scholarly journals Converting Carbon Dioxide to Butyrate with an Engineered Strain of Clostridium ljungdahlii

mBio ◽  
2014 ◽  
Vol 5 (5) ◽  
Author(s):  
Toshiyuki Ueki ◽  
Kelly P. Nevin ◽  
Trevor L. Woodard ◽  
Derek R. Lovley
Keyword(s):  
Carbon Dioxide ◽  
Electron Flow ◽  
Microbial Conversion ◽  
Gene Encoding ◽  
Content Type ◽  
Microbial Electrosynthesis ◽  
Clostridium Ljungdahlii ◽  
Coa Transferase ◽  
Organic Products ◽  
Key Enzyme

ABSTRACTMicrobial conversion of carbon dioxide to organic commodities via syngas metabolism or microbial electrosynthesis is an attractive option for production of renewable biocommodities. The recent development of an initial genetic toolbox for the acetogenClostridium ljungdahliihas suggested thatC. ljungdahliimay be an effective chassis for such conversions. This possibility was evaluated by engineering a strain to produce butyrate, a valuable commodity that is not a natural product ofC. ljungdahliimetabolism. Heterologous genes required for butyrate production from acetyl-coenzyme A (CoA) were identified and introduced initially on plasmids and in subsequent strain designs integrated into theC. ljungdahliichromosome. Iterative strain designs involved increasing translation of a key enzyme by modifying a ribosome binding site, inactivating the gene encoding the first step in the conversion of acetyl-CoA to acetate, disrupting the gene which encodes the primary bifunctional aldehyde/alcohol dehydrogenase for ethanol production, and interrupting the gene for a CoA transferase that potentially represented an alternative route for the production of acetate. These modifications yielded a strain in which ca. 50 or 70% of the carbon and electron flow was diverted to the production of butyrate with H2or CO as the electron donor, respectively. These results demonstrate the possibility of producing high-value commodities from carbon dioxide withC. ljungdahliias the catalyst.IMPORTANCEThe development of a microbial chassis for efficient conversion of carbon dioxide directly to desired organic products would greatly advance the environmentally sustainable production of biofuels and other commodities.Clostridium ljungdahliiis an effective catalyst for microbial electrosynthesis, a technology in which electricity generated with renewable technologies, such as solar or wind, powers the conversion of carbon dioxide and water to organic products. Other electron donors forC. ljungdahliiinclude carbon monoxide, which can be derived from industrial waste gases or the conversion of recalcitrant biomass to syngas, as well as hydrogen, another syngas component. The finding that carbon and electron flow inC. ljungdahliican be diverted from the production of acetate to butyrate synthesis is an important step toward the goal of renewable commodity production from carbon dioxide with this organism.

scholarly journals Lactose-Inducible System for Metabolic Engineering of Clostridium ljungdahlii

2014 ◽  
Vol 80 (8) ◽  
pp. 2410-2416 ◽  
Author(s):  
Areen Banerjee ◽  
Ching Leang ◽  
Toshiyuki Ueki ◽  
Kelly P. Nevin ◽  
Derek R. Lovley
Keyword(s):  
Ethanol Production ◽  
Genetic Manipulation ◽  
Electron Flow ◽  
Model Organism ◽  
Inducible Expression ◽  
Carbon Flow ◽  
Wild Type ◽  
Content Type ◽  
Microbial Electrosynthesis ◽  
Clostridium Ljungdahlii

ABSTRACTThe development of tools for genetic manipulation ofClostridium ljungdahliihas increased its attractiveness as a chassis for autotrophic production of organic commodities and biofuels from syngas and microbial electrosynthesis and established it as a model organism for the study of the basic physiology of acetogenesis. In an attempt to expand the genetic toolbox forC. ljungdahlii, the possibility of adapting a lactose-inducible system for gene expression, previously reported forClostridium perfringens, was investigated. The plasmid pAH2, originally developed forC. perfringenswith agusAreporter gene, functioned as an effective lactose-inducible system inC. ljungdahlii. Lactose induction ofC. ljungdahliicontaining pB1, in which the gene for the aldehyde/alcohol dehydrogenase AdhE1 was downstream of the lactose-inducible promoter, increased expression ofadhE130-fold over the wild-type level, increasing ethanol production 1.5-fold, with a corresponding decrease in acetate production. Lactose-inducible expression ofadhE1in a strain in whichadhE1and theadhE1homologadhE2had been deleted from the chromosome restored ethanol production to levels comparable to those in the wild-type strain. Inducing expression ofadhE2similarly failed to restore ethanol production, suggesting thatadhE1is the homolog responsible for ethanol production. Lactose-inducible expression of the four heterologous genes necessary to convert acetyl coenzyme A (acetyl-CoA) to acetone diverted ca. 60% of carbon flow to acetone production during growth on fructose, and 25% of carbon flow went to acetone when carbon monoxide was the electron donor. These studies demonstrate that the lactose-inducible system described here will be useful for redirecting carbon and electron flow for the biosynthesis of products more valuable than acetate. Furthermore, this tool should aid in optimizing microbial electrosynthesis and for basic studies on the physiology of acetogenesis.

