Analysis of redox responses during TNT transformation by Clostridium acetobutylicum ATCC 824 and mutants exhibiting altered metabolism

Cai, Xianpeng; Servinsky, Matthew D.; Kiel, James T.; Sund, Christian; Bennett, George N.

doi:10.1007/s00253-012-4253-3

Cited by 11 publications

(9 citation statements)

References 37 publications

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“…As expected, increasingly oxidized carbohydrates resulted in the formation of increasingly oxidized products as evidenced by comparing the acetate:butyrate ratios of the fermentations. Measured H 2 :CO 2 ratios for glucose and gluconate were similar to those seen in previous reports [10,31]. The two main sources of reductant in C. acetobutylicum are NADH from lower glycolysis and reduced ferredoxin from PFOR.…”

Section: Resultssupporting

confidence: 87%

“…It is therefore more favorable from the standpoint of ATP yield to use the hydrogenase to reoxidize electron carriers. Electrons can be shuffled between NADH and ferredoxin by the NADH-ferredoxin oxidoreductase, thus it is possible for reducing equivalents formed in lower glycolysis to be oxidized indirectly via the hydrogenase [31,32]. An excess of reductant was present in the glucose and gluconate fermentations and the hydrogenase was saturated, as indicated by H 2 :CO 2 ratios greater than one and the substantial production of butyrate.…”

Section: Resultsmentioning

confidence: 99%

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Fermentation of oxidized hexose derivatives by Clostridium acetobutylicum

Servinsky

Liu²,

Gerlach³

et al. 2014

Microb Cell Fact

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BackgroundClostridium acetobutylicum fermentations are promising for production of commodity chemicals from heterogeneous biomass due to the wide range of substrates the organism can metabolize. Much work has been done to elucidate the pathways for utilization of aldoses, but little is known about metabolism of more oxidized substrates. Two oxidized hexose derivatives, gluconate and galacturonate, are present in low cost feedstocks, and their metabolism will contribute to overall metabolic output of these substrates.ResultsA complete metabolic network for glucose, gluconate, and galacturonate utilization was generated using online databases, previous studies, genomic context, and experimental data. Gluconate appears to be metabolized via the Entner-Doudoroff pathway, and is likely dehydrated to 2-keto-3-deoxy-gluconate before phosphorylation to 2-keto-3-deoxy-6-P-gluconate. Galacturonate appears to be processed via the Ashwell pathway, converging on a common metabolite for gluconate and galacturonate metabolism, 2-keto-3-deoxygluconate. As expected, increasingly oxidized substrates resulted in increasingly oxidized products with galacturonate fermentations being nearly homoacetic. Calculations of expected ATP and reducing equivalent yields and experimental data suggested galacturonate fermentations were reductant limited. Galacturonate fermentation was incomplete, which was not due solely to product inhibition or the inability to utilize low concentrations of galacturonate. Removal of H2 and CO2 by agitation resulted in faster growth, higher cell densities, formation of relatively more oxidized products, and higher product yields for cultures grown on glucose or gluconate. In contrast, cells grown on galacturonate showed reduced growth rates upon agitation, which was likely due to loss in reductant in the form of H2. The growth advantage seen on agitated glucose or gluconate cultures could not be solely attributed to improved ATP economics, thereby indicating other factors are also important.ConclusionsThe metabolic network presented in this work should facilitate similar reconstructions in other organisms, and provides a further understanding of the pathways involved in metabolism of oxidized feedstocks and carbohydrate mixtures. The nearly homoacetic fermentation during growth on galacturonate indicates further optimization of this and related organisms could provide a route to an effective biologically derived acetic acid production platform. Furthermore, the pathways could be targeted to decrease production of undesirable products during fermentations of heterogeneous biomass.

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Section: Resultssupporting

confidence: 87%

Section: Resultsmentioning

confidence: 99%

Fermentation of oxidized hexose derivatives by Clostridium acetobutylicum

Servinsky

Liu²,

Gerlach³

et al. 2014

Microb Cell Fact

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“…The antisense patterns have strong expression levels over the background signal, are not strongly correlated with per‐base sequencing depth levels on the sense strand, and display different stress response profiles than the hydA gene. Natural hydA antisense genes have not been described in C. acetobutylicum , though artificial hydA antisense has been useful for understanding TNT bioremediation in this strain …”

Section: Resultsmentioning

confidence: 99%

RNAseq‐based transcriptome assembly of Clostridium acetobutylicum for functional genome annotation and discovery

