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

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.

Heme biosynthesis and its regulation: towards understanding and improvement of heme biosynthesis in filamentous fungi.

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.

Production of bifunctional proteins by Aspergillus awamori: llama variable heavy chain antibody fragment (V(HH)) R9 coupled to Arthromyces ramosus peroxidase (ARP).

The Arthromyces ramosus peroxidase gene (arp) was genetically fused to either the 5'- or 3'-terminal ends of the gene encoding llama variable heavy chain antibody fragment V(HH) R9, resulting in the fusion expression cassettes ARP-R9 or R9-ARP. Aspergillus awamori transformants were obtained which produced up to 30 mgl(-1) fusion protein in the culture medium. Both fusion proteins showed peroxidase activity in an ABTS activity test. Considerable amounts of fusion protein were detected intracellularly, suggesting that the fungus encounters problems in secreting these kind of proteins. ELISA experiments showed that ARP-R9 was less able to bind its antigen, the azo-dye RR6, as compared to R9-ARP. Furthermore, in contrast to R9-ARP, ARP-R9 bound to RR6 did not show peroxidase activity anymore. These results indicate that fusion of ARP to the C-terminus of the antibody fragment V(HH) R9 (R9-ARP) is the preferred orientation.

Efficient production of Arthromyces ramosus peroxidase by Aspergillus awamori.

The heterologous production of Arthromyces ramosus peroxidase (ARP) was analysed in the filamentous fungus Aspergillus awamori under control of the inducible endoxylanase promoter. Secretion of active ARP was achieved up to 800 mg l(-1) in shake flask cultures. Western blot analysis showed that an rARP product of the correct molecular weight was produced. In contrast to several other studies about heterologous production of heme containing peroxidases, our results suggest that in A. awamori no heme limitation exists during overproduction of ARP.

Genome mining and functional genomics for siderophore production in Aspergillus niger.

Iron is an essential metal for many organisms, but the biologically relevant form of iron is scarce because of rapid oxidation resulting in low solubility. Simultaneously, excessive accumulation of iron is toxic. Consequently, iron uptake is a highly controlled process. In most fungal species, siderophores play a central role in iron handling. Siderophores are small iron-specific chelators that can be secreted to scavenge environmental iron or bind intracellular iron with high affinity. A second high-affinity iron uptake mechanism is reductive iron assimilation (RIA). As shown in Aspergillus fumigatus and Aspergillus nidulans, synthesis of siderophores in Aspergilli is predominantly under control of the transcription factors SreA and HapX, which are connected by a negative transcriptional feedback loop. Abolishing this fine-tuned regulation corroborates iron homeostasis, including heme biosynthesis, which could be biotechnologically of interest, e.g. the heterologous production of heme-dependent peroxidases. Aspergillus niger genome inspection identified orthologues of several genes relevant for RIA and siderophore metabolism, as well as sreA and hapX. Interestingly, genes related to synthesis of the common fungal extracellular siderophore triacetylfusarinine C were absent. Reverse-phase high-performance liquid chromatography (HPLC) confirmed the absence of triacetylfusarinine C, and demonstrated that the major secreted siderophores of A. niger are coprogen B and ferrichrome, which is also the dominant intracellular siderophore. In A. niger wild type grown under iron-replete conditions, the expression of genes involved in coprogen biosynthesis and RIA was low in the exponential growth phase but significantly induced during ascospore germination. Deletion of sreA in A. niger resulted in elevated iron uptake and increased cellular ferrichrome accumulation. Increased sensitivity toward phleomycin and high iron concentration reflected the toxic effects of excessive iron uptake. Moreover, SreA-deficiency resulted in increased accumulation of heme intermediates, but no significant increase in heme content. Together with the upregulation of several heme biosynthesis genes, these results reveal a complex heme regulatory mechanism.

Analysis of the role of the Aspergillus niger aminolevulinic acid synthase (hemA) gene illustrates the difference between regulation of yeast and fungal haem- and sirohaem-dependent pathways.

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 (ΔhemA) was constructed, which showed conditional lethality. Growth of ΔhemA was supported on standard nitrate-containing media with ALA, but not by hemin. Growth of ΔhemA 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 ΔhemA 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 ΔhemA. 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.

Expression and production of llama variable heavy-chain antibody fragments (V(HH)s) by Aspergillus awamori.

We report the expression and production of llama variable heavy-chain antibody fragments (V(HH)s) by Aspergillus awamori. Fragments encoding V(HH)s were cloned in a suitable Aspergillus expression vector and transformants secreting V(HH) fragments were analysed for integrated gene copy-numbers, mRNA levels and protein production. Functional V(HH)s were detected in the culture medium, indicating the feasibility of producing this type of protein in a fungal expression system. Secreted V(HH)s were subjected to (extracellular) degradation, which could be partially prevented by the addition of BSA to the culture medium.