scholarly journals A Genetic System for Clostridium ljungdahlii: a Chassis for Autotrophic Production of Biocommodities and a Model Homoacetogen

2012 ◽  
Vol 79 (4) ◽  
pp. 1102-1109 ◽  
Author(s):  
Ching Leang ◽  
Toshiyuki Ueki ◽  
Kelly P. Nevin ◽  
Derek R. Lovley
Keyword(s):  
Ethanol Production ◽  
Gene Deletion ◽  
Genetic Manipulation ◽  
Electron Flow ◽  
Genetic System ◽  
Content Type ◽  
Suicide Vector ◽  
Double Deletion ◽  
Clostridium Ljungdahlii ◽  
Genomic Studies

ABSTRACTMethods for genetic manipulation ofClostridium ljungdahliiare of interest because of the potential for production of fuels and other biocommodities from carbon dioxide via microbial electrosynthesis or more traditional modes of autotrophy with hydrogen or carbon monoxide as the electron donor. Furthermore, acetogenesis plays an important role in the global carbon cycle. Gene deletion strategies required for physiological studies ofC. ljungdahliihave not previously been demonstrated. An electroporation procedure for introducing plasmids was optimized, and four different replicative origins for plasmid propagation inC. ljungdahliiwere identified. Chromosomal gene deletion via double-crossover homologous recombination with a suicide vector was demonstrated initially with deletion of the gene for FliA, a putative sigma factor involved in flagellar biogenesis and motility inC. ljungdahlii. Deletion offliAyielded a strain that lacked flagella and was not motile. To evaluate the potential utility of gene deletions for functional genomic studies and to redirect carbon and electron flow, the genes for the putative bifunctional aldehyde/alcohol dehydrogenases,adhE1andadhE2, were deleted individually or together. Deletion ofadhE1, but notadhE2, diminished ethanol production with a corresponding carbon recovery in acetate. The double deletion mutant had a phenotype similar to that of theadhE1-deficient strain. Expression ofadhE1intranspartially restored the capacity for ethanol production. These results demonstrate the feasibility of genetic investigations of acetogen physiology and the potential for genetic manipulation ofC. ljungdahliito optimize autotrophic biocommodity production.

scholarly journals Microbial Electrosynthesis: Feeding Microbes Electricity To Convert Carbon Dioxide and Water to Multicarbon Extracellular Organic Compounds

mBio ◽  
2010 ◽  
Vol 1 (2) ◽  
Author(s):  
Kelly P. Nevin ◽  
Trevor L. Woodard ◽  
Ashley E. Franks ◽  
Zarath M. Summers ◽  
Derek R. Lovley
Keyword(s):  
Carbon Dioxide ◽  
Solar Energy ◽  
Organic Compounds ◽  
Graphite Electrode ◽  
Chemical Bonds ◽  
Content Type ◽  
Organic Products ◽  
Microbial Carbon ◽  
New Form ◽  
Sporomusa Ovata

ABSTRACT The possibility of providing the acetogenic microorganism Sporomusa ovata with electrons delivered directly to the cells with a graphite electrode for the reduction of carbon dioxide to organic compounds was investigated. Biofilms of S. ovata growing on graphite cathode surfaces consumed electrons with the reduction of carbon dioxide to acetate and small amounts of 2-oxobutyrate. Electrons appearing in these products accounted for over 85% of the electrons consumed. These results demonstrate that microbial production of multicarbon organic compounds from carbon dioxide and water with electricity as the energy source is feasible. IMPORTANCE Reducing carbon dioxide to multicarbon organic chemicals and fuels with electricity has been identified as an attractive strategy to convert solar energy that is harvested intermittently with photovoltaic technology and store it as covalent chemical bonds. The organic compounds produced can then be distributed via existing infrastructure. Nonbiological electrochemical reduction of carbon dioxide has proven problematic. The results presented here suggest that microbiological catalysts may be a robust alternative, and when coupled with photovoltaics, current-driven microbial carbon dioxide reduction represents a new form of photosynthesis that might convert solar energy to organic products more effectively than traditional biomass-based strategies.

