High‐affinity l‐malate transporter DcuE of Actinobacillus succinogenes catalyses reversible exchange of C4‐dicarboxylates

Abstract

Summary Actinobacillus succinogenes is a natural succinate producer, which is the result of fumarate respiration. Succinate production from anaerobic growth with C4‐dicarboxylates requires transporters catalysing uptake and efflux of C4‐dicarboxylates. Transporter Asuc_1999 (DcuE) found in A. succinogenes belongs to the Dcu family and was considered the main transporter for fumarate respiration. However, deletion of dcuE affected l‐malate uptake of A. succinogenes rather than fumarate uptake. DcuE complemented anaerobic growth of Escherichia coli on l‐malate or fumarate; thus, the transporter was characterized in E. coli heterologously. Time‐dependent uptake and competitive inhibition assays demonstrated that l‐malate is the most preferred substrate for uptake by DcuE. The Vmax of DcuE for l‐malate was 20.04 μmol/gDW·min with Km of 57 μM. The Vmax for l‐malate was comparable to that for fumarate, whereas the Km for l‐malate was 8 times lower than that for fumarate. The catalytic efficiency of DcuE for l‐malate was 7.3‐fold higher than that for fumarate, showing high efficiency and high affinity for l‐malate. Furthermore, DcuE catalysed the reversible exchange of three C4‐dicarboxylates – l‐malate, fumarate and succinate – but the preferred substrate for uptake was l‐malate. Under physiological conditions, the C4‐dicarboxylates were reduced to succinate. Therefore, DcuE is proposed as the l‐malate/succinate antiporter in A. succinogenes.

Asuc_0142 of Actinobacillus succinogenes 130Z is the l-aspartate/C4-dicarboxylate exchanger DcuA

“…For homologous recombination, transformation of pMB179 to A. succinogenes 130Z was performed by electroporation [9]. The cell suspension was cultured on a BHI plate containing sucrose to remove the plasmid and obtain the Δ0142 strain of A.…”

“…DcuE (Asuc_1999) is produced constitutively under anaerobic conditions. This transporter is of the DcuB-type and has a strong preference for l-malate and l-malate/C4DC antiport [9,10]. The second transporter, Asuc_0142, is inducible under anaerobic conditions.…”

“…In A. succinogenes a DcuB-type transporter has been termed DcuE [9, 10]. DcuA, on the other hand, has a preference for l -aspartate and catalyses the l -aspartate/succinate exchange [11].…”

“…DcuB has a high affinity for l -malate and fumarate and is used for the l -malate/succinate or fumarate/succinate exchange. In A. succinogenes a DcuB-type transporter has been termed DcuE [ 9, 10 ]. DcuA, on the other hand, has a preference for l -aspartate and catalyses the l -aspartate/succinate exchange [ 11 ].…”

Metabolic and Microbial Community Engineering for Four-Carbon Dicarboxylic Acid Production from CO2-Derived Glycogen in the Cyanobacterium Synechocystis sp. PCC6803

“…E. coli utilizes citrate, aspartate, tartrate, fumarate, and malate under anaerobic conditions. 41 The fumarate/succinate antiporter Dcu system (dicarboxylate uptake), which is highly conserved in enteric bacteria, including A. succinogenes, 42 is responsible for the uptake of external C4-dicarboxylates, such as fumarate, malate, and aspartate. Fumarase and aspartase catalyze the conversion of Lmalate and aspartate into fumarate, respectively, resulting in fumarate respiration, which generates a transmembrane proton potential to drive ATP synthesis.…”