High-Yield Expressed Human Ferritin Heavy-Chain Nanoparticles in K. marxianus for Functional Food Development

Abstract

The use of Generally Recognized as Safe (GRAS)-grade microbial cell factories to produce recombinant protein-based nutritional products is a promising trend in developing food and health supplements. In this study, GRAS-grade Kluyveromyces marxianus was employed to express recombinant human heavy-chain ferritin (rhFTH), achieving a yield of 11 g/L in a 5 L fermenter, marking the highest yield reported for ferritin nanoparticle proteins to our knowledge. The rhFTH formed 12 nm spherical nanocages capable of ferroxidase activity, which involves converting Fe²⁺ to Fe³⁺ for storage. The rhFTH-containing yeast cell lysates promoted cytokine secretion (tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and -1β (IL-1β)) and enhanced locomotion, pharyngeal pumping frequency, egg-laying capacity, and lifespan under heat and oxidative stress in the RAW264.7 mouse cell line and the C. elegans model, respectively, whereas yeast cell lysate alone had no such effects. These findings suggest that rhFTH boosts immunity, holding promise for developing ferritin-based food and nutritional products and suggesting its adjuvant potential for clinical applications of ferritin-based nanomedicine. The high-yield production of ferritin nanoparticles in K. marxianus offers a valuable source of ferritin for the development of ferritin-based products.

Keywords: Kluyveromyces marxianus; functional foods; nanoparticle; recombinant ferritin.

Figure 1

Expression of rhFTH in K. marxianus. (a) SDS-PAGE and Western blot analyses of rhFTH expression in K. marxianus. The KM and KM-rhFTH strains are K. marxianus transformed with pUKDN125 and pUKDN125-rhFTH, respectively. The red arrow highlights the bands of rhFTH. Rabbit anti-ferritin monoclonal antibody and secondary antibody goat anti-rabbit IgG were used for Western blot. M: PageRuler prestained protein ladder; T: total cell lysate; S: supernatant of cell lysate; P: precipitate of cell lysate. (b) An orthogonal design with three factors at three levels, including (NH₄)₂SO₄ (5, 12, 18 g/L), glucose (10, 40, 60 g/L), and MgSO₄·7H₂O (0.5, 7, 10.5 g/L), was used to test the effects on soluble expression of rhFTH in SM medium in shake flasks at 220 rpm at 30 °C for 72 h. The soluble expression of rhFTH under different conditions was separately compared with that in SM medium. R: Range values of orthogonal design experiments. (c) Comparison of the soluble expressions of rhFTH in SM medium and the optimized SMO medium. (d–f) Effects of different concentrations of Triton X-100, NP-40, and Tween 20 in cell lysis solution on the recovery of soluble rhFTH. Statistical differences were analyzed using t-tests. * p < 0.05; *** p < 0.001; **** p < 0.0001; p > 0.05 (not significant, ns).

Figure 2

Fermentation of KM-rhFTH strain and production of rhFTH in 5 L fermenters. (a–c) The fermentation was carried out in three 5 L fermenters fed with 1000 g (Tank F1), 1300 g (Tank F2), and 1500 g (Tank F3) of glucose. Cells were collected every 12 h, lysed after a 5-fold dilution, then subjected to SDS-PAGE analyses for the expression of soluble rhFTH. (d) The growth curves of KM-rhFTH strain in the three fermenters fed with different amounts of glucose. (e) Cell dry weights at 60 h and soluble yields of rhFTH at 72 h in the three fermenters. (f) Observation of rhFTH in the supernatant of the cell lysate collected from Tank F2 at 72 h by transmission electron microscopy. Bar, 100 nm.

Figure 3

Purification and characterization of rhFTH produced by K. marxianus. (a) SDS-PAGE analysis of rhFTH purified by heat treatment coupled with DEAE chromatography. Lane M: PageRuler prestained protein ladder; Lane 1: Cell lysate supernatant of the KM-rhFTH; Lane 2: Heat-treated supernatant at 75 °C for 10 min; Lane 3: Elution fraction from DEAE column. (b) Native-PAGE. Lane 1: Cell lysate supernatant of the KM-rhFTH strain; Lane 2: Commercial recombinant ferritin expressed in E. coli; Lane 3: Purified rhFTH expressed in K. marxianus; Lane 4: Cell lysate supernatant of the K. marxianus host strain. (c) TEM analysis of purified rhFTH. Scale bar: 50 nm. (d) Iron uptake of the purified rhFTH. The experiments were carried out in solutions containing a fixed concentration of 0.3 mg/mL rhFTH and 0.25, 0.5, and 1 mM FeSO₄, respectively. The reaction was performed at room temperature for a total of 30 min, and absorbance values at A₃₁₀ nm were obtained every 2 s. The initial absorbance was subtracted to obtain ΔA₃₁₀ nm. (e) Analysis of iron content in rhFTH by ICP-MS. (f) Analysis of iron release from rhFTH by the ferrozine method. Incubation with FeSO₄ was conducted at a concentration of 1 mM.

Figure 4

Effects of rhFTH-containing yeast cell lysates on the proliferation and cytokine secretion of RAW264.7 macrophage cell line after 24 h incubation with different treatments. Blank: culture medium; Control: host K. marxianus cell lysate without human ferritin; rhFTH: rhFTH contained in cell lysate; LPS: Lipopolysaccharide at 1 µg/L as the positive control. The biomass of the control host strain was adjusted to an OD₆₀₀ equivalent to that of cell lysates containing 250, 500, 750, and 1000 µg/mL rhFTH. (a) Proliferation of the RAW264.7 cell line. (b) Secretion of IL-6 by the RAW264.7 cell line. (c) Secretion of TNF-α by the RAW264.7 cell line. (d) Secretion of IL-1β by the RAW264.7 cell line. Six replicates were set up for each group. Statistical analysis was performed using t-tests to determine significant differences, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 5

Analysis of biological function of rhFTH-containing yeast cell lysates on the C. elegans model. Blank: culture medium; Control 1: host K. marxianus cell lysate without human ferritin, and the biomass of the control host strain was adjusted to an OD₆₀₀ equivalent to that of cell lysates containing 0.5 mg/mL rhFTH; Control 2: host K. marxianus cell lysate without human ferritin, and the biomass of the control host strain was adjusted to an OD₆₀₀ equivalent to that of cell lysates containing 2.0 mg/mL rhFTH; rhFTH 1: 0.5 mg/mL rhFTH contained in cell lysate; rhFTH 2: 2 mg/mL rhFTH contained in cell lysate. (a) Proportion of well-motile nematode within 30 s at day 0, 5, 10, and 15. (b) Pharyngeal pumping frequency of nematodes within 1 min. (c) Total spawning number of nematodes. (d) Survival curves of nematodes under normal culture conditions (NGM, 20 °C). (e) Survival curves of nematodes under heat stress at 37 °C. (f) Survival curves of nematodes under H₂O₂-induced oxidative stress. (g) T-SOD activity. (h) CAT activity. (i) Quantization of ROS levels. (j) Analysis of the lipofuscin map. Statistical differences were analyzed using t-tests. * p < 0.05; ** p < 0.01; *** p < 0.001.