De novo biosynthesis of the plant-derived aloe-emodin in Aspergillus nidulans via artificial pathway assembly and temporal flux control.

Traditional botanical extraction of medicinal anthraquinones like aloe-emodin is limited by low yields, long growth cycles, and environmental dependence. Microbial biosynthesis provides a sustainable alternative, but de novo reconstruction of aloe-emodin remains difficult due to unresolved pathway steps and poor metabolic flux distribution in engineered hosts. In this study, we engineered Aspergillus nidulans for the de novo biosynthesis of aloe-emodin via modular assembly of an artificial pathway. We first established an emodin-producing chassis by reconstructing a minimal mdp-derived upstream module and enhancing precursor supply, yielding 86.93 mg/L emodin. Introducing the heterologous cytochrome P450 CYP-H6231 effectively catalyzed the hydroxymethylation of emodin to citreorosein. We then extended the pathway by incorporating the reductive tailoring enzymes MdpK, MdpC, and MdpB to form aloe-emodin. While feeding assays supported a citreorosein-linked route to aloe-emodin, constitutive expression of this downstream module caused unbalanced flux and high accumulation of the competing byproduct chrysophanol. To address this, we applied temporal induction to mdpK. Delaying its expression shifted flux partitioning, significantly reducing chrysophanol formation and improving aloe-emodin selectivity. The final engineered strain achieved 278.06 mg/L citreorosein and 70.97 mg/L aloe-emodin during 1-L fed-batch fermentation. This study establishes a de novo microbial route to aloe-emodin and highlights temporal flux control as a practical strategy for assembling complex plant-derived anthraquinone pathways in fungal cell factories.