Supplementary Table 1: BlastP results of reported proteins involved in synthesis of heme against Aspergillus niger CBS 513. 88 ( taxid: 425011). Not present,.no significant blast and no bidirectional match.
Aromatic amino acid biosynthesis in S. Cerevisiae is controlled by a combination of feedback inhibition, activation of enzyme activity, and regulation of enzyme synthesis (CITS:Jones1943992). The carbon flow through the pathways is regulated primarily at the initial step and the branching points by the terminal end-products.
Mito mitochondria, Cyto cytoplasm, Nucl nucleus, O any other location, mTP mitochondrial targeting peptide, SP signal peptide, Loc prediction of localization, RC reliability class, Tplen predicted presequence length. Prediction tools used: WoLF PSORT, MitoProt II, and TargetP (available on and ). Supplementary Table 2: BlastP results of reported proteins involved in regulation of heme against Aspergillus niger CBS 513. 88 ( taxid: 425011).
Not present,.no significant blast and no bidirectional match. Mito mitochondria, Cyto cytoplasm, Nucl nucleus, O any other location, mTP mitochondrial targeting peptide, SP signal peptide, Loc prediction of localization, RC reliability class, Tplen predicted presequence length. Prediction tools used: WoLF PSORT, MitoProt II, and TargetP (available on and ). Supplementary Table 3: BlastP results of reported proteins involved in transport of heme and intermediates against Aspergillus niger CBS 513.
88 ( taxid: 425011). Not present,.no significant blast and no bidirectional match. Mito mitochondria, Cyto cytoplasm, Nucl nucleus, O any other location, mTP mitochondrial targeting peptide, SP signal peptide, Loc prediction of localization, RC reliability class, Tplen predicted presequence length. Prediction tools used: WoLF PSORT, MitoProt II, and TargetP (available on and ). 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.
![Biosynthesis Biosynthesis](/uploads/1/2/5/4/125409299/213805093.png)
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. IntroductionThe production of enzymes by microorganisms as eco-friendly replacements for chemical and polluting industrial processes has gained increasing attention over the years.
The use of enzymes produced by microorganisms as filamentous fungi like Aspergillus sp. Are considered as preferred hosts due to their high capacity for producing secreted proteins. The production of heterologous proteins, however, is often relatively low compared with the amount of homologous protein produced (Punt et al. Several mechanisms are responsible for this low level of protein production which, among others, includes the unfolded protein response (UPR) and ER-associated degradation (ERAD) (Guillemette et al. ), protease activity (Braaksma and Punt ), and limitations at the level of cofactor incorporation (Punt et al.
).Cofactor availability and incorporation has been shown to be a limiting factor in the production of fungal peroxidases which require heme as a cofactor. Peroxidase production can be increased by the supplementation of hemoglobin or hemin to the medium (Conesa et al.; Elrod et al. ); however, the mechanism behind heme uptake is poorly understood and the approach is too costly to be suited for industrial purposes (Elrod et al.
).The preferred situation in order to overcome the limiting effect of heme would be the increase of intracellular heme levels. In animals, fungi, and in prokaryotes belonging to the α-proteobacteria (Ferreira et al.; Panek and O'Brian ), heme is synthesized by eight enzymatic steps (Fig.; Table ), starting with condensation of glycine and succinyl CoA to form 5-aminolevulinic acid (ALA). In the eukaryotes mentioned above, the reaction is mediated by 5-aminolevulinic acid synthase (ALAS) and takes place in the mitochondria. ALA is then transported to the cytosol where it is condensed to porphobilinogen (PBG) by ALA dehydratase (ALAD). Four molecules of PBG are subsequently used to form the unstable hydroxymethylbilane (HMB) by PBG deaminase (PBGD) followed by the cyclization to uroporphyrinogen III (UroIII) by uroporphyrinogen III synthase (UROS). Uroporphyrinogen III is the final common intermediate of heme and siroheme synthesis, siroheme being a cofactor for sulfite and nitrite reductases (Raux et al.
