Heterologous overproduction of 2[4Fe4S]- and [2Fe2S]-type clostridial ferredoxins and [2Fe2S]-type agrobacterial ferredoxin

Huang, Haiyan; Hu, Liejie; Yu, Wenjun; Li, Huili; Tao, Fei; Xie, Huijun; Wang, Shuning

doi:10.1016/j.pep.2015.12.019

Cited by 13 publications

(10 citation statements)

References 53 publications

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“…SDS-PAGE analysis of the ferredoxin partially purified from S. aciditrophicus and ferredoxin recombinantly produced in E. coli had a molecular mass of 12 kDa, roughly twice that predicted from the amino acid sequence (Supplementary Figure S3). This difference suggests that the ferredoxin is a homodimer or there was anomalous migration as a result of interactions of the negatively charged peptides and SDS micelles (Darimont and Sterner, 1994;Huang et al, 2016;Kpebe et al, 2018).…”

Section: Purification and Characterization Of S Aciditrophicus Ferrementioning

confidence: 99%

The Beta Subunit of Non-bifurcating NADH-Dependent [FeFe]-Hydrogenases Differs From Those of Multimeric Electron-Bifurcating [FeFe]-Hydrogenases

et al. 2020

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A non-bifurcating NADH-dependent, dimeric [FeFe]-hydrogenase (HydAB) from Syntrophus aciditrophicus was heterologously produced in Escherichia coli, purified and characterized. Purified recombinant HydAB catalyzed NAD + reduction coupled to hydrogen oxidation and produced hydrogen from NADH without the involvement of ferredoxin. Hydrogen partial pressures (2.2-40.2 Pa) produced by the purified recombinant HydAB at NADH to NAD + ratios of 1-5 were similar to the hydrogen partial pressures generated by pure and cocultures of S. aciditrophicus (5.9-36.6 Pa). Thus, the hydrogen partial pressures observed in metabolizing cultures and cocultures of S. aciditrophicus can be generated by HydAB if S. aciditrophicus maintains NADH to NAD + ratios greater than one. The flavin-containing beta subunits from S. aciditrophicus HydAB and the non-bifurcating NADH-dependent S. wolfei Hyd1ABC share a number of conserved residues with the flavin-containing beta subunits from nonbifurcating NADH-dependent enzymes such as NADH:quinone oxidoreductases and formate dehydrogenases. A number of differences were observed between sequences of these non-bifurcating NADH-dependent enzymes and [FeFe]-hydrogenases and formate dehydrogenases known to catalyze electron bifurcation including differences in the number of [Fe-S] centers and in conserved residues near predicted cofactor binding sites. These differences can be used to distinguish members of these two groups of enzymes and may be relevant to the differences in ferredoxin-dependence and ability to mediate electron-bifurcation. These results show that two phylogenetically distinct syntrophic fatty acid-oxidizing bacteria, Syntrophomonas wolfei a member of the phylum Firmicutes, and S. aciditrophicus, a member of the class Deltaproteobacteria, possess functionally similar [FeFe]-hydrogenases that produce hydrogen from NADH

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Section: Purification and Characterization Of S Aciditrophicus Ferrementioning

confidence: 99%

The Beta Subunit of Non-bifurcating NADH-Dependent [FeFe]-Hydrogenases Differs From Those of Multimeric Electron-Bifurcating [FeFe]-Hydrogenases

et al. 2020

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“…There was no effect from expressing T. saccharolyticum ferredoxin in C. thermocellum . It is known that ferredoxins from one organism can often transfer electrons to proteins from another organism [35], so it would not be surprising if one of the native C. thermocellum ferredoxins was sufficient for electron transfer from T. saccharolyticum Pfor protein.…”

Section: Resultsmentioning

confidence: 99%

Expressing the Thermoanaerobacterium saccharolyticum pforA in engineered Clostridium thermocellum improves ethanol production

Hon

Holwerda

Worthen

et al. 2018

Biotechnol Biofuels

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BackgroundClostridium thermocellum has been the subject of multiple metabolic engineering strategies to improve its ability to ferment cellulose to ethanol, with varying degrees of success. For ethanol production in C. thermocellum, the conversion of pyruvate to acetyl-CoA is catalyzed primarily by the pyruvate ferredoxin oxidoreductase (PFOR) pathway. Thermoanaerobacterium saccharolyticum, which was previously engineered to produce ethanol of high yield (> 80%) and titer (70 g/L), also uses a pyruvate ferredoxin oxidoreductase, pforA, for ethanol production.ResultsHere, we introduced the T. saccharolyticum pforA and ferredoxin into C. thermocellum. The introduction of pforA resulted in significant improvements to ethanol yield and titer in C. thermocellum grown on 50 g/L of cellobiose, but only when four other T. saccharolyticum genes (adhA, nfnA, nfnB, and adhEG544D) were also present. T. saccharolyticum ferredoxin did not have any observable impact on ethanol production. The improvement to ethanol production was sustained even when all annotated native C. thermocellum pfor genes were deleted. On high cellulose concentrations, the maximum ethanol titer achieved by this engineered C. thermocellum strain from 100 g/L Avicel was 25 g/L, compared to 22 g/L for the reference strain, LL1319 (adhA(Tsc)-nfnAB(Tsc)-adhEG544D (Tsc)) under similar conditions. In addition, we also observed that deletion of the C. thermocellum pfor4 results in a significant decrease in isobutanol production.ConclusionsHere, we demonstrate that the pforA gene can improve ethanol production in C. thermocellum as part of the T. saccharolyticum pyruvate-to-ethanol pathway. In our previous strain, high-yield (~ 75% of theoretical) ethanol production could be achieved with at most 20 g/L substrate. In this strain, high-yield ethanol production can be achieved up to 50 g/L substrate. Furthermore, the introduction of pforA increased the maximum titer by 14%.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1245-2) contains supplementary material, which is available to authorized users.

