Engineered Aspergillus niger enables high-yield heterologous kojic acid production

Industrial-scale production of kojic acid (KA)-valued in cosmetics, food, and pharma-has been hindered by limited metabolic engineering strategies for native producers (e.g., Aspergillus oryzae), which largely rely on random mutagenesis. To address this, Aspergillus niger was engineered as a heterologous host. A combinatorial strategy was employed, which included multi-copy integration of His-tagged kojA, overexpression of heterologous transporter AokojT, deletion of repressor nrkC, amplification of global regulator laeA, and expression of Vitreoscilla hemoglobin vgb enhancing oxygen utilization. This integrated approach progressively increased KA titer to 38 g/L in shake flasks. Medium optimization achieved stable pH (∼5.5) without buffering agents, elevating production to 50 g/L. Scale-up in a bioreactor yielded 56 g/L KA (0.58 g/g yield; 8.0 g/L/day productivity), demonstrating strong industrial potential. This engineered strain provides a high-yield platform and offers a transferable metabolic engineering framework for KA overproduction in both heterologous and native fungal producers.