INTRODUCTION

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Iron is an essential trace element for most living organisms. However, its availability is limited by the low solubility of Fe(III) and the ability of intracellular free iron to cause the production of toxic radicals. Iron deficiency is a common and serious nutritional problem that afflicts an estimated 30% of the world's population (32), especially when vegetable-based diets are the primary food source (6, 8). Animal feeds based primarily on cereals often lack iron. Once iron is introduced into a cell, an intracellular storage form of iron is required that is soluble, nontoxic, and bioavailable (11, 28).

Ferritin (apoferritin-Fe complex) is an iron storage protein found in most living organisms (27). Genes encoding this protein have been isolated from various sources including humans, animals, amphibia, plants, fungi, and bacteria (28). Ferritin is a spherical macromolecule with a protein coat of 24 structurally equivalent subunits that can contain up to 4,500 iron atoms as a ferric oxyhydroxide polymer in its central core (29). The major role of ferritin is to provide iron for the synthesis of iron-containing proteins and to prevent damage from free radicals produced by iron-dioxygen interactions (29, 30). In ferritin, there are two main subunits, heavy (also known as heart, or H) and light (as known as liver, or L). Tissue isoferritins have functional differences that may be related to the structural properties of the subunits (1, 26). The H-rich ferritins associate at a lower isoelectric point and lower iron content and accumulate and release iron relatively rapidly (1, 3). Moreover, it has been demonstrated that the Escherichia coli-expressed H subunit of tadpole ferritin assembles into a large homopolymeric ferritin complex that can bind iron (13). There are two forms of iron storage in Saccharomyces cerevisiae: the first is a cytosolic ferritin-like molecule, and the second implies localization of iron to vacuoles where the iron is bound to polyphosphate instead of ferritin (20). The molecular mass of yeast ferritin is estimated to be 274 kDa, with subunits of approximately 11 kDa. Yeast ferritin's iron content is low; i.e., it binds 50 to 100 iron atoms per molecule in a manner essentially independent of intracellular iron concentration, while the concentration of vacuolar iron in yeast is responsive to the cellular environment (20). The simple addition of iron salts as a feed supplement to the diet of domesticated animals results in the production of hexaferric phytate, a relatively unavailable nutritional source of iron (21). However, when ferritin is administered orally to animals, it can prevent iron binding by phytic acid and provide a source of iron for the treatment of anemia (2).

A commercial process for the production of an iron-fortified food additive requires a nonpathogenic organism amenable to large-scale, high-cell-density fermentation, e.g., S. cerevisiae. The food yeast S. cerevisae is a GRAS (generally recognized as safe) organism and can be grown for the production of biomass rich in high-quality proteins and vitamins. As a consequence, it has been used in livestock feeds for fish (salmonids), poultry, and fur-bearing animals and a food supplement for humans (5).

Our objective in this study was to enhance the bioavailable iron-carrying capacity of a currently used feed additive through the heterologous expression of the tadpole ferritin heavy chain in yeast. We tested whether the homopolymeric ferritin could bind iron in yeast and whether the iron content of the recombinant yeast could be further increased in iron-rich media.

	MATERIALS AND METHODS

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Strains and culture conditions. Plasmids were maintained and propagated in E. coli HB101 or DH5 according to Sambrook et al. (24). S. cerevisiae 2805 (MAT pep4::HIS3 prb1 can1 GAL2 his3 ura3-52) was used as a recipient cell for ferritin expression (18).

Vector construction. We cloned the tadpole ferritin heavy-chain gene (TFH) (GenBank accession no. M15655), located 9 bp upstream from the translational start codon to 24 bp downstream of the translational stop codon, between the ADH2-GPD hybrid promoter and the galactose-1-P uridyl transferase (GAL7) terminator (13, 18) of the episomal shuttle vector YEp352 (18). Briefly, a yeast shuttle vector harboring TFH (pYETFAG-1) was constructed as follows: the 564-bp DNA fragment encoding the tadpole ferritin heavy chain was obtained from an HindIII-SmaI double digest of pMYI15, blunt ended, and then ligated into the blunt-ended BamHI site of the pYEAG-TER vector. The yeast hybrid promoter consisted of the upstream activating sequence of the ADH2 (alcohol dehydrogenase II) gene and the GPD (glyceraldehyde-3-phosphate dehydrogenase) TATA element (18). The GAL7 terminator was provided by S. K. Rhee, Korea Research Institute of Biotechnology and Biochemistry.