scholarly journals Electrosynthesis of Organic Compounds from Carbon Dioxide Is Catalyzed by a Diversity of Acetogenic Microorganisms

2011 ◽  
Vol 77 (9) ◽  
pp. 2882-2886 ◽  
Author(s):  
Kelly P. Nevin ◽  
Sarah A. Hensley ◽  
Ashley E. Franks ◽  
Zarath M. Summers ◽  
Jianhong Ou ◽  
...  
Keyword(s):  
Carbon Dioxide ◽  
Organic Compounds ◽  
Electrical Energy ◽  
Content Type ◽  
Transportation Fuels ◽  
Clostridium Ljungdahlii ◽  
Acetogenic Bacteria ◽  
Carbon Carbon Bonds ◽  
Potential Strategy ◽  
Reduce Carbon Dioxide

ABSTRACTMicrobial electrosynthesis, a process in which microorganisms use electrons derived from electrodes to reduce carbon dioxide to multicarbon, extracellular organic compounds, is a potential strategy for capturing electrical energy in carbon-carbon bonds of readily stored and easily distributed products, such as transportation fuels. To date, only one organism, the acetogenSporomusa ovata, has been shown to be capable of electrosynthesis. The purpose of this study was to determine if a wider range of microorganisms is capable of this process. Several other acetogenic bacteria, including two otherSporomusaspecies,Clostridium ljungdahlii,Clostridium aceticum, andMoorella thermoacetica, consumed current with the production of organic acids. In general acetate was the primary product, but 2-oxobutyrate and formate also were formed, with 2-oxobutyrate being the predominant identified product of electrosynthesis byC. aceticum. S. sphaeroides,C. ljungdahlii, andM. thermoaceticahad high (>80%) efficiencies of electrons consumed and recovered in identified products. The acetogenAcetobacterium woodiiwas unable to consume current. These results expand the known range of microorganisms capable of electrosynthesis, providing multiple options for the further optimization of this process.

scholarly journals Autotrophic Carbon Dioxide Fixation via the Calvin-Benson-Bassham Cycle by the Denitrifying Methanotroph “Candidatus Methylomirabilis oxyfera”

2014 ◽  
Vol 80 (8) ◽  
pp. 2451-2460 ◽  
Author(s):  
Olivia Rasigraf ◽  
Dorien M. Kool ◽  
Mike S. M. Jetten ◽  
Jaap S. Sinninghe Damsté ◽  
Katharina F. Ettwig
Keyword(s):  
Carbon Dioxide ◽  
Enrichment Culture ◽  
Carbon Assimilation ◽  
Carbon Dioxide Fixation ◽  
Oxidation Activity ◽  
Bisphosphate Carboxylase ◽  
Content Type ◽  
Whole Cells ◽  
Cell Extracts ◽  
Key Enzyme

ABSTRACTMethane is an important greenhouse gas and the most abundant hydrocarbon in the Earth's atmosphere. Methanotrophic microorganisms can use methane as their sole energy source and play a crucial role in the mitigation of methane emissions in the environment. “CandidatusMethylomirabilis oxyfera” is a recently described intra-aerobic methanotroph that is assumed to use nitric oxide to generate internal oxygen to oxidize methane via the conventional aerobic pathway, including the monooxygenase reaction. Previous genome analysis has suggested that, like the verrucomicrobial methanotrophs, “Ca.Methylomirabilis oxyfera” encodes and transcribes genes for the Calvin-Benson-Bassham (CBB) cycle for carbon assimilation. Here we provide multiple independent lines of evidence for autotrophic carbon dioxide fixation by “Ca.Methylomirabilis oxyfera” via the CBB cycle. The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), a key enzyme of the CBB cycle, in cell extracts from an “Ca.Methylomirabilis oxyfera” enrichment culture was shown to account for up to 10% of the total methane oxidation activity. Labeling studies with whole cells in batch incubations supplied with either13CH4or [13C]bicarbonate revealed that “Ca.Methylomirabilis oxyfera” biomass and lipids became significantly more enriched in13C after incubation with13C-labeled bicarbonate (and unlabeled methane) than after incubation with13C-labeled methane (and unlabeled bicarbonate), providing evidence for autotrophic carbon dioxide fixation. Besides this experimental approach, detailed genomic and transcriptomic analysis demonstrated an operational CBB cycle in “Ca.Methylomirabilis oxyfera.” Altogether, these results show that the CBB cycle is active and plays a major role in carbon assimilation by “Ca.Methylomirabilis oxyfera” bacteria. Our results suggest that autotrophy might be more widespread among methanotrophs than was previously assumed and implies that a methanotrophic community in the environment is not necessarily revealed by13C-depleted lipids.

