Metabolic engineering of Shewanella oneidensis to produce glutamate and itaconic acid.

Shewanella oneidensis is a gram-negative bacterium known for its unique respiratory capabilities, which allow it to utilize a wide range of electron acceptors, including solid substrates such as electrodes. For a future combination of chemical production and electro-fermentation, the goal of this study was to expand its product spectrum. S. oneidensis was metabolically engineered to optimize its glutamate production and to enable production of itaconic acid. By deleting the glutamate importer gltS for a reduced glutamate uptake and pckA/ptA to redirect the carbon flux towards the TCA cycle, a ∆3 mutant was created. In combination with the plasmid pG2 carrying the glutamate dehydrogenase gdhA and a specific glutamate exporter NCgl1221 A111V, a 72-fold increase in glutamate concentration compared to the wild type was achieved. Along with overexpression of gdhA and NCgl1221 A111V, the deletion of gltS and pckA/ptA as well as the deletion of all three genes (∆3) was examined for their impact on growth and lactate consumption. This showed that the redirection of the carbon flux towards the TCA cycle is possible. Furthermore, we were able to produce itaconic acid for the first time with a S. oneidensis strain. A titer of 7 mM was achieved after 48 h. This suggests that genetic optimization with an expression vector carrying a cis-aconitate decarboxylase (cadA) and a aconitate hydratase (acnB) along with the proven redirection of the carbon flux to the TCA cycle enabled the production of itaconic acid, a valuable platform chemical used in the production of a variety of products. KEY POINTS: •Heterologous expression of gdhA and NCgl1221_A111V leads to higher glutamate production. •Deletion of ackA/pta redirects carbon flux towards TCA cycle. •Heterologous expression of cadA and acnB enables itaconic acid production.