118 research outputs found
Identification of a cDNA encoding a novel C18-Î9 polyunsaturated fatty acid-specific elongating activity from the docosahexaenoic acid (DHA)-producing microalga, Isochrysis galbana11The nucleotide sequence reported in this paper has been submitted to the GenBankâą/EBI Data Bank with accession number AF390174.
AbstractIsochrysis galbana, a marine prymnesiophyte microalga, is rich in long chain polyunsaturated fatty acids such as docosahexaenoic acid (C22:6n-3, Î4,7,10,13,16,19). We used a polymerase chain reaction-based strategy to isolate a cDNA, designated IgASE1, encoding a polyunsaturated fatty acid-elongating activity from I. galbana. The coding region of 263 amino acids predicts a protein of 30 kDa that shares only limited homology to animal and fungal proteins with elongating activity. Functional analysis of IgASE1, by expression in Saccharomyces cerevisiae, was used to determine its activity and substrate specificity. Transformed yeast cells specifically elongated the C18-Î9 polyunsaturated fatty acids, linoleic acid (C18:2n-6, Î9,12) and α-linolenic acid (C18:3n-3, Î9,12,15), to eicosadienoic acid (C20:2n-6, Î11,14) and eicosatrienoic acid (C20:3n-3, Î11,14,17), respectively. To our knowledge this is the first time such an elongating activity has been functionally characterised. The results also suggest that a major route for eicosapentaenoic acid (C20:5n-3, Î5,8,11,14,17) and docosahexaenoic acid syntheses in I. galbana may involve a Î8 desaturation pathway
SAXS reveals highly flexible interdomain linkers of tandem acyl carrier proteinâthioesterase domains from a fungal nonâreducing polyketide synthase
Production of 3-Oxo-2-(2'-pentenyl)-cyclopentane-1-octanoic Acid in the Fungus Aspergillus oryzae:A Step Towards Heterologous Production of Pyrethrins in Fungi
Cleaning the Cellular Factory:Deletion of McrA in Aspergillus oryzae NSAR1 and the generation of a novel kojic acid deficient strain for cleaner heterologous production of secondary metabolites
The use of filamentous fungi as cellular factories, where natural product pathways can be refactored and expressed in a host strain, continues to aid the field of natural product discovery. Much work has been done to develop host strains which are genetically tractable, and for which there are multiple selectable markers and controllable expression systems. To fully exploit these strains, it is beneficial to understand their natural metabolic capabilities, as such knowledge can rule out host metabolites from analysis of transgenic lines and highlight any potential interplay between endogenous and exogenous pathways. Additionally, once identified, the deletion of secondary metabolite pathways from host strains can simplify the detection and purification of heterologous compounds. To this end, secondary metabolite production in Aspergillus oryzae strain NSAR1 has been investigated via the deletion of the newly discovered negative regulator of secondary metabolism, mcrA (multicluster regulator A). In all ascomycetes previously studied mcrA deletion led to an increase in secondary metabolite production. Surprisingly, the only detectable phenotypic change in NSAR1 was a doubling in the yields of kojic acid, with no novel secondary metabolites produced. This supports the previous claim that secondary metabolite production has been repressed in A. oryzae and demonstrates that such repression is not McrA-mediated. Strain NSAR1 was then modified by employing CRISPR-Cas9 technology to disrupt the production of kojic acid, generating the novel strain NSARÎK, which combines the various beneficial traits of NSAR1 with a uniquely clean secondary metabolite background
Identification and manipulation of the pleuromutilin gene cluster from Clitopilus passeckerianus for increased rapid antibiotic production
Semi-synthetic derivatives of the tricyclic diterpene antibiotic pleuromutilin from the basidiomycete Clitopilus passeckerianus are important in combatting bacterial infections in human and veterinary medicine. These compounds belong to the only new class of antibiotics for human applications, with novel mode of action and lack of cross-resistance, representing a class with great potential. Basidiomycete fungi, being dikaryotic, are not generally amenable to strain improvement. We report identification of the seven-gene pleuromutilin gene cluster and verify that using various targeted approaches aimed at increasing antibiotic production in C. passeckerianus, no improvement in yield was achieved. The seven-gene pleuromutilin cluster was reconstructed within Aspergillus oryzae giving production of pleuromutilin in an ascomycete, with a significant increase (2106%) in production. This is the first gene cluster from a basidiomycete to be successfully expressed in an ascomycete, and paves the way for the exploitation of a metabolically rich but traditionally overlooked group of fungi
Heterologous expression of the avirulence gene ACE1 from the fungal rice pathogen Magnaporthe oryzae
The ACE1 and RAP1 genes from the avirulence signalling gene cluster of the rice blast fungus Magnaporthe oryzae were expressed in Aspergillus oryzae and M. oryzae itself. Expression of ACE1 alone produced a polyenyl pyrone (magnaporthepyrone), which is regioselectively epoxidised and hydrolysed to give different diols, 6 and 7, in the two host organisms. Analysis of the three introns present in ACE1 determined that A. oryzae does not process intron 2 correctly, while M. oryzae processes all introns correctly in both appressoria and mycelia. Co-expression of ACE1 and RAP1 in A. oryzae produced an amide 8 which is similar to the PKS-NRPS derived backbone of the cytochalasans. Biological testing on rice leaves showed that neither the diols 6 and 7, nor amide 8 was responsible for the observed ACE1 mediated avirulence, however, gene cluster analysis suggests that the true avirulence signalling compound may be a tyrosine-derived cytochalasan compound.Government of Egypt ScholarshipThe School of Chemistry at the University of Bristol and the Mark Evans ScholarshipKano State Government NigeriaMacArthur FoundationBayero UniversityNigerian Petroleum Technology FundMalaysian Govenment ScholarshipEP/F066104/
