Tailoring key enzymes for renewable and high-level itaconic acid production using genetic Escherichia coli via whole-cell bioconversion

Renewable chemical productions through carbon-neutral design are widely concerned in recent years. Among all, itaconic acid (IA) is one of the most important building block chemicals from biorefinery. However, IA fermentation by the eukaryotic Aspergillus terreus is time-consuming and less productive. The whole-cell (WC) bioconversion, proposed as an alternative approach by transforming citrate into IA via two key enzymes of aconitase (ACN, EC 4.2.1.3) and cis-aconitate decarboxylase (CAD, EC 4.1.1.6), is attractive. In this study, we screened the best genes from genes library, studied the kinetics parameters of ACN from Corynebacterium glutamicum (Cg) and CAD from Aspergillus terreus (At), thus achieving the maximum IA production. The catalytic activity of CgAcnA was 39-fold of AtCadA, indicating CAD was the rate-determining step. For metal ions effect, copper and ferric ions inhibited 95% and 59% enzyme activity when both enzymes co-worked together. Finally, the engineered Escherichia coli expressing dual genes and cultured in glycerol-included medium reached the highest IA titer of 67 g/L and productivity of 8.375 g/L/h, which demonstrates as a promising renewable process.

Automated summary

Renewable chemical production through carbon-neutral design has received significant attention. In this study, the WC bioconversion of citrate into IA using key enzymes ACN and CAD showed promising results, with CAD identified as the rate-determining step. Metal ions like copper and ferric ions were found to inhibit enzyme activity when both enzymes co-worked. The engineered E. coli expressing dual genes achieved a high IA titer and productivity, demonstrating a promising renewable process.