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“…For anaerobic purification of CcoG, all buffers were degassed using argon gas and transferred to an anaerobic chamber 1 d prior to usage, and all steps were carried out in sealed vessels or in an anaerobic chamber. An epitope-tagged version of R. capsulatus CcoG and its mutated variants was overproduced in the E. coli BL21 strain carrying the plasmids pBADccoG Myc-His , pBADccoG Myc-His (ΔCu), and pBADccoG Myc-His (ΔFeS) (SI Appendix, Table S2) together with the plasmid pRKISC, kindly provided by Y. Takahashi, Saitama University, Saitama, Japan, harboring the endogenous Fe-S cluster biogenesis (Isc) system, to enhance the Fe-S cluster incorporation (52). Cells were induced with 0.5% L-Ara and 0.1% IPTG at an OD 600 of 0.5 for 3.5 h and membranes were prepared using a French pressure cell at 16,000 psi in 25 mM Tris•HCl (pH 7.5), 300 mM NaCl, 1 mM Pefabloc, 0.5 mM aminocaproic acid, 30 μg/mL lysozyme, and 30 μg/mL DNase I.…”

Section: Methodsmentioning

confidence: 99%

The cbb ₃ -type cytochrome oxidase assembly factor CcoG is a widely distributed cupric reductase

Marckmann

Trasnea

Schimpf

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Copper (Cu)-containing proteins execute essential functions in prokaryotic and eukaryotic cells, but their biogenesis is challenged by high Cu toxicity and the preferential presence of Cu(II) under aerobic conditions, while Cu(I) is the preferred substrate for Cu chaperones and Cu-transport proteins. These proteins form a coordinated network that prevents Cu accumulation, which would lead to toxic effects such as Fenton-like reactions and mismetalation of other metalloproteins. Simultaneously, Cu-transport proteins and Cu chaperones sustain Cu(I) supply for cuproprotein biogenesis and are therefore essential for the biogenesis of Cu-containing proteins. In eukaryotes, Cu(I) is supplied for import and trafficking by cell-surface exposed metalloreductases, but specific cupric reductases have not been identified in bacteria. It was generally assumed that the reducing environment of the bacterial cytoplasm would suffice to provide sufficient Cu(I) for detoxification and cuproprotein synthesis. Here, we identify the proposed cbb3-type cytochrome c oxidase (cbb3-Cox) assembly factor CcoG as a cupric reductase that binds Cu via conserved cysteine motifs and contains 2 low-potential [4Fe-4S] clusters required for Cu(II) reduction. Deletion of ccoG or mutation of the cysteine residues results in defective cbb3-Cox assembly and Cu sensitivity. Furthermore, anaerobically purified CcoG catalyzes Cu(II) but not Fe(III) reduction in vitro using an artificial electron donor. Thus, CcoG is a bacterial cupric reductase and a founding member of a widespread class of enzymes that generate Cu(I) in the bacterial cytosol by using [4Fe-4S] clusters.

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Heterologous overproduction of 2[4Fe4S]- and [2Fe2S]-type clostridial ferredoxins and [2Fe2S]-type agrobacterial ferredoxin

Cited by 13 publications

References 53 publications

The Beta Subunit of Non-bifurcating NADH-Dependent [FeFe]-Hydrogenases Differs From Those of Multimeric Electron-Bifurcating [FeFe]-Hydrogenases

The Beta Subunit of Non-bifurcating NADH-Dependent [FeFe]-Hydrogenases Differs From Those of Multimeric Electron-Bifurcating [FeFe]-Hydrogenases

Expressing the Thermoanaerobacterium saccharolyticum pforA in engineered Clostridium thermocellum improves ethanol production

The cbb ₃ -type cytochrome oxidase assembly factor CcoG is a widely distributed cupric reductase

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Heterologous overproduction of 2[4Fe4S]- and [2Fe2S]-type clostridial ferredoxins and [2Fe2S]-type agrobacterial ferredoxin

Cited by 13 publications

References 53 publications

The Beta Subunit of Non-bifurcating NADH-Dependent [FeFe]-Hydrogenases Differs From Those of Multimeric Electron-Bifurcating [FeFe]-Hydrogenases

The Beta Subunit of Non-bifurcating NADH-Dependent [FeFe]-Hydrogenases Differs From Those of Multimeric Electron-Bifurcating [FeFe]-Hydrogenases

Expressing the Thermoanaerobacterium saccharolyticum pforA in engineered Clostridium thermocellum improves ethanol production

The cbb 3 -type cytochrome oxidase assembly factor CcoG is a widely distributed cupric reductase

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The cbb ₃ -type cytochrome oxidase assembly factor CcoG is a widely distributed cupric reductase