Model-Based Metabolic Engineering of Escherichia coli for High Yield Itaconic Acid Production

Harder, B. J., Max Planck Institute for Dynamics of Complex Technical Systems
Bettenbrock, K., Max Planck Institute for Dynamics of Complex Technical Systems

Itaconic acid is a high potential platform chemical which is currently industrially produced by Aspergillus terreus. It is primarily used for polymer synthesis and has the potential to replace petrochemically derived methacrylic acid. Heterologous production of itaconic acid with Escherichia coli could help to overcome limitations of A. terreus regarding slow growth and high sensitivity to oxygen supply. However, the performance achieved so far with E. coli strains is still low.

To enable heterologous itaconic acid production we introduced a plasmid carrying the codon-optimized genes of the cis-aconitic acid decarboxylase from Aspergillus terreus and the citrate synthase of Corynebacterium glutamicum into E.coli MG1655. We then applied a model-based approach to construct a high yield producer strain. Based on the concept of minimal cut sets, we identified intervention strategies that guarantee high itaconic acid yield while still allowing growth. One cut set was selected as starting point and some of the corresponding genes were iteratively knocked-out. As a conceptual novelty, we pursued an adaptive approach allowing changes in the model and initially calculated intervention strategy if a genetic modification induces changes in byproduct formation. Using this approach, we iteratively implemented 5 interventions leading to high yield itaconic acid production in Minimal Medium with glucose as substrate supplemented with small amounts of glutamic acid. In shake flask cultivations of the derived E. coli strain we achieved an itaconic acid titer of 2.2 g/l with an excellent yield of 0.77 mol/mol. In a fed-batch cultivation, this strain produced 32 g/l itaconic acid with an overall yield of 0.68 mol/mol and a peak productivity of 0.45 g/l/h. These values are by far the highest that have ever been achieved for heterologous itaconic acid production and indicate that realistic applications come into reach.

Our results demonstrate that itaconic acid can be produced with high yield and titer by a dedicated E. coli mutant strain. Furthermore it shows that constrained minimal cut sets, applied in an iterative manner, provide a suitable computational tool for in silico strain design and to identify intervention strategies for redirecting carbon flux to high product formation.