Genetic design of co-expressing a novel aconitase with cis-aconitate decarboxylase and chaperone GroELS for high-level itaconic acid production

Abstract

Itaconic acid (IA) is regarded as one of the 12 important building blocks as recognized by US Department of Energy. Thus, the production of IA is crucial and urgent. In this study, we attempted to explore a new aconitase from an acidophilic E. coli Nissle 1917, denoted as ENAcnA, and was compared to Corynebacterium glutamicum for the first time. The native ENAcnA has 10 amino acid mutations and the supposed critical mutations at N397S, Q409L, S522G, and G826E in the aconitase family domain contributed to the high hydratase activity at the low pH condition. Co-expression of ENAcnA and cis-aconitate decarboxylase from Aspergillus terreus in the recombinant E. coli BL21(DE3) resulted in IA titer and productivity of 80.4 g/L and 10.1 g/L/h, respectively. Integration of GroELS chaperon to chromosome of BL21(DE3) for generating BD::7G strain even enhanced IA titer by 13 %. The cold treatment of whole-cell biocatalyst substantially assisted the mass transfer of substrate through the cell membrane, and consequently achieved the maximum IA productivity. The discovery of ENAcnA, chaperon assistance in host, and cold treatment of whole cell biocatalysts contributed to high-level IA production up to 95.5 g/L for the industrial scale.