Construction of expression vectors for protein production in Gluconobacter oxydans

Kallnik, Verena; Meyer, María; Deppenmeier, Uwe; Schweiger, Paul

doi:10.1016/j.jbiotec.2010.10.069

Cited by 43 publications

(27 citation statements)

References 34 publications

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“…For the complementation of lldEFAD2, fragment lldEFAD2 was PCR amplified by using lldE-pBBR-F/lldEFAD2-pBBR-R as primers. The resulting PCR product was cloned into pBBR1MCS-5, a broad-host-range plasmid (61). The resulting plasmid was conjugated into P. putida KT2440 ΔlldE ΔglcD via a triparental mating method, as previously described (6).…”

Section: Discussionmentioning

confidence: 99%

A Bacterial Multidomain NAD-Independent d -Lactate Dehydrogenase Utilizes Flavin Adenine Dinucleotide and Fe-S Clusters as Cofactors and Quinone as an Electron Acceptor for d -Lactate Oxidization

et al. 2017

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Bacterial membrane-associated NAD-independent d-lactate dehydrogenase (Fe-S d-iLDH) oxidizes d-lactate into pyruvate. A sequence analysis of the enzyme reveals that it contains an Fe-S oxidoreductase domain in addition to a flavin adenine dinucleotide (FAD)-containing dehydrogenase domain, which differs from other typical d-iLDHs. Fe-S d-iLDH from KT2440 was purified as a His-tagged protein and characterized in detail. This monomeric enzyme exhibited activities with l-lactate and several d-2-hydroxyacids. Quinone was shown to be the preferred electron acceptor of the enzyme. The two domains of the enzyme were then heterologously expressed and purified separately. The Fe-S cluster-binding motifs predicted by sequence alignment were preliminarily verified by site-directed mutagenesis of the Fe-S oxidoreductase domain. The FAD-containing dehydrogenase domain retained 2-hydroxyacid-oxidizing activity, although it decreased compared to the full Fe-S d-iLDH. Compared to the intact enzyme, the FAD-containing dehydrogenase domain showed increased catalytic efficiency with cytochrome as the electron acceptor, but it completely lost the ability to use coenzyme Q Additionally, the FAD-containing dehydrogenase domain was no longer associated with the cell membrane, and it could not support the utilization of d-lactate as a carbon source. Based on the results obtained, we conclude that the Fe-S oxidoreductase domain functions as an electron transfer component to facilitate the utilization of quinone as an electron acceptor by Fe-S d-iLDH, and it helps the enzyme associate with the cell membrane. These functions make the Fe-S oxidoreductase domain crucial for the d-lactate utilization function of Fe-S d-iLDH. Lactate metabolism plays versatile roles in most domains of life. Lactate utilization processes depend on certain enzymes to oxidize lactate to pyruvate. In recent years, novel bacterial lactate-oxidizing enzymes have been continually reported, including the unique NAD-independent d-lactate dehydrogenase that contains an Fe-S oxidoreductase domain besides the typical flavin-containing domain (Fe-S d-iLDH). Although Fe-S d-iLDH is widely distributed among bacterial species, the investigation of it is insufficient. Fe-S d-iLDH from KT2440, which is the major d-lactate-oxidizing enzyme for the strain, might be a representative of this type of enzyme. A study of it will be helpful in understanding the detailed mechanisms underlying the lactate utilization processes.

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Section: Discussionmentioning

confidence: 99%

A Bacterial Multidomain NAD-Independent d -Lactate Dehydrogenase Utilizes Flavin Adenine Dinucleotide and Fe-S Clusters as Cofactors and Quinone as an Electron Acceptor for d -Lactate Oxidization

et al. 2017

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“…When the hdr-like gene cluster from dsrE to lbpA (Fig. 1) was reintroduced in trans on broad host range plasmid pBBR1p264 [50] such that it was expressed under a strong constitutive promoter from Gluconobacter oxydans, thiosulfate oxidation and sulfate formation capacities were completely restored (Fig. 6a).…”

Section: Thiosulfate Oxidation In H Denitrificans Wild-type and Mutamentioning

confidence: 99%

A novel bacterial sulfur oxidation pathway provides a new link between the cycles of organic and inorganic sulfur compounds

