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.

Comprehensive investigation of succinic acid production by Actinobacillus succinogenes: a promising native succinic acid producer

“…Results from model predictions generated via flux balance analysis were in accordance with experimental data on mixed acid fermentation. 75 Elimination of metabolic pathways competing with succinic acid production and the incorporation of non-native metabolic pathways would lead to high succinic acid production with high yield and productivity. However, it has been demonstrated that the removal of competitive carbon pathways is insufficient to enhance carbon flux to succinic acid, although effective reduction in byproducts was observed in engineered strains.…”

Insights on the Advancements of In Silico Metabolic Studies of Succinic Acid Producing Microorganisms: A Review with Emphasis on Actinobacillus succinogenes

“…Comprehensive carbon metabolism model (375 reactions) to analyze the metabolism and predict knockout strategies for maximum SA production with maintaining the cell growth 2018 [95] A. succinogenes Genome-scale metabolic model to evaluate the metabolic capability of the strain to produce SA under various conditions 2018 [30] Zymomonas mobilis Genome-scale metabolic model to characterize SA-producing capability and comparatively identify gene deletions for enhanced SA production 2018 [96] E. coli Optimization modeling to identify near-optimal knockout genes for the maximum production of SA 2020 [97] Aspergillus niger…”

“…Hence, the development of models with more inclusive and higher predictive power was needed. Nag et al developed an extended intermediate model that contained nucleic acid, amino acid, lipid and glycogen metabolisms in addition to the central carbon metabolism [95]. However, this model still did not explicitly incorporate all the known metabolic pathways of A. succinogenes to have a comprehensive understanding of the strain's metabolism.…”

Bio‐based succinic acid: an overview of strain development, substrate utilization, and downstream purification

“…To elaborate the metabolic machinery of A. succinogenes clearly, 13 C metabolic flux analyses, flow calculation, several central carbon metabolism models, and the major biomass components models were used recently . Subsequently, several efficient expression methods like pLGZ920 plasmid and electroporation markerless knockout methods were proposed .…”