Insights into Actinobacillus succinogenes fermentative metabolism

Bio-based succinate production is receiving increasing attention as a potential intermediary feedstock for replacing a large petrochemical-based bulk chemical market. The economical and environmental benefits of a bio-based succinate industry have motivated research and development of organisms that produce succinate. Actinobacillus succinogenes is one of the best succinate-producing organisms ever reported, however it also produces unwanted formate, acetate, and ethanol. Thus, it is desirable to engineer A. succinogenes metabolism to produce succinate as the sole fermentation product. Metabolic engineering of A. succinogenes has been impeded by a relatively limited knowledge of its physiology and metabolism, and a lack of genetic tools.; 13C-metabolic flux analysis methods were chosen to obtain a detailed knowledge of A. succinogenes metabolic pathways, the fluxes through these pathways, and how these fluxes respond to perturbations. 13C metabolic flux analysis is informative when different pathways result in different labeling patterns in metabolic products (e.g., proteinaceous amino acids). A chemically defined A. succinogenes growth medium, which included only the required amino acids (i.e., cysteine, methionine and glutamate), was developed to ensure that amino acids are labeled from 13C-labeled substrate. Glutamate auxotrophy was determined to be due to an inability to made alpha-ketoglutarate, indicating at least two missing tricarboxylic acid cycle-associated activities. A 13C-labelling experiment was performed with [1-13C] glucose in the defined medium to delineate A. succinogenes metabolic pathways and quantify in vivo fluxes. NADPH was produced by pyruvate and formate dehydrogenases coupled with transhydrogenase and by malic enzyme rather than by the oxidative pentose phosphate pathway. A C4-decarboxylating activity shunted flux from the succinate-producing C4 pathway to the formate-, acetate-, and ethanol-producing C3 pathway, likely through both malic enzyme and oxaloacetate decarboxylase. 13C-labelling experiments were then conducted under either N2 or H2 atmospheres with a mixture of [1-13C]glucose, [U-13 C]glucose, and either 25 or 100 mM NaHCO3. The labeling conditions revealed exchange fluxes in the C3 pathway and between the C3 and C4 pathways. The effects of NaHCO3 and H, concentrations on A. succinogenes fermentations went beyond their roles as C4 pathway substrates for succinate production. NaHCO3 concentration affected the amount of flux shunted from the C4 to the C3 pathway. Fluxes within the C3 pathway changed to compensate for different reductant demands at different NaHCO3 concentrations. H2 also affected the C3 pathway fluxes by alleviating the need for NADH production by pyruvate and formate dehydrogenases. 13C-metabolic flux analyses were complemented by A. succinogenes draft genome sequence information. The genome sequence was also analyzed for its industrially relevant features. In summary, A. succinogenes metabolism is complex with flux distribution to succinate versus alternative fermentation products occurring at four different nodes. The metabolism is also flexible as fluxes change to meet the reductant demands at different NaHCO3 and H2 concentrations.