High-level itaconic acid (IA) production using engineered Escherichia coli Lemo21(DE3) toward sustainable biorefinery

Itaconic acid (IA) serves as a prominent building block for polyamides as sustainable material. In vivo IA pro-duction is facing the competing side reactions, byproducts accumulation, and long cultivation time. Therefore, the utilization of whole-cell biocatalysts to carry out production from citrate is an alternative approach to sidestep the current limitations. In this study, in vitro reaction of IA was obtained 72.44 g/L by using engineered E.coli Lemo21(DE3) harboring the aconitase (Acn, EC 4.2.1.3) and cis-aconitate decarboxylase (CadA, EC 4.1.1.6) which was cultured in glycerol-based minimal medium. IA productivity enhancement was observed after cold-treating the biocatalysts in -80 degrees C for 24 h prior to the reaction, reaching 81.6 g/L. On the other hand, a new seeding strategy in Terrific Broth (TB) as a nutritionally rich medium was employed to maintain the bio-catalysts stability up to 30 days. Finally, the highest IA titer of 98.17 g/L was attained using L21::7G chassis, that has a pLemo plasmid and integration of GroELS to the chromosome. The high-level of IA production along with the biocatalyst reutilization enables the economic viability toward a sustainable biorefinery.

Automated summary

Using whole-cell biocatalysts to produce itaconic acid (IA) from citrate is an alternative approach to overcome the limitations of in vivo IA production. By employing engineered E.coli Lemo21(DE3) with aconitase (Acn) and cis-aconitate decarboxylase (CadA), IA titer of 72.44 g/L was achieved, which increased to 81.6 g/L after cold-treating the biocatalysts. Furthermore, a new seeding strategy in Terrific Broth (TB) maintained the biocatalysts stability for up to 30 days and the highest IA titer of 98.17 g/L was obtained using the L21::7G chassis.