The synthesis of heme continues by decarboxylation of all four acetic side chains to methyl groups by uroporphyrinogen III decarboxylase (UROD) to form coproporphyrinogen III (Copro). With the exception of Saccharomyces cerevisiae, Copro is subsequently transported back to the mitochondria. The synthesis is continued by coproporphyrinogen III oxidase (CPO) with the formation of protoporphyrinogen IX (PP’genIX). Next, protoporphyrinogen oxidase (PPO) mediates a six-electron oxidation forming protoporphyrin IX (PPIX). The final product, heme, is formed by ferrochelatase (FC), which mediates the insertion of ferrous iron in PPIX (Elrod et al.; Moretti et al.
Structural features of heme biosynthesis enzymes (Layer et al. ) and iron utilization and regulation in fungi (Haas et al. ) have recently been reviewed extensively and will only be discussed briefly hereafter. Chemical heme biosynthesis pathway. Biosynthesis is initiated in mitochondria with the condensation of glycine and succinyl CoA to 5′-aminolevulinic acid (ALA) by 5′-aminolevulinic acid synthase (ALAS).
ALA is subsequently exported into the cytosol to be processed to uroporphyrinogen III (UroIII), the final common intermediate between heme and siroheme synthesis. For heme biosynthesis, UroIII is decarboxylated by UroIII decarboxylase (UROD) to coproporphyrinogen III, which in turn is redirected to mitochondria.
Heme biosynthesis is finalized in mitochondria in three subsequent enzymatic reactions. Siroheme synthesis also derives from uroporphyrinogen III synthesis. Siroheme is synthesized in four subsequent reactions by one multifunctional (CysG in E. Coli) or two enzymes (Met1p and Met8p in S. ALAS 5′-aminolevulinic acid synthase, ALAD 5′-aminolevulinic acid dehydratase, PBGD porphobilinogen deaminase, UROS uroporphyrinogen III synthase, UROD uroporphyrinogen III decarboxylase, CPO coproporphyrinogen III oxidase, PPO protoporphyrinogen oxidase, FC ferrochelatase. Enzyme (Elrod et al. )Enzyme abbreviationProtein (version)A.
Niger hypothetical proteinE-valueTargetPWoLF PSORTMitoProt IIE. Prediction tools used: WoLF PSORT, MitoProt II, and TargetP (available on and )N. Not present,.no significant blast and no bidirectional match, Mito mitochondria, Cyto cytoplasm, Nucl nucleus, O any other locationIncreased peroxidase production can be achieved by overproducing heme biosynthesis genes. However, this approach does not outperform external hemin addition in experiments aimed at the improved production of secreted proteins from Aspergillus oryzae (Elrod et al. This is most likely caused by the strict regulation of the heme biosynthetic pathway on multiple levels. Heme is an essential molecule for the cell due to its involvement in many essential processes. However, elevated levels of free heme and accumulation of its porphyrin intermediates are toxic to the cell.
Porphyrins absorb light leading to photosensitization and consequently cellular damage due to singlet oxygen release. The toxic effect of heme is mainly mediated by the iron catalyzing the Fenton reaction, generating hydroxyl radicals and subsequently damages DNA, membrane lipids, proteins, etc.
(Krishnamurthy et al.; Hamza; Hou et al. Therefore, in order to increase intracellular levels of heme to levels appropriate for overproduction of peroxidases, insight into the complete biosynthetic pathway and its regulation is necessary.
This review will outline the heme biosynthetic pathway and discuss potential rate-limiting steps. The final aim of this research would be further understanding and resolution of bottlenecks in hemoprotein synthesis in filamentous fungi.
5-Aminolevulinic acid dehydratase (ALAD)After the formation of ALA in the mitochondria, it is exported to the cytosol, where heme synthesis continues with the formation of PBG by ALAD (Elrod et al. ).ALAD is present in the cytosol as a homo-octomer. Based on metal requirement, the ALAD can be divided into two classes: the zinc-dependent ALAD which is present in animals, yeast, and bacteria, and the magnesium-dependent ALAD which is present among plants (Senior et al.
).In the zinc-dependent class, two zinc ions are bound at separate sites, termed α and β. Cerevisiae and Escherichia coli, a zinc ion can be replaced by a magnesium ion as long as there is one zinc ion bound to the β site (Senior et al. Furthermore, the enzyme is sensitive to inhibition by lead, which displaces the zinc ions (Erskine et al. ), and is sensitive to oxidation of the enzymes thiol groups.