Northern blot analysis. Transformants were grown in YEPD medium, and total RNA was prepared (17). Following electrophoresis with a 1.2% agarose gel in the presence of formaldehyde and transfer to a nylon membrane, Northern blots were probed with ³²P-labeled TFH (24).

Preparation of cell crude extract of yeast. Cells were grown for 3 days, harvested, washed twice with extraction buffer (50 mM Tris-HCl, 2 mM EDTA), and ground three times in a bead beater (Biospec Products, Inc., Bartlesville, Okla.) for 1 min. The lysate was centrifuged (10 min at 10,000 × g), and the supernatant was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (see below). As ferritin is thermostable (13) and as the host strain is protease deficient, all preparation steps were performed at room temperature (~25°C).

PAGE. SDS-PAGE was conducted according to the method of Laemmli (15) with a 12% acrylamide separating gel and 5% acrylamide stacking gel, each containing 1% SDS. Samples were heated at 100°C for 10 min in 125 mM Tris-glycine buffer, pH 6.8, containing 1% SDS and 1% -mercaptoethanol (10). Electrophoresis was carried out at a constant current of 10 mA for 3 h with a running buffer of 25 mM Tris-glycine, pH 8.8, containing 0.1% SDS. After electrophoresis, the gel was stained with 0.025% Coomassie brilliant blue R-250.

Western blot analysis. The cell extract resolved by SDS-PAGE was blotted onto a nitrocellulose filter. After being blocked, the filter was incubated with an anti-E. coli expressed TFH antiserum (13), followed by anti-rabbit immunoglobulin G conjugated to horseradish peroxidase as a secondary antibody. 4-Chloro-1-naphthol was used as a substrate for colorimetric detection with horseradish peroxidase (24).

Atomic absorption spectrometry. We conducted an iron uptake assay with samples grown in ura selective medium supplemented with ferric citrate. Cells were grown in YEPD medium supplemented with ferric citrate at 30°C for 4 days, harvested by centrifugation (10 min; 3,500 × g), washed three times with H₂O, and then dried at 50°C for 48 h. The dried cells were digested with 6 ml of 14 M nitric and 10 M perchloric acids (2:1, vol/vol) in a glass tube at 250°C for 8 h. The concentration of iron was determined by atomic absorption spectrometry (AAS 208; Hitachi, Inc., Ibaraki, Japan) (16).

	RESULTS

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Expression of TFH gene in recombinant yeast. From Northern blot analysis of transformants, we found that the introduced TFH gene was expressed under the control of the hybrid promoter. Transformant TYETFAG-1 was arbitrarily selected for further experiments from 10 candidates which showed a similar level of expression (Fig. 1). Plasmid stability was good, and more than 80% of plated cells appeared to carry the plasmid 72 h after liquid cultivation, while no loss of plasmid was observed under selective conditions (data not shown).

View larger version (63K): [in this window] [in a new window]   FIG. 1.   Northern blot analysis of TFH. (A) Lane 1 contains the 0.56-kb cDNA fragment of TFH as a positive control. Lanes 2 to 4 contain total RNA from the recipient strain, the transformant without ethanol induction, and the transformant with ethanol induction, respectively. Panels B and C show the gel stained with ethidium bromide and Northern blot analysis of GPD, respectively, indicating that an equal amount of RNA has been loaded for each sample.

Western blot analysis of recombinant TFH. Different banding patterns were observed from preparations obtained from horse spleen ferritin, E. coli-derived TFH, and TYETFAG-1, but not from the recipient cell (Fig. 2). Although the antibody obtained from the previous study is polyclonal and the specificity is not high enough to show a single band, the distinctive band obtained with an antibody against the E. coli-derived TFH appears to be specific to TFH because the molecular mass of the yeast ferritin-like subunit is smaller, 11 kDa, than that of native TFH, 20.5 kDa. The molecular mass of yeast-derived TFH was estimated to be 24 kDa.

View larger version (87K): [in this window] [in a new window]   FIG. 2.   Western blot analysis of recombinant TFH. (A) Coomassie blue-stained SDS-PAGE of the yeast cell extract. (B) Antigen-antibody reaction of the corresponding twin gel of yeast cell extract. Lanes 1, size marker; lanes 2, 2 µg of horse spleen ferritin; lanes 3, 10 µl (1.0 µg µl1) of E. coli-derived TFH; lanes 4, 15 µ1 (1.0 µg µl1) of concentrated cell extract of the host cell; lanes 5, 15 µl (1.0 µg µl1) of concentrated cell extract of the transformant; lanes 6, 30 µl (1.0 µg µl1) of concentrated cell extract of the transformant. The numbers on the left refer to estimated sizes in kilodaltons.