2018

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Dimethylsulfide (DMS) plays a globally significant role in carbon and sulfur cycling and impacts Earth's climate because its oxidation products serve as nuclei for cloud formation. While the initial steps of aerobic DMS degradation and the fate of its carbon atoms are reasonably well documented, oxidation of the contained sulfur is largely unexplored. Here, we identified a novel pathway of sulfur compound oxidation in the ubiquitously occurring DMS-degrader Hyphomicrobium denitrificans X that links the oxidation of the volatile organosulfur compound with that of the inorganic sulfur compound thiosulfate. DMS is first transformed to methanethiol from which sulfide is released and fully oxidized to sulfate. Comparative proteomics indicated thiosulfate as an intermediate of this pathway and pointed at a heterodisulfide reductase (Hdr)-like system acting as a sulfur-oxidizing entity. Indeed, marker exchange mutagenesis of hdr-like genes disrupted the ability of H. denitrificans to metabolize DMS and also prevented formation of sulfate from thiosulfate provided as an additional electron source during chemoorganoheterotrophic growth. Complementation with the hdr-like genes under a constitutive promoter rescued the phenotype on thiosulfate as well as on DMS. The production of sulfate from an organosulfur precursor via the Hdr-like system is previously undocumented and provides a new shunt in the biogeochemical sulfur cycle. Furthermore, our findings fill a long-standing knowledge gap in microbial dissimilatory sulfur metabolism because the Hdr-like pathway is abundant not only in chemoheterotrophs, but also in a wide range of chemo- and photolithoautotrophic sulfur oxidizers acting as key players in global sulfur cycling.

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“…For the complementation of gox1432 deletion mutants, the vector pBBR. p264.ST was used (Kallnik et al 2010). Gene gox1432 including its native ribosomal binding site was amplified using the pBBR.p264.gox1432_Fw and pBBR.p264.…”

Section: Standard Molecular Biology Techniques and Cloningmentioning

confidence: 99%

Role of mannitol dehydrogenases in osmoprotection of Gluconobacter oxydans

Zahid¹,

Deppenmeier²

2016

Appl Microbiol Biotechnol

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Gluconobacter (G.) oxydans is able to incompletely oxidize various sugars and polyols for the production of biotechnologically important compound. Recently, we have shown that the organism produces and accumulates mannitol as compatible solute under osmotic stress conditions. The present study describes the role of two cytoplasmic mannitol dehydrogenases for osmotolerance of G. oxydans. It was shown that Gox1432 is a NADP-dependent mannitol dehydrogenase (EC 1.1.1.138), while Gox0849 uses NAD as cofactor (EC 1.1.1.67). The corresponding genes were deleted and the mutants were analyzed for growth under osmotic stress and non-stress conditions. A severe growth defect was detected for Δgox1432 when grown in high osmotic media, while the deletion of gox0849 had no effect when cells were exposed to 450 mM sucrose in the medium. Furthermore, the intracellular mannitol content was reduced in the mutant lacking the NADP-dependent enzyme Gox1432 in comparison to the parental strain and the Δgox0849 mutant under stress conditions. In addition, transcriptional analysis revealed that Gox1432 is more important for mannitol production in G. oxydans than Gox0849 as the transcript abundance of gene gox1432 was 30-fold higher than of gox0849. In accordance, the activity of the NADH-dependent enzyme Gox0849 in the cell cytoplasm was 10-fold lower in comparison to the NADPH-dependent mannitol dehydrogenase Gox1432. Overexpression of gox1432 in the corresponding deletion mutant restored growth of the cells under osmotic stress, further strengthening the importance of the NADP-dependent mannitol dehydrogenase for osmotolerance in G. oxydans. These findings provide detailed insights into the molecular mechanism of mannitol-mediated osmoprotection in G. oxydans and are helpful engineering strains with improved osmotolerance for biotechnological applications.

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Construction of expression vectors for protein production in Gluconobacter oxydans

Cited by 43 publications

References 34 publications

A Bacterial Multidomain NAD-Independent d -Lactate Dehydrogenase Utilizes Flavin Adenine Dinucleotide and Fe-S Clusters as Cofactors and Quinone as an Electron Acceptor for d -Lactate Oxidization

A Bacterial Multidomain NAD-Independent d -Lactate Dehydrogenase Utilizes Flavin Adenine Dinucleotide and Fe-S Clusters as Cofactors and Quinone as an Electron Acceptor for d -Lactate Oxidization

A novel bacterial sulfur oxidation pathway provides a new link between the cycles of organic and inorganic sulfur compounds

Role of mannitol dehydrogenases in osmoprotection of Gluconobacter oxydans

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