Engineering a versatile Aspergillus niger platform for efficient and scalable biosynthesis of polyketides.

Polyketides are a diverse class of natural products with broad pharmacological activities and substantial industrial relevance; however, their scalable biosynthesis is often constrained by inefficient heterologous expression platforms. In this study, a systematic evaluation of multiple microbial hosts identified Aspergillus niger HL-1 as an efficient fungal chassis for polyketide production. Subcellular compartmentalization analysis revealed that cytosolic biosynthesis significantly outperformed peroxisomal localization, leading to a 5.88-fold increase in triacetic acid lactone (TAL) production. To further enhance production, intracellular acetyl-CoA and malonyl-CoA supplies were engineered and integrated with compartmentalized metabolic rewiring to redirect carbon flux toward polyketide biosynthesis. In addition, increasing the copy number of polyketide synthase genes further improved titers. As a result, monacolin J and TAL reached 1.68 g/L and 7.50 g/L in shake-flask cultures, respectively, and were further elevated to 2.61 g/L and 25.08 g/L in a 1.5-L bioreactor. Finally, a versatile type III polyketide synthase (PKS) expression platform was established, enabling the functional expression of ten heterologous PKSs. By further disrupting the endogenous katA gene to decelerate p-coumaric acid consumption, the competitive catabolic pathway was successfully blocked, yielding a naringenin shake-flask titer of 384.91 mg/L from p-coumaric acid. This work establishes a robust and scalable fungal platform for the efficient production of both complex polyketides and flavonoids, providing a promising strategy for biotechnological applications.