced 3.8 mg/L of glutarate together with 27.7 mg/L of glutaconate in anaerobic culture mode. The glutarate production increased by approximately 50% through the mutation of Ter from Treponema denticola . The results demonstrated biosynthesis of glutarate via a non-natural synthetic pathway, which may enable its biobased production from renewable resources.

Tobacco Rattle Virus-Based Silencing of Enoyl-CoA Reductase Gene and Its Role in Resistance Against Cotton Wilt Disease.

A Tobacco rattle virus (TRV)-based virus-induced gene silencing assay was employed as a reverse genetic approach to study gene function in cotton (Gossypium hirsutum). This approach was used to investigate the function of the Enoyl-CoA reductase (GhECR) gene in pathogen defense. Amino acid sequence alignment of Arabidopsis ECR with homologous sequence from G. hirsutum, G. arboreum, G. herbaceum and G. barbadense showed that ECRs are highly conserved among these species. TRV-based silencing of GhECR gene in G. hirsutum induced a cell death/necrotic lesion-like phenotype. Reverse transcription polymerase chain reaction (RT-PCR) and real-time quantitative PCR showed reduced GhECR mRNA levels in TRV inoculated plants. Three isolates of Verticillium dahliae (V. dahliae) and Fusarium oxysporumf. sp.vasinfectum(FOV) were used to infect GhECR-silenced plants. Out of 6 races of 2 pathogens, down regulation of GhECR gene resulted in reduced resistance. This is the first report showing that cotton GhECR gene is involved in resistance to different strains of V. dahliae and FOV.

Non-electron transfer chain mitochondrial defects differently regulate HIF-1α degradation and transcription

Mitochondria are the main consumers of molecular O2 in a cell as well as an abundant source of reactive oxygen species (ROS). Both, molecular oxygen and ROS are powerful regulators of the hypoxia-inducible factor-1α-subunit (HIF-α). While a number of mechanisms in the oxygen-dependent HIF-α regulation are quite well known, the view with respect to mitochondria is less clear. Several approaches using pharmacological or genetic tools targeting the mitochondrial electron transport chain (ETC) indicated that ROS, mainly formed at the Rieske cluster of complex III of the ETC, are drivers of HIF-1α activation. However, studies investigating non-ETC located mitochondrial defects and their effects on HIF-1α regulation are scarce, if at all existing. Thus, in the present study we examined three cell lines with non-ETC mitochondrial defects and focused on HIF-1α degradation and transcription, target gene expression, as well as ROS levels. We found that cells lacking the key enzyme 2-enoyl thioester reductase/mitochondrial enoyl-CoA reductase (MECR), and cells lacking manganese superoxide dismutase (MnSOD) showed a reduced induction of HIF-1α under long-term (20h) hypoxia. By contrast, cells lacking the mitochondrial DNA depletion syndrome channel protein Mpv17 displayed enhanced levels of HIF-1α already under normoxic conditions. Further, we show that ROS do not exert a uniform pattern when mediating their effects on HIF-1α, although all mitochondrial defects in the used cell types increased ROS formation. Moreover, all defects caused a different HIF-1α regulation via promoting HIF-1α degradation as well as via changes in HIF-1α transcription. Thereby, MECR- and MnSOD-deficient cells showed a reduction in HIF-1α mRNA levels whereas the Mpv17 lacking cells displayed enhanced HIF-1α mRNA levels under normoxia and hypoxia. Altogether, our study shows for the first time that mitochondrial defects which are not related to the ETC and Krebs cycle contribute differently to HIF-1α regulation by affecting HIF-1α degradation and HIF-1α transcription where ROS play not a major role.

Subcellular localization of the five members of the human steroid 5α-reductase family

In humans the steroid 5α-reductase (SRD5A) family comprises five integral membrane enzymes that carry out reduction of a double bond in lipidic substrates: Δ4-3-keto steroids, polyprenol and trans-enoyl CoA. The best-characterized reaction is the conversion of testosterone into the more potent dihydrotestosterone carried out by SRD5A1-2. Some controversy exists on their possible nuclear or endoplasmic reticulum localization.We report the cloning and transient expression in HeLa cells of the five members of the human steroid 5α-reductase family as both N- and C-terminus green fluorescent protein tagged protein constructs. Following the intrinsic fluorescence of the tag, we have determined that the subcellular localization of these enzymes is in the endoplasmic reticulum, upon expression in HeLa cells. The presence of the tag at either end of the polypeptide chain can affect protein expression and, in the case of trans enoyl-CoA reductase, it induces the formation of protein aggregates.