Systematic metabolic engineering for trans-aconitic acid production in Aspergillus niger

Trans-aconitate (TAA) is a valuable bio-based chemical for nematicides and polymer synthesis. We repurposed the industrial citrate producer Aspergillus niger for efficient TAA biosynthesis. Heterologous AexA enabled extracellular aconitate export. However, overexpressing aconitases from various sources, targeted to the cytoplasm or mitochondria, failed to increase titers. Although mitochondria proved the optimal site for cis-aconitate accumulation, the bottleneck likely persisted due to thermodynamic constraints. To overcome this, we designed a push–pull strategy: a pull module using mitochondrial MttA to export cis-aconitate to the cytosol, where TbrA isomerizes it to TAA, coupled with a push module enhancing cytosolic acetyl-CoA via the phosphoketolase pathway. Multicopy tbrA expression was critical. The final strain produced 34.30 g/L total aconitate (>90% TAA) in a 5-L bioreactor within 6 days. This work highlights a compartmentalized push–pull strategy exploiting mitochondrial-cytosolic organization to convert a citrate chassis into a high-yield TAA cell factory.