Ralston

Papoutsakis

2018

AIChE Journal

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Accurate genome annotations are essential in modern biology and biotechnology, yet they are still largely based on genome sequencing and comparative analyses. We show that the Clostridium acetobutylicum genome annotation can be markedly improved by integrating bioinformatic predictions with RNA sequencing (RNAseq) data. Samples were acquired under butanol, butyrate, and unstressed treatments across various growth conditions. Analysis of an initial assembly revealed errors due to background signals and limitations of assembly algorithms. Hurdles for RNAseq transcriptome mapping include optimizing library complexity and sequencing depth, yet most studies report low sequencing depth and ignore the effect of ribosomal RNA abundance. An integrative analysis was developed to combine motif predictions, single‐nucleotide resolution sequencing depth, and library complexity to resolve difficulties in assembly curation. This minimized false positive error and determined gene boundaries, in some cases, to the exact base‐pair of prior studies. This is the first strand‐specific transcriptome assembly in a Clostridium organism. © 2018 American Institute of Chemical Engineers AIChE J, 64: 4271–4280, 2018

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“…While it has been demonstrated that the [FeFe]-hydrogenase oxidizes H2 in vitro, the physiologically relevant need for ferredoxin oxidation indicates that its primary role is H2 production [10]. During acidogenesis, the H2/CO2 ratio is approximately 1.2, but this decreases to ~0.7 during solventogenesis as a result of rerouting the electron flow from H2 to alcohol formation [5,6]. Reduction of carbohydrates and carboxylic acids to alcohols during solventogenesis changes the requirements for reduced cofactors, …”

Section: Discussionmentioning

confidence: 99%

“…Hydrogenases can produce and/or consume H 2 , depending on metabolic state and/or the electron carrying cofactors used. In C. acetobutylicum, the majority of hydrogenase studies have focused on the [FeFe]-hydrogenases, which are important for ferredoxin recycling and the reduction of xenobiotic compounds [1,5]. The saccharolytic nature of C. acetobutylicum and the associated high metabolic flux through PFOR leads to a strong demand for ferredoxin oxidation by the [FeFe]-hydrogenase in growth conditions important for solvent formation [40].…”

Section: Discussionmentioning

confidence: 99%

Hydrogen-Cycling during Solventogenesis in Clostridium acetobutylicum American Type Culture Collection (ATCC) 824 Requires the [NiFe]-Hydrogenase for Energy Conservation

et al. 2018

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Clostridium acetobutylicum has traditionally been used for production of acetone, butanol, and ethanol (ABE). Butanol is a commodity chemical due in part to its suitability as a biofuel; however, the current yield of this product from biological systems is not economically feasible as an alternative fuel source. Understanding solvent phase physiology, solvent tolerance, and their genetic underpinning is key for future strain optimization of the bacterium. This study shows the importance of a [NiFe]-hydrogenase in solvent phase physiology. C. acetobutylicum genes ca_c0810 and ca_c0811, annotated as a HypF and HypD maturation factor, were found to be required for [NiFe]-hydrogenase activity. They were shown to be part of a polycistronic operon with other hyp genes. Hydrogenase activity assays of the ∆hypF/hypD mutant showed an almost complete inactivation of the [NiFe]-hydrogenase. Metabolic studies comparing ∆hypF/hypD and wild type (WT) strains in planktonic and sessile conditions indicated the hydrogenase was important for solvent phase metabolism. For the mutant, reabsorption of acetate and butyrate was inhibited during solventogenesis in planktonic cultures, and less ABE was produced. During sessile growth, the ∆hypF/hypD mutant had higher initial acetone: butanol ratios, which is consistent with the inability to obtain reduced cofactors via H 2 uptake. In sessile conditions, the ∆hypF/hypD mutant was inhibited in early solventogenesis, but it appeared to remodel its metabolism and produced mainly butanol in late solventogenesis without the uptake of acids. Energy filtered transmission electron microscopy (EFTEM) mapped Pd(II) reduction via [NiFe]-hydrogenase induced H 2 oxidation at the extracelluar side of the membrane on WT cells. A decrease of Pd(0) deposits on ∆hypF/hypD comparatively to WT indicates that the [NiFe]-hydrogenase contributed to the Pd(II) reduction. Calculations of reaction potentials during acidogenesis and solventogenesis predict the [NiFe]-hydrogenase can couple NAD + reduction with membrane transport of electrons. Extracellular oxidation of H 2 combined with the potential for electron transport across the membrane indicate that the [NiFe}-hydrogenase contributes to proton motive force maintenance via hydrogen cycling.

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Analysis of redox responses during TNT transformation by Clostridium acetobutylicum ATCC 824 and mutants exhibiting altered metabolism

Cited by 11 publications

References 37 publications

Fermentation of oxidized hexose derivatives by Clostridium acetobutylicum

Fermentation of oxidized hexose derivatives by Clostridium acetobutylicum

RNAseq‐based transcriptome assembly of Clostridium acetobutylicum for functional genome annotation and discovery

Hydrogen-Cycling during Solventogenesis in Clostridium acetobutylicum American Type Culture Collection (ATCC) 824 Requires the [NiFe]-Hydrogenase for Energy Conservation

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