scholarly journals The Role of the Histone Methyltransferase PfSET10 in Antigenic Variation by Malaria Parasites: a Cautionary Tale

mSphere ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Che J. Ngwa ◽  
Mackensie R. Gross ◽  
Jean-Pierre Musabyimana ◽  
Gabriele Pradel ◽  
Kirk W. Deitsch
Keyword(s):  
Gene Expression ◽  
New Technologies ◽  
Antigenic Variation ◽  
Histone Methyltransferase ◽  
Research Focus ◽  
Gene Encoding ◽  
Content Type ◽  
High Profile ◽  
Key Enzyme ◽  
Parasite Viability

ABSTRACT The virulence of the malaria parasite Plasmodium falciparum is due in large part to its ability to avoid immune destruction through antigenic variation. This results from changes in expression within the multicopy var gene family that encodes the surface antigen P. falciparum erythrocyte protein one (PfEMP1). Understanding the mechanisms underlying this process has been a high-profile research focus for many years. The histone methyltransferase PfSET10 was previously identified as a key enzyme required both for parasite viability and for regulating var gene expression, thus making it a prominent target for developing antimalarial intervention strategies and the subject of considerable research focus. Here, however, we show that disruption of the gene encoding PfSET10 is not lethal and has no effect on var gene expression, in sharp contrast with previously published reports. The contradictory findings highlight the importance of reevaluating previous conclusions when new technologies become available and suggest the possibility of a previously unappreciated plasticity in epigenetic gene regulation in P. falciparum. IMPORTANCE The identification of specific epigenetic regulatory proteins in infectious organisms has become a high-profile research topic and a focus for several drug development initiatives. However, studies that define specific roles for different epigenetic modifiers occasionally report differing results, and we similarly provide evidence regarding the histone methyltransferase PfSET10 that is in stark contrast with previously published results. We believe that the conflicting results, rather than suggesting erroneous conclusions, instead reflect the importance of revisiting previous conclusions using newly developed methodologies, as well as caution in interpreting seemingly contrary results in fields that are known to display considerable plasticity, for example metabolism and epigenetics.

scholarly journals Draft Genome Sequence of Haloferax volcanii SS0101, Isolated from Salt Farms in Samut Sakhon, Thailand

2019 ◽  
Vol 8 (46) ◽  
Author(s):  
Suwanan Wanthongcharoen ◽  
Wariya Yamprayoonswat ◽  
Pattarawan Ruangsuj ◽  
Nuttida Thongpramul ◽  
Watthanachai Jumpathong ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Draft Genome ◽  
Haloferax Volcanii ◽  
Draft Genome Sequence ◽  
Halophilic Archaeon ◽  
Gene Encoding ◽  
Content Type ◽  
Key Enzyme

Haloferax volcanii SS0101 is a halophilic archaeon isolated from salt farms in Thailand. The genome sequence of H. volcanii SS0101 contains a gene encoding capreomycidine synthase, a key enzyme for capreomycidine biosynthesis. This 3.8-Mb draft genome sequence of H. volcanii SS0101 will provide the tools for investigating genes involved in capeomycidine production in haloarchaea.

scholarly journals New Model for Electron Flow for Sulfate Reduction in Desulfovibrio alaskensis G20

2013 ◽  
Vol 80 (3) ◽  
pp. 855-868 ◽  
Author(s):  
Kimberly L. Keller ◽  
Barbara J. Rapp-Giles ◽  
Elizabeth S. Semkiw ◽  
Iris Porat ◽  
Steven D. Brown ◽  
...  
Keyword(s):  
Sulfate Reduction ◽  
Redox Regulation ◽  
Electron Flow ◽  
Regulation Of Gene Expression ◽  
Type I ◽  
Gene Encoding ◽  
Content Type ◽  
Sulfate Reducing ◽  
Proteomic Analyses ◽  
Physiological Tests