Oxidation of the thiol groups subsequently leads to a decrease in activity and a stoichiometric loss of bound metal ions, demonstrating that cysteines are required for zinc binding (Senior et al. Regulation of PBGDIn S.
Cerevisiae, following ALAD, PBGD might play an additional rate-limiting role in heme synthesis (Hoffman et al. Like HEM1, expression of the yeast PBGD encoding gene HEM3 appears constitutive but is actually activated by the Hap4p/CBC complex and repressed on fermentable carbon sources (Keng et al. The CBC complex also appears to be involved in HEM3 regulation in Schizosaccharomyces pombe in response to iron deprivation. Despite the absence of a canonical CCAAT consensus in its promoter, an approximate 3.5-fold Php4-dependent de-repression was observed (Mercier et al.
Recently, it was also demonstrated in hepatic cells that PBGD transcripts are reduced under hypoxia (Vargas et al. Uroporphyrinogen III synthase (UROS) and regulationThe formation of HMB is followed by subsequent flipping of the D-ring during ring closure by UROS, yielding UroIII (Elrod et al. HMB is an unstable molecule and can be non-enzymatically converted to uroporphyrinogen I (Jordan and Berry; Mathews et al. However, this product is not physiologically relevant. UroIII forms the final common intermediate in the formation of all tetrapyrroles (Schubert et al.
).Currently, other than the observed Php4-dependent repression in S. Pombe (Mercier et al. ) and hypoxia-dependent repression in human hepatic UROS (Vargas et al. ), no data is available upon regulation of UROS.
On the other hand, UROS is not expected to become rate limiting as its substrate is unstable and its product is required for both heme synthesis as well as siroheme synthesis, which forms a branch point in the pathway. Coproporphyrinogen III oxidase (CPO)Eukaryotic CPO (Elrod et al.
) forms the first oxygen requiring reaction within the heme biosynthetic pathway. Oxygen is required in the oxidative decarboxylation at the 2- and 4-carboxyethyl side chains in Copro to yield two vinyl groups in PP’genIX (Zagorec et al. ).CPO is located in the cytosol in yeast but is associated with the mitochondrial outer membrane in higher eukaryotes (Dailey ), the only difference being the presence or absence of the mitochondrial targeting sequence in their sequence (Phillips et al. ).Although eukaryotic CPO has been reported to have an obligate requirement for molecular oxygen (Zagorec et al.; Dailey ), S. Cerevisiae still synthesizes 3–7% of its normal heme levels during anaerobic conditions, and oxygen is not an obligatory electron acceptor for heme synthesis during stress conditions in this organism (Hoffman et al. Protoporphyrinogen oxidase (PPO) and regulationThe heme synthesis continues by a six-electron oxidation of PP’genIX to yield PPIX (Elrod et al.
This oxygen-dependent reaction in eukaryotes is catalyzed in three independent cycles by PPO (Dailey ).In eukaryotes, the protein is associated with the cytoplasmic side of the mitochondrial inner membrane as a monomer or as a homodimer, and requires flavin adenine dinucleotide (FAD) as a cofactor. The 17 N-terminal amino acids of human PPO are sufficient for proper targeting, but this sequence is not processed upon translocation. The N-terminal sequence contains an approximately 60 amino acids long dinucleotide-binding motif essential for binding of FAD (Dailey and Dailey; Dailey et al. ).Knowledge of PPO regulation is limited, but the CBC complex might be involved in regulation of PPO as well. A 2–3-fold increase in activity was observed when S.
Cerevisiae was grown on ethanol or galactose compared to growth on glucose (Camadro and Labbe ). Pombe, a 2-fold induction in mRNA was observed 1.5 h after a switch to oxygen-limiting conditions, but PPO activity was not determined (Todd et al. ), while in S. Cerevisiae no increase of PPO activity was observed under anaerobic conditions (Zagorec et al. For human PPO, it has been suggested that, for its housekeeping functions, PPO activity levels are in excess (Dailey et al. ); therefore, PPO is not likely to become a rate-limiting factor.
