Nutritional requirements and strain heterogeneity in Ashbya gossypii

Colony radial growth rates and specific growth rates of three related Ashbya gossypii strains ATCC10895, IMI31268, MUCL29450 and an unrelated strain, CBS109.26, were measured on various carbon and nitrogen sources at pH 4.5 and pH 6.5 to elucidate physiological growth requirements and strain differences. All strains grew on yeast extract or ammonium as nitrogen sources, but not on nitrate. Substantial growth at pH 4.5 was observed only on complex medium. D-Glucose, glycerol and starch were utilised as carbon sources. Ethanol was produced during growth on glycerol. Conversion of xylose into xylitol demonstrates that the xylose reductase is active. Phenotypic differences between related strains were greater than expected. We demonstrate that A. gossypii utilizes ammonium as sole nitrogen source at pH 6.5, facilitating further physiological studies using chemically defined media in the future.The financial support of Fundacao para a Ciencia e a Tecnologia (FCT), Portugal, is acknowledged, project AshByofactory PTDC/EBB-EBI/101985/2008 and grant SFRH/BD/30229/2006 to O. Ribeiro

The peroxin PEX14 of Neurospora crassa is essential for the biogenesis of both glyoxysomes and Woronin bodies

In the filamentous fungus Neurospora crassa, glyoxysomes and Woronin bodies coexist in the same cell. Because several glyoxysomal matrix proteins and also HEX1, the dominant protein of Woronin bodies, possess typical peroxisomal targeting signals, the question arises as to how protein targeting to these distinct yet related types of microbodies is achieved. Here we analyzed the function of the Neurospora ortholog of PEX14, an essential component of the peroxisomal import machinery. PEX14 interacted with both targeting signal receptors and was localized to glyoxysomes but was virtually absent from Woronin bodies. Nonetheless, a pex14 Delta mutant not only failed to grow on fatty acids because of a defect in glyoxysomal beta-oxidation but also suffered from cytoplasmic bleeding, indicative of a defect in Woronin body-dependent septal pore plugging. Inspection of pex14 Delta mutant hyphae by fluorescence and electron microscopy indeed revealed the absence of Woronin bodies. When these cells were subjected to subcellular fractionation, HEX1 was completely mislocalized to the cytosol. Expression of GFP-HEX1 in wild-type mycelia caused the staining of Woronin bodies and also of glyoxysomes in a targeting signal-dependent manner. Our data support the view that Woronin bodies emerge from glyoxysomes through import of HEX1 and subsequent fission

Die peroxisomale Isocitrat-Dyhydrogenase aus Ashbya gossypii - ihre Bedeutung für Wachstum und Riboflavinbildung

The filamentous fungus Ashbya gossypii is used for industrial vitamin B2 (riboflavin) production. Because of their yield-enhancing effect plant oils are the preferred substrates for the biotechnical process. Besides the isocitrate lyase (ICL) as the key enzyme of the glyoxylate cycle, essential for the degradation of fatty acids, an NADP-specific isocitrate dehydrogenase (ICDH) is localized in peroxisomes of the fungus. The present work addresses the function of this ICDH in the metabolism of A . gossypii concerning riboflavin formation and growth. The AgIDP3 gene was shown to encode an NADP-specific ICDH by heterologous complementation of a Saccharomyces cerevisiae strain with dysfunctional mitochondrial ICDH isoenzymes . Disruption of the AgIDP3 gene in A. gossypii resulted in the loss of about 90 % of enzyme activity in crude extracts . Using both digitonin-mediated protoplast permeabilization and percoll gradient centrifugation it was demonstrated, firstly, that AgIDP3 coded for a peroxisomal ICDH from A. gossypii. Secondly, the remaining activity was assigned to a mitochondrial NADP-specific isoenzyme. Under all tested conditions no cytosolic NADP-specific ICDH activity was detectable. The investigation of the AgIDP3 mRNA amounts in comparison to that of the ICL-encoding AgICL1 gene of A . gossypii by means of Nothern blot analysis revealed a regulation of the peroxisomal ICDH and ICL at the transcriptional level . In contrast to the AgICL1 gene the glucose repression of the AgIDP3 gene was less stringent and the carbon sources ethanol and acetate only had the same derepressing effect as glycerol . The strong induction of gene expression during cultivation on soybean oil indicated a function of the peroxisomal ICDH within the fat metabolism. In fact, when growing on polyunsaturated fatty acids AgIDP3 knockout mutants exhibited lower growth raten compared to the wild type . Additionally, auxiliary enzymes needed for the degradation of unsaturated fatty acids, like the 2342-enoyl-CoA isomerase and the NADPH-oxidizing 2,4-dienoyl-CoA reductase, were localized in peroxisomes of A. gossypii by percoll gradient centrifugation. So that as in S. cerevisiae the peroxisomal ICDH of A . gossypii serves as a regenerator of peroxisomal reduction equivalents. As the substrate of the isocitrate-converting enzymes in peroxisomes isocitrate has to be shuttled into this compartment, since citrate synthase activity was solely found in mitochondria of A. gossypii. Activities of the a-ketoglutarate-converting NAD-specific glutamate dehydrogenase and the a-ketoglutarate dehydrogenase complex were strictly localized in the cytosol and mitochondria, respectively . Thus an involvement of the peroxisomal ICDH of A . gossypii within the glutamate biosynthesis seems unlikely. Interestingly, the function of the NADP-specific ICDH in peroxisomes of A. .gossypii is not restricted to the regeneration of NADPH for the degradation of fatty acids . Moreover, disruption of the AgIDP3 gene resulted in a decrease of riboflavin formation of about 44 % and 75 % compared to the wild type during growth on glucose and soybean oil, respectively . On the other hand, the overexpression of the AgIDP3 gene under control of the constitutive TEF promotor and terminator from A . gossypii led to more than threefold higher NADP-specific ICDH activity on glucose as well as on soybean oil. Since this overexpression resulted in higher riboflavin formation of about 10 % and 66 % on these two carbon sources, respectively, riboflavin formation seems to be limited by the availability of reduction equivalents . Whether NADPH is used directly by one of the six enzymes specific for riboflavin formation, e .g. the NADPHspecific reductase which reduces a ribosyl-pyrimidin intermediate, or by any of the more than 100 enzymes indirectly involved in riboflavin synthesis, needs to be further investigate

