Evaluation of pyruvate decarboxylase-negative strains for the production of succinic acid.

Dicarboxylic acids are important bio-based building blocks, and is postulated to be an advantageous host for their fermentative production. Here, we engineered a pyruvate decarboxylase-negative strain for succinic acid production to exploit its promising properties, that is, lack of ethanol production and accumulation of the precursor pyruvate. The metabolic engineering steps included genomic integration of a biosynthesis pathway based on the reductive branch of the tricarboxylic acid cycle and a dicarboxylic acid transporter. Further modifications were the combined deletion of and and multi-copy integration of the native gene, encoding a pyruvate carboxylase required to drain pyruvate into the synthesis pathway. The effect of increased redox cofactor supply was tested by modulating oxygen limitation and supplementing formate. The physiologic analysis of the differently engineered strains focused on elucidating metabolic bottlenecks. The data not only highlight the importance of a balanced activity of pathway enzymes and selective export systems but also shows the importance to find an optimal trade-off between redox cofactor supply and energy availability in the form of ATP.