Quinone-dependent D-lactate dehydrogenase Dld (Cg1027) is essential for growth of Corynebacterium glutamicum on D-lactate

Kato, Osamu; Youn, Jung-Won; Stansen, K Corinna; Matsui, Daisuke; Oikawa, Tadao; Wendisch, Volker F.

doi:10.1186/1471-2180-10-321

Cited by 33 publications

(24 citation statements)

References 63 publications

(65 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In C. glutamicum, proton motive force is generated largely via cytochrome bc 1 -aa 3 supercomplex and/or less efficiently by cytochrome bd oxidase (66). NDH-II-defective strains have been described; they oxidize NADH by the coupling of NAD-dependent lactate dehydrogenase with quinone-dependent lactate dehydrogenase LldD (48) or Dld (67) and/or by the coupling of NAD-dependent malate dehydrogenase and quinone-dependent malate oxidoreductase (68) to compensate for the lack of NDH-II in C. glutamicum (66). It is conceivable that suppressor mutations leading to NADP-dependent lactate dehydrogenase or NADP-dependent malate dehydrogenase activity might have relieved growth inhibition by excess NADPH; however, these have not been observed in this study.…”

Section: Discussionmentioning

confidence: 99%

Metabolic Engineering of an ATP-Neutral Embden-Meyerhof-Parnas Pathway in Corynebacterium glutamicum: Growth Restoration by an Adaptive Point Mutation in NADH Dehydrogenase

Reddy

Lindner

Wendisch

2015

Appl Environ Microbiol

View full text Add to dashboard Cite

Corynebacterium glutamicum uses the Embden-Meyerhof-Parnas pathway of glycolysis and gains 2 mol of ATP per mol of glucose by substrate-level phosphorylation (SLP). To engineer glycolysis without net ATP formation by SLP, endogenous phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was replaced by nonphosphorylating NADPdependent glyceraldehyde-3-phosphate dehydrogenase (GapN) from Clostridium acetobutylicum, which irreversibly converts glyceraldehyde-3-phosphate (GAP) to 3-phosphoglycerate (3-PG) without generating ATP. As shown recently (S. Takeno, R. Murata, R. Kobayashi, S. Mitsuhashi, and M. Ikeda, Appl Environ Microbiol 76:7154 -7160, 2010, http://dx.doi.org/10.1128/AEM.01464-10), this ATP-neutral, NADPH-generating glycolytic pathway did not allow for the growth of Corynebacterium glutamicum with glucose as the sole carbon source unless hitherto unknown suppressor mutations occurred; however, these mutations were not disclosed. In the present study, a suppressor mutation was identified, and it was shown that heterologous expression of udhA encoding soluble transhydrogenase from Escherichia coli partly restored growth, suggesting that growth was inhibited by NADPH accumulation. Moreover, genome sequence analysis of second-site suppressor mutants that were able to grow faster with glucose revealed a single point mutation in the gene of non-proton-pumping NADH:ubiquinone oxidoreductase (NDH-II) leading to the amino acid change D213G, which was shared by these suppressor mutants. Since related NDH-II enzymes accepting NADPH as the substrate possess asparagine or glutamine residues at this position, D213G, D213N, and D213Q variants of C. glutamicum NDH-II were constructed and were shown to oxidize NADPH in addition to NADH. Taking these findings together, ATP-neutral glycolysis by the replacement of endogenous NAD-dependent GAPDH with NADP-dependent GapN became possible via oxidation of NADPH formed in this pathway by mutant NADPH-accepting NDH-II D213G and thus by coupling to electron transport phosphorylation (ETP).A TP generation occurs naturally by substrate-level phosphorylation (SLP), electron transport phosphorylation (ETP), photophosphorylation, and decarboxylation phosphorylation. The Embden-Meyerhof-Parnas (EMP) pathway of glycolysis supplies ATP by SLP in the absence of terminal electron acceptors or anaerobic conditions, as well as supplying direct precursors for biomass formation (1). Bacteria and archaea possess one or more of multiple biologically feasible routes for glucose catabolism, such as the EMP pathway, the Entner-Doudoroff (ED) pathway, and the phosphoketolase pathway, which yield zero to 3 ATP molecules per glucose molecule and exist in different variants. In natural habitats, a trade-off between the rate and the yield of ATP production is observed for heterotrophic organisms; faster but less efficient ATP production may be advantageous under conditions characterized by a low abundance of growth substrates (2, 3). For example, Zymomonas mobilis ferments suga...

show abstract

Section: Discussionmentioning

confidence: 99%

Metabolic Engineering of an ATP-Neutral Embden-Meyerhof-Parnas Pathway in Corynebacterium glutamicum: Growth Restoration by an Adaptive Point Mutation in NADH Dehydrogenase

Reddy

Lindner

Wendisch

2015

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…Kanamycin-resistant transformants were the result of chromosomal integration by single crossover events, as verified by Southern blotting. Inactivation of the lldD gene was confirmed by the absence of LLD activity (Kato et al 2010) in the membrane fraction of the RES::pAG1001 strain.…”

