Enhancing Heme Production in Aspergillus Niger Through Metabolic Engineering for the Development of Functional Fungal Meat Analogue.

Aspergillus niger, a generally recognized as safe (GRAS) filamentous fungus, is a promising chassis for edible mycelia but lacks meat-like sensory properties. Here, modular metabolic engineering including relieving feedback inhibition (ΔHRM), overexpressing rate-limiting enzymes, and enhancing iron uptake (ΔsreA) was employed to enhance heme biosynthesis, endowing mycelia with a meat-red color. Untargeted metabolomics uncovered a heme tolerance mechanism via precursor efflux mediated by ABC transporters. Heterologous expression of hemoglobins and a P450 enzyme further improved heme utilization, and genome-wide variations analysis uncovered adaptive responses to oxidative and endoplasmic reticulum (ER) folding stress under high-heme pressure. Notably, this enhanced heme supply substantially boosted functional metabolite production, increasing monacolin J yield by 9.99-fold and ergothioneine yield by 1.11-fold. The engineered mycelium closely resembled commercial plant-based meat in color. This study provides a feasible approach to developing A. niger as a sustainable chassis for functional meat alternatives.