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The role of coproporphyrinogen III oxidase and ferrochelatase genes in heme biosynthesis and regulation in Aspergillus niger

  • Genomics, transcriptomics, proteomics
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Abstract

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.

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Acknowledgments

The authors thank Ellen Lagendijk, Nina Madl, Petra Höfler, Sanne Westhoff, and Janneke Teunissen for technical assistance. This work is funded by the Sixth Framework Program (FP6-2004-NMP-NI-4): “White Biotechnology for added value products from renewable plant polymers: Design of tailor-made biocatalysts and new Industrial bioprocesses” (Biorenew), contract no.: 026456, and by the Austrian Science Fund (FWF): P22406-B18.

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  1. Sandra de Weert

    Present address: Koppert Biological Systems, Veilingweg14, 2650 AD, Berkel en Rodenrijs, The Netherlands

Authors and Affiliations

  1. Institute of Biology Leiden, Molecular Microbiology and Biotechnology, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands

    Angelique C. W. Franken, Cees A. M. J. J. van den Hondel, Arthur F. J. Ram, Sandra de Weert & Peter J. Punt

  2. Biological Chemistry/Biocenter, Innsbruck Medical University, Fritz-Pregl-Str. 3, 6020, Innsbruck, Austria

    Ernst R. Werner

  3. Division of Molecular Biology/Biocenter, Innsbruck Medical University, Innrain 80-82, 6020, Innsbruck, Austria

    Hubertus Haas

  4. HAN Biocentre, Laan van Scheut 2, 6525 EM, Nijmegen, The Netherlands

    B. Christien Lokman & Cees A. M. J. J. van den Hondel

  5. Kluyver Centre for Genomics of Industrial Fermentation, P.O. Box 5057, 2600 GA, Delft, The Netherlands

    Cees A. M. J. J. van den Hondel & Arthur F. J. Ram

  6. TNO Microbiology & Systems Biology, Utrechtseweg 48, 3704 HE, Zeist, The Netherlands

    Peter J. Punt

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  1. Angelique C. W. Franken
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Correspondence to Peter J. Punt.

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Franken, A.C.W., Werner, E.R., Haas, H. et al. The role of coproporphyrinogen III oxidase and ferrochelatase genes in heme biosynthesis and regulation in Aspergillus niger . Appl Microbiol Biotechnol 97, 9773–9785 (2013). https://doi.org/10.1007/s00253-013-5274-2

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  • DOI: https://doi.org/10.1007/s00253-013-5274-2

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