Phylogenetic Analyses Reveal Monophyletic Origin of the Ergot Alkaloid Gene dmaW in Fungi

Ergot alkaloids are indole-derived mycotoxins that are important in agriculture and medicine. Ergot alkaloids are produced by a few representatives of two distantly related fungal lineages, the Clavicipitaceae and the Trichocomaceae. Comparison of the ergot alkaloid gene clusters from these two lineages revealed differences in the relative positions and orientations of several genes. The question arose: is ergot alkaloid biosynthetic capability from a common origin? We used a molecular phylogenetic approach to gain insights into the evolution of ergot alkaloid biosynthesis. The 4-γ,γ-dimethylallyltryptophan synthase gene, dmaW, encodes the first step in the pathway. Amino acid sequences deduced from dmaW and homologs were submitted to phylogenetic analysis, and the results indicated that dmaW of Aspergillus fumigatus (mitosporic Trichocomaceae) has the same origin as corresponding genes from clavicipitaceous fungi. Relationships of authentic dmaW genes suggest that they originated from multiple gene duplications with subsequent losses of original or duplicate versions in some lineages

Phylogenetic Analyses Reveal Monophyletic Origin of the Ergot Alkaloid Gene \u3cem\u3edmaW\u3c/em\u3e in Fungi

Ergot alkaloids are indole-derived mycotoxins that are important in agriculture and medicine. Ergot alkaloids are produced by a few representatives of two distantly related fungal lineages, the Clavicipitaceae and the Trichocomaceae. Comparison of the ergot alkaloid gene clusters from these two lineages revealed differences in the relative positions and orientations of several genes. The question arose: is ergot alkaloid biosynthetic capability from a common origin? We used a molecular phylogenetic approach to gain insights into the evolution of ergot alkaloid biosynthesis. The 4-γ,γ-dimethylallyltryptophan synthase gene, dmaW, encodes the first step in the pathway. Amino acid sequences deduced from dmaW and homologs were submitted to phylogenetic analysis, and the results indicated that dmaW of Aspergillus fumigatus (mitosporic Trichocomaceae) has the same origin as corresponding genes from clavicipitaceous fungi. Relationships of authentic dmaW genes suggest that they originated from multiple gene duplications with subsequent losses of original or duplicate versions in some lineages

The North American toxic fungal pathogen G3 Claviceps purpurea (Fries) Tulasne is established in the German Wadden Sea.

Three lineages (G1, G2 and G3) within the toxic invasive ergot fungus Claviceps purpurea (Fries) Tulasne are known; these should be recognized as unique species, or at least as varieties. On 2 November 2011, a very dense, well established population of G3 C. purpurea was found on the common cord-grass Spartina anglica C.E. Hubbard at two localities on the German North Sea coast in the Wadden Sea (Caciliengroden and Hooksiel). It is most likely that G3 C. purpurea has a North American origin and entered German coastal waters by floating sclerotia from Irish, British, or Benelux waters, where it was previously found. However, introduction via ships’ ballast water coming from their native or introduced ranges is also plausible. Furthermore imports of G3 sclerotia via seed mussels collected from wild subtidal banks in Irish, British and Dutch coastal waters and released into the German Wadden Sea can currently not be excluded. Risks from this highly toxic fungus for human, grazing animals and the marine environment have been identified but not yet quantified in terms of impact. A suitable monitoring programme should be implemented to detect any unwanted impacts caused by G3 C. purpurea in German salt marshes at an early phase

Doctor of Philosophy

dissertationFungal polyketides are a complex class of natural products with diverse scaffolds. The biosynthesis of these molecules involves the iterative condensation of acetate units using a minimal set of domains on a single polypeptide. The different levels of reduction at each iterative step generates the structural diversity observed via the stuttering action of these domains. For some polyketides, further structural complexity is introduced via amidation by a nonribosomal peptide synthetase (NRPS) module fused to the polyketide synthase (PKS). The NRPS contains a condensation domain (C) that catalyzes the coupling of the polyketide to a specific amino acid. Studies by others have suggested that PKS function is independent of NRPS activity and thus, this system is amenable to combinatorial biosynthesis to generate analogues with different polyketide chains and amino acids by performing module swaps. Forging intermodular interactions and understanding C domain selectivity for substrates is key to successful engineering of these analogues. Herein, I investigate the impact of these factors on the ability to synthesize unnatural products by this route. Studying these components required the overexpression of chimeric gene constructs, which was anticipated to result in low compound yields. Therefore, a novel platform to express fungal genes with yields exceeding 1 g per kg media was developed and validated through the characterization of a silent pathway. Application to the PKS-NRPS combinatorial biosynthesis problem led to the discovery that C domains are highly selective for closely related substrates in the presence of favorable PKS/NRPS interactions

BIODIESEL PRODUCTION FROM PALM OIL MILL EFFLUENT BY USING THERMOMYCES LANUGINOSUS LIPASE AS A CATALYST

Biodiesel production via enzymatic transesterification using Palm Oil Mill Effluent (POME) as the raw material and Thermomyces lanuginosus lipase (TLL) as the catalyst is proposed in this study. POME is one of the waste water discharged from the sterilization process, crude oil clarification process and cracked mixture separation process of palm oil in mills. POME contains a high amount of organic matter, oil and grease, total solids and suspended solids. POME could be an alternative raw material for producing biodiesel because of its high oil and fat content that could be converted into biodiesel. High Free Fatty Acid (FFA) makes the conversion seems impossible because if using chemical transesterification, it will form saponification. Enzymatic transesterification is the solution for this problem. In this study, the conditions for biodiesel production included lipase content of 0.3%, a methanol/oil molar ratio of 6:1, a water content of 4.7%, a stirring speed of 500 rpm, a reaction temperature of 30 °C and a reaction time of 24 hour. The biodiesel yield was achieved at 81.87%%