Ferrochelatase (FC)FC forms the final step in the heme biosynthesis pathway and catalyzes the insertion of ferrous iron into PPIX (Elrod et al. ).In eukaryotes, the apoprotein, synthesized in the cytosol, is translocated to its final destination, the mitochondrial matrix. Translocation of ferrochelatase is an energy-dependent process, which involves removal of the N-terminal leader sequence and assembly of the 2Fe–2S cluster (Dailey et al. FC is found as a homodimer associated with the mitochondrial inner membrane, with its active site facing the matrix space (Dailey et al.; Ferreira ). It has been proposed that FC could form a complex with PPO (Dailey ). The localization of FC is surprising since its product is mostly used in cytoplasmic proteins like P450s and cytochrome b5 or in respiratory cytochromes located on the cytoplasmic side of the mitochondrial inner membrane (Prasad and Dailey ).FCs are clearly similar in structure and gross catalytic properties among all species analyzed, although primary sequences show less than 10% identity between bacterial and higher eukaryotes (Dailey et al. Regulation of FCIn addition to the other regulated enzymes within the heme biosynthesis pathway, FC could also be subject to regulation.
Studies by Taketani et al. in human erythroleukemia cells showed that the amount of FC and activity as well as heme content decreased upon a decrease in the available iron, though mRNA levels and PPIX content were unchanged. Addition of ferric ion–nitrilotriacetate Fe (III) NTA restored ferrochelatase activity.
The additive effect of iron was tested as well and a 1.5-fold increase in FC activity was observed upon the addition of 100 μM Fe (III) NTA, and higher concentrations resulted in a decrease of FC activity. Furthermore, E. Coli FC was insensitive to treatment with iron chelators. Coli does not contain 2Fe–2S clusters, and the human FC apoprotein is sensitive to proteolytic degradation. Therefore, it is suggested that FC is under the positive control of intracellular iron and that this possibly correlates with the formation of the 2Fe–2S cluster (Taketani et al. Transporters of hemeNext to heme synthesis, transport of heme or its intermediates might also be a rate-limiting factor in heme biosynthesis and/or in production of hemoproteins.
Directed transport is probably required to provide different organelles like the ER, nucleus, and peroxisomes with sufficient amounts of heme. Due to the hydrophobic and reactive nature of heme, “free” diffusion through the cytosol is not likely (Hamza ).Research on transport of heme and its intermediates is only beginning to emerge in eukaryotes, and a few transporters have been identified in mammalian cells: Heme Carrier Protein 1 (HCP1), Feline Leukemia Virus C Receptor (FLVCR), and Breast Cancer Resistance Protein (BCRP) (Hamza and references therein). Next to these heme transporters, ABCB6 was found for the transport of Copro (reviewed by Krishnamurthy et al. Mitochondrial porphyrin importABCB6 was first found to be localized on the mitochondrial outer membrane and has the highest affinity for Copro but can also transport other porphyrins. Due to this affinity, ABCB6 can shuttle heme back into the mitochondrial intermembrane space where it might be able to regulate ALAS import. Furthermore, expression of ABCB6 is induced by heme and its intermediates. Overexpression of ABCB6 resulted in increased expression of several rate-limiting enzymes (CPO, ALAD, and ALAS) in the heme biosynthetic pathway, resulting in higher heme levels (Krishnamurthy et al., ).
However, ABCB6 is also reported to be localized in the Golgi apparatus (Tsuchida et al. ) and the plasma membrane (Paterson et al. In the latter study, two different versions of ABCB6 were detected: one is located at the mitochondrial outer membrane (ABCB6-L), and the other is modified posttranslationally and resides at the plasma membrane (ABCB6-H). ABCB6-H appears functionally similar to ABCB6-L and may be involved in the cellular efflux of porphyrins from the cell (Paterson et al. ).The 2-oxoglutarate carrier (OGC) is responsible for the mitochondrial transporter of 2-oxoglutarate but exhibits similar features to ABCB6. Like ABCB6, OCG also has a general affinity for porphyrins and is able to translocate porphyrins and heme into the mitochondrial matrix.
