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

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

Importance of malate synthase in the glyoxylate cycle of Ashbya gossypii for the efficient production of riboflavin

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