Xylanase from Fusarium heterosporum: Properties and influence of thiol compounds on xylanase activity

The properties of xylanase purified from Fusarium heterosporum that was grown in barley-brewing residue under solid-state fermentation and the effects of thiol compounds on the reactivation of the metal ion-inhibited xylanase were investigated. Xylanase was purified to homogeneity by ion exchange chromatography, and its molecular mass was estimated to be 19.5 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH for the xylanase was 5.0, and it was stable in acidic pH (4.5 to 5.5), where it retained more than 87% of its activity after 24 h. The optimum temperature was 50°C, and it had a half-life of 53 min at 45°C. The apparent Km and Vmax values for the xylanase were 5.63 mg/ml and 800 μmol/mg/min, respectively. Ba2+, Ca2+, Mg2+ and the thiol compounds β-mercaptoethanol and dithiothreitol (DTT) enhanced xylanase activity, while Hg2+, Pb2+ and Zn2+ strongly inhibited enzyme activity. Furthermore, this xylanase had an alternative mode of regulation in the presence of thiol compounds because the enzyme was able to recover its catalytic activity after inhibition by heavy metal ions.Keywords: Hemicellulase, fungus, solid-state fermentation, barley brewing residue, thiol compoundsAfrican Journal of Biotechnology, Vol. 13(9), pp. 1047-1055, 26 February, 201

TINJAUAN, D-ASAM AMINO OKSIDASE DARI MIKROBA: PRODUKSI DAN APLIKASI

D-amino acid oxidase (DAAO) is a flavin adenine dinucleotide-containing enzyme that catalyzes the oxidative deamination of amino acid D-isomers with high stereospecificity, which results in α-keto acids, ammonia and hydrogen peroxide. Having high stereospecificity, DAAO is used in a variety of applications such as drug, biocatalyst, biosensor and preparation of transgenic plants. DAAO is widespread in nature, found in microorganisms to mammals. Microbial DAAO is considered more important than mammalian DAAO for biotechnology application. DAAO production in submerged fermentation is influenced by several factors, such as carbon source, nitrogen source, inducer, dissolve oxygen, temperature and pH. The influence of those factors on DAAO production by microbial origin, DAAO production by microbial recombinant, and its application in biotechnology are discussed in this review.Keywords: Enzyme, DAAO, D-amino acid, production, application ABSTRAKEnzim D-asam amino oksidase (DAAO) merupakan enzim yang mengandung Flavin Adenine Dinucleotide yang bekerja mengkatalisis reaksi oksidasi deaminasi D-asam amino dengan stereospesifisitas yang tinggi menghasilkan α-asam keto, amonia dan hidrogen peroksida. Karena mempunyai karakteristik sreteospesifisitas yang tinggi, enzim DAAO banyak digunakan untuk berbagai aplikasi seperti obat, biokatalis, biosensor dan penyiapan tanaman transgenik. Enzim ini dapat dihasilkan oleh organisme mulai dari bakteri hingga mamalia, namun untuk aplikasi dibidang bioteknologi, enzim DAAO yang berasal dari mikroorganisme dipandang lebih penting dari pada yang berasal dari mamalia. Produksi enzim dari DAAO dari mikroorganisme dalam kultur cair dipengaruhi oleh beberapa faktor seperti sumber karbon, nitrogen, senyawa penginduksi, oksigen terlarut, temperatur dan pH medium. Pengaruh dari faktor-faktor tersebut terhadap produksi enzim DAAO, produksi enzim DAAO menggunakan mikroba rekombinan serta aplikasinya dalam bidang bioteknologi dibahas dalam tinjauan.Kata Kunci: Enzim, DAAO, D-asam amino, produksi, aplikas

Drying techniques for the visualisation of agarose-based chromatography media by scanning electron microscopy

The drying of chromatography resins prior to scanning electron microscopy is critical to image resolution and hence understanding of the bead structure at sub-micron level. Achieving suitable drying conditions is especially important with agarose-based chromatography resins, as over-drying may cause artefact formation, bead damage and alterations to ultrastructural properties; and under-drying does not provide sufficient resolution for visualisation under SEM. This paper compares and contrasts the effects of two drying techniques, critical point drying and freeze drying, on the morphology of two agarose based resins (MabSelect(TM) : dw ~ 85 µm and Capto(TM) Adhere: dw ~75 µm) and provides a complete method for both. The results show that critical point drying provides better drying and subsequently clearer ultrastructural visualisation of both resins under SEM. Under this protocol both the polymer fibres (thickness ~20 nm) and the pore sizes (diameter ~100 nm) are clearly visible. Freeze drying is shown to cause bead damage to both resins, but to different extents. MabSelect resin encounters extensive bead fragmentation, whilst Capto Adhere resin undergoes partial bead disintegration, corresponding with the greater extent of agarose crosslinking and strength of this resin. While freeze drying appears to be the less favourable option for ultrastructural visualisation of chromatography resin, it should be noted that the extent of fracturing caused by the freeze drying process may provide some insight into the mechanical properties of agarose-based chromatography media

Ergot of nut sedge in South Africa

Please read the abstract in the section 00front of this documentThesis (PhD)--University of Pretoria, 2008.Microbiology and Plant Pathologyunrestricte