Iterative Optimization of Exogenous Genes and Redirection of Metabolic Flux for Enhanced Itaconate Biosynthesis in Engineered Escherichia Coli

Background: Itaconate or itaconic acid (IA), a sustainable and valuable platform chemical, has garnered significant attention due to its potential as an alternative petroleum -derived compound. In the past, Aspergillus terreus offered a high titer of IA, but it still faced the challenges of low productivity. To overcome the problem, a fast-growing Escherichia coli BW25113 was involved consolidating the itaconate production pathway, eliminating byproduct -forming pathways, and enhancing intermediate levels to facilitate large-scale production. Method: E. coli was engineered through the deletion of isocitrate decarboxylase (Icd) and the overexpression of pyruvate carboxylase (CgPyc), cis-aconitate decarboxylase (AtCadA) and aconitate hydratase (ENAcnA) to facilitate the IA biosynthesis pathway. A B0015 terminator was encoded behind AtCadA to improve transcription efficiency as well as pox B gene was deleted to accelerate carbon influx towards the tricarboxylic acid cycle instead of forming the byproduct acetate. A semi -continuous batch fermentation approach was employed for large-scale IA biosynthesis to address ATP insufficiency in long -termed fermentation. Significant finding: The engineered strain AB15C/ Delta icd improved IA titer by 20 % compared to the control strain. By deleting the acetate forming pathway under acidic condition, AB15C/ Delta icd Delta poxB increased itaconate production. Overexpression of ENAcnA in AB15CEN*/ Delta icd Delta poxB strain resulted in 13.8 g/L of IA. Finally, a semi-continuous batch fermentation was implemented, leading to an IA accumulation of 35.5 g/L after 4 batches.