Enhanced citric acid production via aconitase co-inhibition strategy using L-cysteine HCl-resistant Aspergillus niger NA-CYS3

Abstract The present study presents a new approach to boost citric acid production by combining genetic improvement and enzyme inhibition in the fungus Aspergillus niger . A mutant strain, named NA-CYS3, was developed by chemically treating the wild-type strain to make it resistant to L-cysteine HCl, a chemical that normally limits growth. This mutant showed better tolerance and higher productivity. Optimal fermentation conditions including medium volume (50 mL), acidity (pH: 4.5), inoculum size (10%), incubation time (144 h), and temperature (30 °C) were optimized to maximize citric acid yield. Under these conditions, NA-CYS3 produced 26.35 g/L, a 2.7-fold increase in substrate use and higher yield compared to the wild-type strain. To further increase production, the activity of aconitase, an enzyme involved in the citric acid cycle, was partially blocked by adding two inhibitors, potassium ferrocyanide ( K₄Fe(CN)₆ ; 0.004%) and methanol (1 mL), shortly after fermentation started. This “aconitase co-inhibition strategy” slowed down certain metabolic steps, leading NA-CYS3 to produce nearly 49 g/L of citric acid, significantly more than the wild-type’s 35.65 g/L. Kinetic parameters (Yp/s, Qp, qp) further confirmed its superior production capacity. These findings demonstrate that combining strain mutation with targeted enzyme inhibition can greatly enhance citric acid biosynthesis. The NA-CYS3 strain shows promise for efficient and large-scale industrial production of citric acid.