Browsing by Author "Punt, P.J."

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The role of Coproporphyrinogen III oxidase and Ferrochelatase genes in heme biosynthesis and regulation in Aspergillus niger
Hondel, C.A. van den ; Punt, P.J. ; Haas, H. ; Ram, Arthur F.J. ; Werner, M. ; Weert, S. de ; Franken, A.C. ; Lokman, B.C.
2013, Article / Letter to editor (Applied Microbiology and Biotechnology, vol. 97, iss. 22, (2013), pp. 9773-9785)Heme is a suggested limiting factor in peroxidase production by Aspergillus spp., which are well-known suitable hosts for heterologous protein production. In this study, the role of genes coding for coproporphyrinogen III oxidase (hemF) and ferrochelatase (hemH) was analyzed by means of deletion and overexpression to obtain more insight in fungal heme biosynthesis and regulation. These enzymes represent steps in the heme biosynthetic pathway downstream of the siroheme branch and are suggested to play a role in regulation of the pathway. Based on genome mining, both enzymes deviate in cellular localization and protein domain structure from their Saccharomyces cerevisiae counterparts. The lethal phenotype of deletion of hemF or hemH could be remediated by heme supplementation confirming that Aspergillus niger is capable of hemin uptake. Nevertheless, both gene deletion mutants showed an extremely impaired growth even with hemin supplementation which could be slightly improved by media modifications and the use of hemoglobin as heme source. The hyphae of the mutant strains displayed pinkish coloration and red autofluorescence under UV indicative of cellular porphyrin accumulation. HPLC analysis confirmed accumulation of specific porphyrins, thereby confirming the function of the two proteins in heme biosynthesis. Overexpression of hemH, but not hemF or the aminolevulinic acid synthase encoding hemA, modestly increased the cellular heme content, which was apparently insufficient to increase activity of endogenous peroxidase and cytochrome P450 enzyme activities. Overexpression of all three genes increased the cellular accumulation of porphyrin intermediates suggesting regulatory mechanisms operating in the final steps of the fungal heme biosynthesis pathway.
Analysis of the role of the A. niger aminolevulinic acid synthase (hemA) gene illustrates the difference between regulation of yeast and fungal heme and siroheme dependent pathways
Ram, Arthur F.J. ; Hondel, C.A. van den ; Lokman, B.C. ; Punt, P.J. ; Weert, S. de ; Franken, A.C.
2012, Article / Letter to editor (FEMS Microbiol Letters, vol. 335, iss. 2, (2012), pp. 104-112)To increase knowledge on haem biosynthesis in filamentous fungi like Aspergillus niger, pathway-specific gene expression in response to haem and haem intermediates was analysed. This analysis showed that iron, 5-aminolevulinic acid (ALA) and possibly haem control haem biosynthesis mostly via modulating expression of hemA [coding for 5-aminolevulinic acid synthase (ALAS)]. A hemA deletion mutant (?hemA) was constructed, which showed conditional lethality. Growth of ?hemA was supported on standard nitrate-containing media with ALA, but not by hemin. Growth of ?hemA could be sustained in the presence of hemin in combination with ammonium instead of nitrate as N-source. Our results suggest that a branch-off within the haem biosynthesis pathway required for sirohaem synthesis is responsible for lack of growth of ?hemA in media containing nitrate as sole N-source, because of the requirement of sirohaem for nitrate assimilation, as a cofactor of nitrite reductase. In contrast to the situation in Saccharomyces cerevisiae, cysteine, but not methionine, was found to further improve growth of ?hemA. These results demonstrate that A. niger can use exogenous hemin for its cellular processes. They also illustrate important differences in regulation of haem biosynthesis and in the role of haem and sirohaem in A. niger compared to S. cerevisiae.
Heme biosynthesis and its regulation : Toward understanding and improvement of heme biosynthesis in filamentous fungi.
Weert, S. de ; Punt, P.J. ; Lokman, B.C. ; Hondel, C.A. van den ; Franken, A.C. ; Ram, Arthur F.J. ; Ram, A.F.
2011, Article / Letter to editor (Applied Microbiology and Biotechnology, vol. 91, iss. 3, (2011), pp. 447-460)Heme biosynthesis in fungal host strains has acquired considerable interest in relation to the production of secreted heme-containing peroxidases. Class II peroxidase enzymes have been suggested as eco-friendly replacements of polluting chemical processes in industry. These peroxidases are naturally produced in small amounts by basidiomycetes. Filamentous fungi like Aspergillus sp. are considered as suitable hosts for protein production due to their high capacity of protein secretion. For the purpose of peroxidase production, heme is considered a putative limiting factor. However, heme addition is not appropriate in large-scale production processes due to its high hydrophobicity and cost price. The preferred situation in order to overcome the limiting effect of heme would be to increase intracellular heme levels. This requires a thorough insight into the biosynthetic pathway and its regulation. In this review, the heme biosynthetic pathway is discussed with regards to synthesis, regulation, and transport. Although the heme biosynthetic pathway is a highly conserved and tightly regulated pathway, the mode of regulation does not appear to be conserved among eukaryotes. However, common factors like feedback inhibition and regulation by heme, iron, and oxygen appear to be involved in regulation of the heme biosynthesis pathway in most organisms. Therefore, they are the initial targets to be investigated in Aspergillus niger.

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