Global epidemiology of hyperthyroidism and hypothyroidism
Thyroid hormones are essential for growth, neuronal development, reproduction and regulation of energy metabolism. Hypothyroidism and hyperthyroidism are common conditions with potentially devastating health consequences that affect all populations worldwide. Iodine nutrition is a key determinant of thyroid disease risk; however, other factors, such as ageing, smoking status, genetic susceptibility, ethnicity, endocrine disruptors and the advent of novel therapeutics, including immune checkpoint inhibitors, also influence thyroid disease epidemiology. In the developed world, the prevalence of undiagnosed thyroid disease is likely falling owing to widespread thyroid function testing and relatively low thresholds for treatment initiation. However, continued vigilance against iodine deficiency remains essential in developed countries, particularly in Europe. In this report, we review the global incidence and prevalence of hyperthyroidism and hypothyroidism, highlighting geographical differences and the effect of environmental factors, such as iodine supplementation, on these data. We also highlight the pressing need for detailed epidemiological surveys of thyroid dysfunction and iodine status in developing countries
Isolation of a Î5-fatty acid desaturase gene from Mortierella alpina
Arachidonic acid (C20:4 Î5,8,11,14)) is a polyunsaturated fatty acid synthesized by the ?-fatty acid desaturation of di-homo-?-linolenic acid (C20:3 Î8,11,14)). In mammals, it is known to be a precursor of the prostaglandins and the leukotrienes but it is also accumulated by the filamentous fungus Mortierella alpina. We have isolated a cDNA encoding the Î5-fatty acid desaturase from M. alpina via a polymerase chain reaction- based strategy using primers designed to the conserved histidine box regions of microsomaL desaturases, and confirmed its function by expression in the yeast Saccharomyces cerevisiae. Analysis of the lipids from the transformed yeast demonstrated the accumulation of arachidonic acid. The M. alpina Î5- desaturase is the first example of a cloned Î5-desaturase, and differs from other fungal desaturases previously characterized by the presence of an N- terminal domain related to cytochrome b5
Assignment of a dubious gene cluster to melanin biosynthesis in the tomato fungal pathogen Cladosporium fulvum
Pigments and phytotoxins are crucial for the survival and spread of plant pathogenic fungi. The genome of the tomato biotrophic fungal pathogen Cladosporium fulvum contains a predicted gene cluster (CfPKS1, CfPRF1, CfRDT1 and CfTSF1) that is syntenic with the characterized elsinochrome toxin gene cluster in the citrus pathogen ElsinoĂ« fawcettii. However, a previous phylogenetic analysis suggested that CfPks1 might instead be involved in pigment production. Here, we report the characterization of the CfPKS1 gene cluster to resolve this ambiguity. Activation of the regulator CfTSF1 specifically induced the expression of CfPKS1 and CfRDT1, but not of CfPRF1. These co-regulated genes that define the CfPKS1 gene cluster are orthologous to genes involved in 1,3-dihydroxynaphthalene (DHN) melanin biosynthesis in other fungi. Heterologous expression of CfPKS1 in Aspergillus oryzae yielded 1,3,6,8-tetrahydroxynaphthalene, a typical precursor of DHN melanin. Îcfpks1 deletion mutants showed similar altered pigmentation to wild type treated with DHN melanin inhibitors. These mutants remained virulent on tomato, showing this gene cluster is not involved in pathogenicity. Altogether, our results showed that the CfPKS1 gene cluster is involved in the production of DHN melanin and suggests that elsinochrome production in E. fawcettii likely involves another gene cluster.</p
Auxin transport through non-hair cells sustains root-hair development.
The plant hormone auxin controls root epidermal cell development in a concentration-dependent manner. Root hairs are produced on a subset of epidermal cells as they increase in distance from the root tip. Auxin is required for their initiation and continued growth, but little is known about its distribution in this region of the root. Contrary to the expectation that hair cells might require active auxin influx to ensure auxin supply, we did not detect the auxin-influx transporter AUX1 in root-hair cells. A high level of AUX1 expression was detected in adjacent non-hair cell files. Non-hair cells were necessary to achieve wild-type root-hair length, although an auxin response was not required in these cells. Three-dimensional modelling of auxin flow in the root tip suggests that AUX1-dependent transport through non-hair cells maintains an auxin supply to developing hair cells as they increase in distance from the root tip, and sustains root-hair outgrowth. Experimental data support the hypothesis that instead of moving uniformly though the epidermal cell layer, auxin is mainly transported through canals that extend longitudinally into the tissue
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