Section: Dna Isolation Manipulation and Transfermentioning

confidence: 82%

“…The protein encoded by lldD (gene symbol cg3227, EC 1.1.2.3) utilises L-lactate, and the second is a D-lactate-specific enzyme encoded by dld (gene symbol cg1027, EC 1.1.2.4). Each has been shown to be necessary for growth of C. glutamicum on L-lactate and D-lactate, respectively, as the sole carbon source (Stansen et al 2005;Kato et al 2010). Both are believed to be flavin-dependent and have been kinetically characterised to some extent, the authors reporting sub-millimolar K m values for lactate (LLD: K m 0.51 mM for L-lactate Stansen et al 2005; DLD: K m 0.61 mM for D-lactate Kato et al 2010).…”

Section: Introductionmentioning

confidence: 95%

Kinetic characterisation of recombinant Corynebacterium glutamicum NAD+-dependent LDH over-expressed in E. coli and its rescue of an lldD − phenotype in C. glutamicum: the issue of reversibility re-examined

et al. 2011

View full text Add to dashboard Cite

The ldh gene of Corynebacterium glutamicum ATCC 13032 (gene symbol cg3219, encoding a 314 residue NAD+-dependent L-(+)-lactate dehydrogenase, EC 1.1.1.27) was cloned into the expression vector pKK388-1 and over-expressed in an ldhA-null E. coli TG1 strain upon isopropyl-β-D-thiogalactopyranoside (IPTG) induction. The recombinant protein (referred to here as CgLDH) was purified by a combination of dye-ligand and ion-exchange chromatography. Though active in its absence, CgLDH activity is enhanced 17- to 20-fold in the presence of the allosteric activator D-fructose-1,6-bisphosphate (Fru-1,6-P2). Contrary to a previous report, CgLDH has readily measurable reaction rates in both directions, with Vmax for the reduction of pyruvate being approximately tenfold that of the value for L-lactate oxidation at pH 7.5. No deviation from Michaelis-Menten kinetics was observed in the presence of Fru-1,6-P2, while a sigmoidal response (indicative of positive cooperativity) was seen towards L-lactate without Fru-1,6-P2. Strikingly, when introduced into an lldD- strain of C. glutamicum, constitutively expressed CgLDH enables the organism to grow on L-lactate as the sole carbon source.

show abstract

“…The nLDHs often play important roles in anaerobic fermentation to produce lactate from pyruvate (14,15). The iLDHs usually serve as the key enzymes to support the growth of microorganisms on lactate (9,(11)(12)(13). Furthermore, the iLDHs can be divided into D-iLDHs and L-iLDHs according to their chiral specificity.…”

mentioning

confidence: 99%

“…Many bacteria, such as Escherichia coli (9,10), Corynebacterium glutamicum (11), and Pseudomonas stutzeri (12), are able to utilize lactate for growth. During the lactate utilization process in these bacteria, lactate is first oxidized to pyruvate; pyruvate is then used as both a carbon and an energy source to support the growth of these bacteria.…”

mentioning

confidence: 99%

Utilization of d -Lactate as an Energy Source Supports the Growth of Gluconobacter oxydans

Sheng

Zhang

et al. 2015

Appl Environ Microbiol

View full text Add to dashboard Cite

b D-Lactate was identified as one of the few available organic acids that supported the growth of Gluconobacter oxydans 621H in this study. Interestingly, the strain used D-lactate as an energy source but not as a carbon source, unlike other lactate-utilizing bacteria. The enzymatic basis for the growth of G. oxydans 621H on D-lactate was therefore investigated. Although two putative NAD-independent D-lactate dehydrogenases, GOX1253 and GOX2071, were capable of oxidizing D-lactate, GOX1253 was the only enzyme able to support the D-lactate-driven growth of the strain. GOX1253 was characterized as a membrane-bound dehydrogenase with high activity toward D-lactate, while GOX2071 was characterized as a soluble oxidase with broad substrate specificity toward D-2-hydroxy acids. The latter used molecular oxygen as a direct electron acceptor, a feature that has not been reported previously in D-lactate-oxidizing enzymes. This study not only clarifies the mechanism for the growth of G. oxydans on D-lactate, but also provides new insights for applications of the important industrial microbe and the novel D-lactate oxidase.

show abstract

Quinone-dependent D-lactate dehydrogenase Dld (Cg1027) is essential for growth of Corynebacterium glutamicum on D-lactate

Cited by 33 publications

References 63 publications

Metabolic Engineering of an ATP-Neutral Embden-Meyerhof-Parnas Pathway in Corynebacterium glutamicum: Growth Restoration by an Adaptive Point Mutation in NADH Dehydrogenase

Metabolic Engineering of an ATP-Neutral Embden-Meyerhof-Parnas Pathway in Corynebacterium glutamicum: Growth Restoration by an Adaptive Point Mutation in NADH Dehydrogenase

Kinetic characterisation of recombinant Corynebacterium glutamicum NAD+-dependent LDH over-expressed in E. coli and its rescue of an lldD − phenotype in C. glutamicum: the issue of reversibility re-examined

Utilization of d -Lactate as an Energy Source Supports the Growth of Gluconobacter oxydans

Contact Info

Product

Resources

About