Understanding and engineering hydrogen production in anaerobic gut fungi

Anaerobic gut fungi (AGF) decompose lignocellulosic substrates into fermentable sugars through the production of powerful enzymes, and they are attractive targets for waste to fuel applications. A recently constructed genome-scale metabolic model of AGF provides a framework to guide strain engineering efforts, especially to control the fungal fermentation byproducts formate, acetate, ethanol, lactate, succinate, and H₂. However, the model’s accuracy is compromised because the core metabolic pathways in the fungal hydrogenosome (a hydrogen producing organelle) are unknown. In particular, the enzyme(s) that regenerate oxidized NAD (NAD⁺) and ferredoxin/flavodoxin and the enzyme(s) that catalyze H₂ production are not known. Here we show that a combination of enzymes including [FeFe]-hydrogenase (HydA) and NADH dehydrogenase (NuoEF) are the key metabolic drivers of energy production in the model fungus, Caecomyces churrovis. Through comparative genomics, we identified the genes encoding enzyme HydA ([FeFe]-hydrogenase) and NuoEF (NADH dehydrogenase) in C. churrovis. Our proteomic analysis of isolated hydrogenosomes confirmed the existence of these enzymes, among many other hydrogenosomal proteins. Enzyme assays of the heterologous generated HydA validated activity of H₂:methyl viologen oxidoreductase (mean ± standard error of mean, 463.70 ± 113.59 mU/mg). Similarly, high NADH:methyl viologen oxidoreductase activity was detected with the heterologous generated NuoEF (3140.57 ± 351.98 mU/mg). The mixture of HydA and NuoEF reduced NAD⁺ and ferredoxin using H₂ as electron donor, behaving as a bifurcating hydrogenase. This work is the first demonstration that flavin-based electron bifurcation occurs in eukaryotes. This bifurcating enzyme could be targeted for future genetic engineering to modulate fermentation products of AGF.