A global transcriptome analysis of the natural succinate producer Actinobacillus succinogenes revealed that 353 genes were differentially expressed when grown on various carbon and energy sources, which were categorized into six functional groups. We then analyzed the expression pattern of 37 potential C4‐dicarboxylate transporters in detail. A total of six transporters were considered potential fumarate transporters: three transporters, Asuc_1999 (Dcu), Asuc_0304 (DASS), and Asuc_0270‐0273 (TRAP), were constitutively expressed, whereas three others, Asuc_1568 (DASS), Asuc_1482 (DASS), and Asuc_0142 (Dcu), were differentially expressed during growth on fumarate. Transport assays under anaerobic conditions with [14C]fumarate and [14C]succinate were performed to experimentally verify that A. succinogenes possesses multiple C4‐dicarboxlayte transport systems with different substrate affinities. Upon uptake of 5 mmol/L fumarate, the systems had substrate specificity for fumarate, oxaloacetate, and malate, but not for succinate. Uptake was optimal at pH 7, and was dependent on both proton and sodium gradients. Asuc_1999 was suspected to be a major C4‐dicarboxylate transporter because of its noticeably high and constitutive expression. An Asuc_1999 deletion (∆1999) decreased fumarate uptake significantly at approximately 5 mmol/L fumarate, which was complemented by the introduction of Asuc_1999. Asuc_1999 expressed in Escherichia coli catalyzed fumarate uptake at a level of 21.6 μmol·gDW
−1·min−1. These results suggest that C4‐dicarboxylate transport in A. succinogenes is mediated by multiple transporters, which transport various types and concentrations of C4‐dicarboxylates.
Actinobacillus succinogenes, which is known to produce large amounts of succinate during fermentation of hexoses, was able to grow on C4-dicarboxylates such as fumarate under aerobic and anaerobic conditions. Anaerobic growth on fumarate was stimulated by glycerol and the major product was succinate, indicating the involvement of fumarate respiration similar to succinate production from glucose. The aerobic growth on C4-dicarboxylates and the transport proteins involved were studied. Fumarate was oxidized to acetate. The genome of A. succinogenes encodes six proteins with similarity to secondary C4-dicarboxylate transporters, including transporters of the Dcu (C4-dicarboxylate uptake), DcuC (C4-dicarboxylate uptake C), DASS (divalent anion : sodium symporter) and TDT (tellurite resistance dicarboxylate transporter) family. From the cloned genes, Asuc_0304 of the DASS family protein was able to restore aerobic growth on C4-dicarboxylates in a C4-dicarboxylate-transport-negative Escherichia coli strain. The strain regained succinate or fumarate uptake, which was dependent on the electrochemical proton potential and the presence of Na+. The transport had an optimum pH ~7, indicating transport of the dianionic C4-dicarboxylates. Transport competition experiments suggested substrate specificity for fumarate and succinate. The transport characteristics for C4-dicarboxylate uptake by cells of aerobically grown A. succinogenes were similar to those of Asuc_0304 expressed in E. coli, suggesting that Asuc_0304 has an important role in aerobic fumarate uptake in A. succinogenes. Asuc_0304 has sequence similarity to bacterial Na+-dicarboxylate cotransporters and contains the carboxylate-binding signature. Asuc_0304 was named SdcA (sodium-coupled C4-dicarboxylate transporter from
A
. succinogenes).
Corynebacterium glutamicum is an industrial strain used for the production of valuable chemicals such as L-lysine and L-glutamate. Although C. glutamicum has various industrial applications, a limited number of tunable systems are available to engineer it for efficient production of platform chemicals. Therefore, in this study, we developed a novel tunable promoter system based on repeats of the Vitreoscilla hemoglobin promoter (Pvgb). Tunable expression of green fluorescent protein (GFP) was investigated under one, four, and eight repeats of Pvgb (Pvgb, Pvgb4, and Pvgb8). The intensity of fluorescence in recombinant C. glutamicum strains increased as the number of Pvgb increased from single to eight (Pvgb8) repeats. Furthermore, we demonstrated the application of the new Pvgb promoter-based vector system as a platform for metabolic engineering of C. glutamicum by investigating 5-aminovaleric acid (5-AVA) and gamma-aminobutyric acid (GABA) production in several C. glutamicum strains. The profile of 5-AVA and GABA production by the recombinant strains were evaluated to investigate the tunable expression of key enzymes such as DavBA and GadBmut. We observed that 5-AVA and GABA production by the recombinant strains increased as the number of Pvgb used for the expression of key proteins increased. The recombinant C. glutamicum strain expressing DavBA could produce higher amounts of 5-AVA under the control of Pvgb8 (3.69 ± 0.07 g/L) than the one under the control of Pvgb (3.43 ± 0.10 g/L). The average gamma-aminobutyric acid production also increased in all the tested strains as the number of Pvgb used for GadBmut expression increased from single (4.81–5.31 g/L) to eight repeats (4.94–5.58 g/L).
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