2.3.3. Iron Tolerance and Resistance

*S. cerevisiae* achieves iron resistance through the downregulation of iron import systems via Aft1, or activation of vacuolar transporter Ccc1 [64,249]. Ccc1 is regulated by the iron sensitive transcription factor Yap5; removal of *YAP5* increases iron sensitivity, while its overexpression dramatically reduces cytosolic iron [120]. It may be worth the effort to investigate how the overexpression of *CCC1* affects iron resistance and the vacuolar ability to store excess iron in order to prevent toxicity. Another vacuolar gene, *VMA13*, might also play a potentially novel role in iron tolerance [250]. Vma13 is commonly known as a vacuolar H<sup>+</sup> -ATPase subunit that plays a role in vacuolar acidification; however, a study involving *vma13*∆ mutants showed that they experienced increased sensitivity to iron deprivation, suggesting Vma13 plays a role in iron import [250]. The function of

*VMA13* in iron homeostasis combined with its role in vacuolar acidification should be studied to determine if mutants can also help increase iron resistance. Another method of iron resistance in *S. cerevisiae* is the expression of ferritin related genes. Ferritin is an iron storage protein found in many other eukaryotes, but is not native to fungi [122–124]. Its effects on increased iron resistance and storage capacity in yeast has been investigated and results indicate that the expression of human, soybean, and tadpole ferritin genes (*HuFH*, SFerH1/SFerH2, and *TFH*, respectively) resulted in the increased ability of yeast to store and carry higher concentration of iron [122–124]. Llanos et al. showed the ability of soybean ferritin genes, SFerH1 and SFerH2, to increase iron resistance in *ccc1*∆ mutants [122]. This is significant because, even without the natural vacuolar detoxification system, yeast cells with soybean ferritin were still able to store increased concentrations of iron and evade toxicity.

Fewer studies report on iron resistance in other fungi, but several inferences can be made based on knowledge of iron homeostasis. In *S. pombe*, ferrichome production, excretion, and subsequent uptake are used to acquire extracellular Fe3+ in iron-deficient conditions [59,63,236,237,251]. The engineering of cells to overexpress Sib2 and Sib1 could potentially serve as extra storage vesicles for any excess cytosolic iron acquired by the cell [59,63,236,237,251]. It is not clear how ferrichrome is excreted from the cell after production, therefore this exact mechanism would first need to be identified and wellstudied to determine if inhibiting excretion would have any other adverse effects on cellular health. *U. maydis* also biosynthesizes siderophores (hydroxamate) via *sid1* for iron uptake which could also be investigated for increased production for storage of excess iron [60,121].