Iron tolerance of recombinant yeast. After 48 h of incubation on ura selective media, transformants could grow on a medium containing 20 mM ferric citrate, but the untransformed host could grow only on a medium containing 10 mM ferric citrate. Small colonies of transformants and host cells appeared on plates containing 30 and 20 mM ferric citrate, respectively, after prolonged incubation (>72 h). However, no growth of the recipient cells was ever detected on plates with 30 mM ferric citrate.

Iron-binding capability of recombinant TFH. The iron-binding capability of the expressed TFH was analyzed with Prussian blue staining (Fig. 3). After being stained with ferrocyanide, a characteristic blue band was observed in cell extracts from transformants, indicating that the TFH subunits had assembled into a functional protein that could bind irons. No staining was observed in the recipient cell (Fig. 3).

View larger version (74K): [in this window] [in a new window]   FIG. 3.   Prussian blue staining of TFH. (A) Coomassie blue-stained SDS-PAGE of the yeast cell extract. (B) Prussian blue-stained PAGE after preincubation of the yeast cell extract with 1 mM ferrous ammonium sulfate. Lanes: 1, horse spleen ferritin; 2, E. coli-derived TFH; 3, concentrated cell extract of the host cell; 4, concentrated cell extract of the transformant.

Atomic absorption spectrometry. Iron uptake and cell growth were measured for the recipient strain and the transformant grown at various concentrations of ferric citrate (Table 1). Cells grown in a high-iron-content environment took up significantly more iron. Iron content of the recipient strain increased up to 33-fold in iron-loaded cells. Relative to the untransformed strain, the transformed strain had 160 and 140% increase in iron per gram (dry cell weight) at 10 and 20 mM ferric citrate in culture medium, respectively. Growth of both strains was restricted as the concentration of ferric citrate increased, and the difference between the recipient strain and transformant was statistically significant (P < 0.05) at 20 mM ferric citrate.

	DISCUSSION

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S. cerevisiae is a well-established host for the production of heterologous proteins (4, 23). Iron has two functions, one as an essential element and the other as a potential toxic radical. Although the effect of Fe radicals on cell damage is widely accepted (22), more than 90% of cases of anemia are caused by iron deficiency (32). Ferritin is an efficient way of storing iron as well as increasing its bioavailability (9).

Eukaryotic ferritin subunits are processed to mature proteins by posttranslational modification (19), but prokaryotic and in vitro translation systems do not process these subunits properly (13, 14, 31). The yeast-derived TFH (24 kDa) in this study was larger than either the native TFH protein (20.5 kDa) (7) or the E. coli-derived TFH (22 kDa). These results suggested that the yeast-derived TFH subunits were not processed correctly. As there is no canonical glycosylation site (Asn-X-Ser/Thr) in TFH (23), this result suggests that a posttranslational modification, other than glycosylation, may occur in yeast. However, Prussian blue staining indicated that the yeast-derived TFH appeared to assemble into a large holoprotein and could store iron. The yeast-derived ferritin also migrates somewhat more slowly on a nondenaturing gel than does the E. coli-derived ferritin (Fig. 3), which is known to be a larger holoprotein than the native tadpole ferritin (13).

Previous studies have shown that vacuolar iron content increases when yeast is subjected to a high concentration of iron, although the cytosolic iron concentration of yeast contributed by a ferritin-like molecule is independent of its environment (20). In our study, the efficacy of iron uptake was significantly increased due to the heterologous expression of TFH and reached two and a half times that of the host strain grown under the same conditions. Although the increase in the total iron concentration in the transformant was relatively modest, growth of the transfomant was robust, suggesting that the difference in iron concentration is due mainly to the presence of TFH, which maintains iron in a nontoxic state. Thus, the bioavailability of iron should be dramatically increased in food and feed supplement with these engineered yeasts.

If ferritin is administered orally to animals, it can provide a source of iron for the treatment of anemia (2). This finding suggests that increasing the amount of iron-containing ferritin in animal feeds might reduce dietary iron deficiency and, perhaps, improve the dietary quality of livestock as an iron-fortified food for human consumption (9). Since S. cerevisiae, as a GRAS organism, has been used for many years as a feed additive and as a food supplement, we expect that yeast iron-fortified food produced by strains such as this could alleviate some problems due to iron deficiency in both humans and animals.