Production of redox-active organic electrolyte candidates using the oleaginous yeast Yarrowia lipolytica as a heterologous cell factory

Fungal quinone polyketides are promising bio-electrolyte candidates for aqueous organic redox flow batteries (AORFBs) but are difficult to produce efficiently and at scale in native filamentous fungi. Here, we engineered Yarrowia lipolytica as a heterologous host to biosynthesize toluquinol and gentisyl alcohol by reconstructing and validating the biosynthetic gene cluster from Aspergillus hancockii. Comparative pathway analysis revealed loss of activity in one of the annotated CYP450 monooxygenases, the function of which was restored through orthology-guided sequence truncation, although at lower efficiency than that of the corresponding ortholog from Aspergillus clavatus. We achieved titers of 1.1 g/L toluquinol and 1.4 g/L gentisyl alcohol from the strains expressing the A. hancockii pathway in shake flask cultures. Furthermore, titers of up to 1.8 g/L gentisyl alcohol were achieved upon expression of a hybrid pathway incorporating the CYP450 from A. clavatus, thereby underscoring the possibility of improving product formation through complementation with cross-species enzymes. Subsequent cyclic voltammetry confirmed the redox activity of heterologously produced quinones and revealed a quasi-reversible redox process. Overall, these results establish a scalable heterologous production platform for fungal quinones with redox-active properties, streamlining their systematic evaluation as AORFBs electrolytes.