However, unlike ABCB6, OCG is a mitochondrial inner membrane transporter (Kabe et al. Transporters in fungiA screen to identify genes involved in heme uptake in Candida albicans resulted in the identification of a fungal gene family involved in FAD transport into the endoplasmic reticulum (ER). Analysis of Flavin Carrier Protein 1 (Flc1) showed that Flc1p normally resides in the ER.
Although heme transport is not the primary function of Flc1p, it does significantly contribute to heme uptake in C. However, whether Flc1p is directly or indirectly involved in heme uptake remains to be determined. Two homologues are also present in C. Albicans ( CaFLC2 and CaFLC3) which are strongly predicted to have similar, if not identical, biochemical activities. Overexpression of S.
Cerevisiae FLC1 and FLC2, but not FLC3, improved heme uptake (Protchenko et al. ).Recently, a transporter, Pug1p (porphyrin uptake gene), was identified in S. Cerevisiae which is located exclusively at the plasma membrane. Overexpression of Pug1p increased utilization of PPIX but reduced uptake of exogenous heme in a heme-deficient strain. Pug1p belongs to a family of fungal proteins which may possibly function as efflux channels or pumps induced under conditions of enhanced uptake and dysregulation of intracellular small molecules. The authors suggest that Pug1p may function to facilitate excretion of excess porphyrins under conditions of porphyrin accumulation, for instance during hypoxia (Protchenko et al.
Despite the transporters described above, little is known about eukaryotic heme transport, and many questions remain to be answered. Siroheme side branchAs mentioned earlier, UroIII is a common intermediate for both heme and siroheme synthesis. Therefore, feedback regulation on the heme biosynthesis pathway originating from siroheme synthesis may be considered. We will therefore also briefly discuss the current knowledge of siroheme biosynthesis.Siroheme is a heme-like prosthetic group for sulfite and nitrite reductases which are involved in the reduction of sulfite and nitrite to sulfide and ammonia, respectively (Schubert et al. Via sulfite reductases, the siroheme pathway is also required for the synthesis of methionine and cysteine (Keng and Guarente ). It is synthesized from UroIII in four enzymatic steps: two S-adenosyl- l-methionine-dependent transmethylations, a dehydrogenation, and a ferrochelation (Raux et al. Coli, one protein (cysG) performs all these enzymatic reactions (Leustek et al.
), whereas in S. Cerevisiae these steps are performed by Met1p and Met8p. Met1p performs the transmethylation reaction and Met8p is responsible for both the dehydrogenation and ferrochelation reactions. As the N terminus of Met1p is not required for any enzymatic transformations of siroheme synthesis, its function remains unknown.
It may act in some regulatory fashion to mediate control over the branch point in S. Cerevisiae (Raux et al. (51K, pdf)BlastP results of reported proteins involved in synthesis of heme against Aspergillus niger CBS 513. 88 ( taxid: 425011). Not present,.no significant blast and no bidirectional match. Mito mitochondria, Cyto cytoplasm, Nucl nucleus, O any other location, mTP mitochondrial targeting peptide, SP signal peptide, Loc prediction of localization, RC reliability class, Tplen predicted presequence length. Prediction tools used: WoLF PSORT, MitoProt II, and TargetP (available on and ).
(48K, pdf)BlastP results of reported proteins involved in regulation of heme against Aspergillus niger CBS 513. 88 ( taxid: 425011). Not present,.no significant blast and no bidirectional match. Mito mitochondria, Cyto cytoplasm, Nucl nucleus, O any other location, mTP mitochondrial targeting peptide, SP signal peptide, Loc prediction of localization, RC reliability class, Tplen predicted presequence length. Prediction tools used: WoLF PSORT, MitoProt II, and TargetP (available on and ). (46K, pdf)BlastP results of reported proteins involved in transport of heme and intermediates against Aspergillus niger CBS 513.
88 ( taxid: 425011). Not present,.no significant blast and no bidirectional match.
Mito mitochondria, Cyto cytoplasm, Nucl nucleus, O any other location, mTP mitochondrial targeting peptide, SP signal peptide, Loc prediction of localization, RC reliability class, Tplen predicted presequence length. Prediction tools used: WoLF PSORT, MitoProt II, and TargetP (available on and ). This work is funded by the Sixth Framework Programme (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). The authors want to acknowledge very useful comments on the manuscript by the anonymous reviewer.Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.