Metabolic Engineering of Anaerobic Itaconate Production in E. coli
Itaconic acid is a potential new chemical building block. Industrial application is limited because of the high production costs. Currently itaconic acid is produced by Aspergillus terreus. The pathway yield of itaconic acid in this fungus is 1 mol/mol glucose, 25% lower than the maximum theoretical yield. This results in oxygen consumption, which has a negative effect on productivity.
To increase both yield and productivity, itaconate production should be performed without oxygen input. This requires the development of metabolic networks in which cofactor regeneration is possible without respiration. The mixed-acid fermentation pathway of E. coli was engineered to this end. Cis-aconitate decarboxylase (CadA), the key enzyme of itaconate production, of Aspergillus terreus was introduced in E. coli. To boost precursor availability, citrate synthase and aconitase from Corynebacterium glutamicum were introduced. To reduce byproduct formation both phosphotransacetylase and lactate dehydrogenase were knocked out. This resulted in anaerobic production of itaconic acid. Ethanol (+formate) and succinate production were used for cofactor regeneration.
It appeared that itaconate production was limited by CadA activity. The immunoresponse gene (Irg1) of mouse also catalyses the decarboxylation of cis-aconitate into aconitate in vitro. Replacing the CadA gene with the Irg1 gene resulted in itaconate production in vivo, at a similar rate as CadA. Using Irg1 genes from other origins may give the opportunity to increase itaconate production.
Although the mixed-acid-pathway does allow anaerobic itaconate production, the concurrent production of by products for cofactor regeneration may limit its commercial applicability. Two alternative pathways were designed showing a pathway yield identical to the maximum theoretical yield, combining the TCA cycle with either a reverse glyoxylate cycle or a reverse TCA cycle. Progress made with these pathways and their applicability for the production of other chemicals will be discussed.