Developing an oxygen sensitive iron sulphur cluster expression system based on proteins from anaerobic protozoa

Cell factories are widely used to produce microbial metabolites on an industrial scale, offering a practical alternative to traditional production methods. However, some enzymes, particularly iron–sulphur (Fe-S) enzymes, remain difficult to work with due to their sensitivity to oxygen. These enzymes are crucial in many metabolic processes, but the instability of their Fe-S clusters in aerobic conditions limits their use in engineered systems. This project explored whether the Fe-S cluster assembly protein SufCB, from the protozoan Blastocystis Nand II, could be used in Saccharomyces cerevisiae to improve Fe-S enzyme function. Specifically, we investigated whether SufCB could interact with the yeast protein Nbp35 in a similar way to the native protein Cfd1, potentially forming a complex involved in the cytosolic Fe-S assembly (CIA) pathway. To do this, we used bacterial culturing, molecular cloning (via Gibson assembly), and protein extraction techniques. Western blotting was used to detect expression and investigate possible interactions. While the interaction between SufCB and Nbp35 could not be fully confirmed, the results suggest that SufCB may play a role in the CIA pathway, and that further work is needed to explore this possibility in more detail.