Natural Fiber-Stabilized Geopolymer Foams—A Review

The development of sustainable, environmentally friendly insulation materials with a reduced carbon footprint is attracting increased interest. One alternative to conventional insulation materials are foamed geopolymers. Similar to foamed concrete, the mechanical properties of geopolymer foams can also be improved by using fibers for reinforcement. This paper presents an overview of the latest research findings in the field of fiber-reinforced geopolymer foam concrete with special focus on natural fibers reinforcement. Furthermore, some basic and background information of natural fibers and geopolymer foams are reported. In most of the research, foams are produced either through chemical foaming with hydrogen peroxide or aluminum powder, or through mechanical foaming which includes a foaming agent. However, previous reviews have not sufficiently addresses the fabrication of geopolymer foams by syntactic foams. Finally, recent efforts to reduce the fiber degradation in geopolymer concrete are discussed along with challenges for natural fiber reinforced-geopolymer foam concrete

Die peroxisomale Isocitrat-Dehydrogenase aus Ashbya gossypii - ihre Bedeutung fuer Wachstum und Riboflavinbildung

The filamentous fungus Ashbya gossypii is used for industrial vitamin B_2 (riboflavin) production. Because of their yield-enhancing effect plant oils are the preferred substrates for the biotechnical process. Besides the isocitrate lyase (ICL) as the key enzyme of the glyoxylate cycle, essential for the degradation of fatty acids, an NADP-specific isocitrate dehydrogenase (ICDH) is localized in preoxisomes of the fungus. The present work addresses the function of this ICDH in the metabolism of A. gossypii concerning riboflavin formation and growth. The AgIDP3 gene was shown to encode an NADP-specific ICDH by heterologous complementation of a Saccharomyces cerevisiae strain with dysfunctional mitochondrial ICDH isoenzymes. Disruption of the AgIDP3 gene in A. gossypii resulted in the loss of about 90% of enzyme activity in crude extracts. Using both digitonin-mediated protoplast permeabilization and percoll gradient centrifugation it was demonstrated, firstly, that AgIDP3 coded for a peroxisomal ICDH from A. gossypii. Secondly, the remaining activity was assigned to a mitochondrial NADP-specific isoenzyme. Under all tested conditions no cytosolic NADP-specific ICDH activity was detectable. (orig.)Available from TIB Hannover: RA 831(3724) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman

Isocitrate iyase of Ashbya gossypii : transcriptional regulation and peroxisomal localization

AbstractThe isocitrate lyase-encoding gene AgICL1 from the filamentous hemiascomycete Ashbya gossypii was isolated by heterologous complementation of a Saccharomyces cerevisiae icl1d mutant. The open reading frame of 1680 bp encoded a protein of 560 amino acids with a calculated molecular weight of 62 584. Disruption of the AgICL1 gene led to complete loss of AgIcl1p activity and inability to grow on oleic acid as sole carbon source. Compartmentation of AgIcl1p in peroxisomes was demonstrated both by Percoll density gradient centrifugation and by immunogold labeling of ultrathin sections using specific antibodies. This fitted with the peroxisomal targeting signal AKL predicted from the C-terminal DNA sequence. Northern blot analysis with mycelium grown on different carbon sources as well as AgICL1 promoter replacement with the constitutive AgTEF promoter revealed a regulation at the transcriptional level. AgICL1 was subject to glucose repression, derepressed by glycerol, partially induced by the C2 compounds ethanol and acetate, and fully induced by soybean oil