A Na+-coupled C4-dicarboxylate transporter (Asuc_0304) and aerobic growth of Actinobacillus succinogenes on C4-dicarboxylates

Abstract

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).

Enterobacter sp. LU1 as a novel succinic acid producer – co‐utilization of glycerol and lactose

“…By contrast, glycerol does not suppress the metabolism of fatty acids in Y. lipolytica yeast grown on a mixture of free saturated fatty acids and glycerol (Papanikolaou et al ., ) or in Y. lipolytica cultivated on rapeseed and sunflower oils (Kamzolova et al ., , ). The improvement in glycerol utilization in the presence of fumaric acid was thoroughly described in previous studies on glycerol fermentation (Ryu et al ., ; Na Rhie et al ., ). Malic acid, which is a direct metabolic precursor of fumaric acid in bacterial metabolism, also improved glycerol utilization.…”

Structural basis for the reaction cycle of DASS dicarboxylate transporters

“…Transformed cells were grown in LB broth with ampicillin to an OD 595 ~0.8, then diluted to an OD 595 of 0.1. Dilute cells were inoculated into eM9 media supplemented with 50 mM αKG ( Rhie et al, 2014 ), and cell growth monitored using a Tecan SPECTRAFluor Plus microplate reader (Männedorf, Switzerland) incubated at 37°C.…”

“…Transformed cells were grown in LB broth with ampicillin to an OD 595 ~ 0.8, then diluted to an OD 595 of 0.1. Dilute cells were inoculated into eM9 media supplemented with mM KG50 , and cell growth monitored using a Tecan SPECTRAFluor Plus microplate reader (Männedorf, Switzerland) incubated at 37 ºC.LaINDY expression and purificationLaINDY was expressed by autoinduction51 at 25 ºC overnight in the E. coli strain BL21 DE3 transformed with the pET-LaINDY plasmid. Cells were harvested and lysed in a buffer of 50 mM Tris pH 8.0, 400 mM NaCl, 10 mM Imidazole.…”

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

“…l ‐Malate can replace fumarate, which is dehydrated by fumarase (Asuc_0956) to fumarate (Janausch et al ., ; McKinlay et al ., ; Unden et al ., ). A. succinogenes grows well anaerobically on fumarate or l ‐malate when glycerol is supplied as an electron donor (Rhie et al ., ). The growth by fumarate respiration with external fumarate or l ‐malate requires transport systems catalysing the uptake and efflux of C 4 ‐dicarboxylates.…”