ABSTRACTTo understand the energy conversion activities of the anaerobic sulfate-reducing bacteria, it is necessary to identify the components involved in electron flow. The importance of the abundant type I tetraheme cytochromec3(TpIc3) as an electron carrier during sulfate respiration was questioned by the previous isolation of a null mutation in the gene encoding TpIc3,cycA, inDesulfovibrio alaskensisG20. Whereas respiratory growth of the CycA mutant with lactate and sulfate was little affected, growth with pyruvate and sulfate was significantly impaired. We have explored the phenotype of the CycA mutant through physiological tests and transcriptomic and proteomic analyses. Data reported here show that electrons from pyruvate oxidation do not reach adenylyl sulfate reductase, the enzyme catalyzing the first redox reaction during sulfate reduction, in the absence of either CycA or the type I cytochromec3:menaquinone oxidoreductase transmembrane complex, QrcABCD. In contrast to the wild type, the CycA and QrcA mutants did not grow with H2or formate and sulfate as the electron acceptor. Transcriptomic and proteomic analyses of the CycA mutant showed that transcripts and enzymes for the pathway from pyruvate to succinate were strongly decreased in the CycA mutant regardless of the growth mode. Neither the CycA nor the QrcA mutant grew on fumarate alone, consistent with the omics results and a redox regulation of gene expression. We conclude that TpIc3and the Qrc complex areD. alaskensiscomponents essential for the transfer of electrons released in the periplasm to reach the cytoplasmic adenylyl sulfate reductase and present a model that may explain the CycA phenotype through confurcation of electrons.

scholarly journals Novel Transcriptional Regulons for Autotrophic Cycle Genes in Crenarchaeota

2015 ◽  
Vol 197 (14) ◽  
pp. 2383-2391 ◽  
Author(s):  
Semen A. Leyn ◽  
Irina A. Rodionova ◽  
Xiaoqing Li ◽  
Dmitry A. Rodionov
Keyword(s):  
Carbon Dioxide ◽  
Transcriptional Regulation ◽  
Comparative Genomics ◽  
Dna Binding ◽  
Transcriptional Control ◽  
Binding Assays ◽  
Promoter Regions ◽  
Content Type ◽  
Genome Context

ABSTRACTAutotrophic microorganisms are able to utilize carbon dioxide as their only carbon source, or, alternatively, many of them can grow heterotrophically on organics. Different variants of autotrophic pathways have been identified in various lineages of the phylumCrenarchaeota. Aerobic members of the orderSulfolobalesutilize the hydroxypropionate-hydroxybutyrate cycle (HHC) to fix inorganic carbon, whereas anaerobicThermoprotealesuse the dicarboxylate-hydroxybutyrate cycle (DHC). Knowledge of transcriptional regulation of autotrophic pathways inArchaeais limited. We applied a comparative genomics approach to predict novel autotrophic regulons in theCrenarchaeota. We report identification of two novel DNA motifs associated with the autotrophic pathway genes in theSulfolobales(HHC box) andThermoproteales(DHC box). Based on genome context evidence, the HHC box regulon was attributed to a novel transcription factor from the TrmB family named HhcR. Orthologs of HhcR are present in allSulfolobalesgenomes but were not found in other lineages. A predicted HHC box regulatory motif was confirmed byin vitrobinding assays with the recombinant HhcR protein fromMetallosphaera yellowstonensis. For the DHC box regulon, we assigned a different potential regulator, named DhcR, which is restricted to the orderThermoproteales. DhcR inThermoproteus neutrophilus(Tneu_0751) was previously identified as a DNA-binding protein with high affinity for the promoter regions of two autotrophic operons. The global HhcR and DhcR regulons reconstructed by comparative genomics were reconciled with available omics data inMetallosphaeraandThermoproteusspp. The identified regulons constitute two novel mechanisms for transcriptional control of autotrophic pathways in theCrenarchaeota.IMPORTANCELittle is known about transcriptional regulation of carbon dioxide fixation pathways inArchaea. We previously applied the comparative genomics approach for reconstruction of DtxR family regulons in diverse lineages ofArchaea. Here, we utilize similar computational approaches to identify novel regulatory motifs for genes that are autotrophically induced in microorganisms from two lineages ofCrenarchaeotaand to reconstruct the respective regulons. The predicted novel regulons in archaeal genomes control the majority of autotrophic pathway genes and also other carbon and energy metabolism genes. The HhcR regulon was experimentally validated by DNA-binding assays inMetallosphaeraspp. Novel regulons described for the first time in this work provide a basis for understanding the mechanisms of transcriptional regulation of autotrophic pathways inArchaea.

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