Prediction of reaction knockouts to maximize succinate production by Actinobacillus succinogenes.

Abstract

Succinate is a precursor of multiple commodity chemicals and bio-based succinate production is an active area of industrial bioengineering research. One of the most important microbial strains for bio-based production of succinate is the capnophilic gram-negative bacterium Actinobacillus succinogenes, which naturally produces succinate by a mixed-acid fermentative pathway. To engineer A. succinogenes to improve succinate yields during mixed acid fermentation, it is important to have a detailed understanding of the metabolic flux distribution in A. succinogenes when grown in suitable media. To this end, we have developed a detailed stoichiometric model of the A. succinogenes central metabolism that includes the biosynthetic pathways for the main components of biomass—namely glycogen, amino acids, DNA, RNA, lipids and UDP-N-Acetyl-α-D-glucosamine. We have validated our model by comparing model predictions generated via flux balance analysis with experimental results on mixed acid fermentation. Moreover, we have used the model to predict single and double reaction knockouts to maximize succinate production while maintaining growth viability. According to our model, succinate production can be maximized by knocking out either of the reactions catalyzed by the PTA (phosphate acetyltransferase) and ACK (acetyl kinase) enzymes, whereas the double knockouts of PEPCK (phosphoenolpyruvate carboxykinase) and PTA or PEPCK and ACK enzymes are the most effective in increasing succinate production.