Genome‐scale model for <i>Clostridium acetobutylicum</i>: Part II. Development of specific proton flux states and numerically determined sub‐systems

Senger, Ryan S.; Papoutsakis, Eleftherios T.

doi:10.1002/bit.22009

Cited by 72 publications

(62 citation statements)

References 63 publications

(104 reference statements)

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“…Additionally, because acid production was observed in the C. acetobutylicum knockout strains as well as in our C. beijerinckii mutants, we searched for evidence that alternate acetate routes could exist in C. acetobutylicum as well. Cysteine synthase was found in both published genome-scale models (43)(44)(45) and in the MetaCyc annotation database (46). Aldehyde dehydrogenase was not found in either model or in the KEGG (47) or MetaCyc annotation database, and evidence for acetylornithine deacetylase was found only in the MetaCyc annotation database.…”

Section: Discussionmentioning

confidence: 99%

Development of a Gene Knockout System Using Mobile Group II Introns (Targetron) and Genetic Disruption of Acid Production Pathways in Clostridium beijerinckii

Wang

Milne

et al. 2013

Appl Environ Microbiol

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h Clostridium beijerinckii is a well-known solvent-producing microorganism with great potential for biofuel and biochemical production. To better understand and improve the biochemical pathway to solvents, the development of genetic tools for engineering C. beijerinckii is highly desired. Based on mobile group II intron technology, a targetron gene knockout system was developed for C. beijerinckii in this study. This system was successfully employed to disrupt acid production pathways in C. beijerinckii, leading to pta (encoding phosphotransacetylase)-and buk (encoding butyrate kinase)-negative mutants. In addition to experimental characterization, the mutant phenotypes were analyzed in the context of our C. beijerinckii genome-scale model. Compared to those of the parental strain (C. beijerinckii 8052), acetate production in the pta mutant was substantially reduced and butyrate production was remarkably increased, while solvent production was dependent on the growth medium. The pta mutant also produced much higher levels of lactate, suggesting that disrupting pta influenced the energy generation and electron flow pathways. In contrast, acetate and butyrate production in the buk mutant was generally similar to that of the wild type, but solvent production was consistently 20 to 30% higher and glucose consumption was more rapid and complete. Our results suggest that the acid and solvent production of C. beijerinckii can be effectively altered by disrupting the acid production pathways. As the gene disruption method developed in this study does not leave any antibiotic marker in a disrupted allele, multiple and high-throughput gene disruption is feasible for elucidating genotype and phenotype relationships in C. beijerinckii.

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Section: Discussionmentioning

confidence: 99%

Development of a Gene Knockout System Using Mobile Group II Introns (Targetron) and Genetic Disruption of Acid Production Pathways in Clostridium beijerinckii

Wang

Milne

et al. 2013

Appl Environ Microbiol

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“…Due to the absence of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, the pentose phosphate pathway in C. acetobutylicum mainly serves to synthesize biomass precursors, ribose-5-phosphate and erythrose-4-phosphate (33). Regulation of pentose phosphate pathway genes by AraR allows the cells to activate this pathway and catabolize arabinose efficiently when arabinose is available in the culture.…”

Section: Discussionmentioning

confidence: 99%

Ribulokinase and Transcriptional Regulation of Arabinose Metabolism in Clostridium acetobutylicum

Zhang

Leyn

et al. 2011

Journal of Bacteriology

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The transcription factor AraR controls utilization of L-arabinose in Bacillus subtilis. In this study, we combined a comparative genomic reconstruction of AraR regulons in nine Clostridium species with detailed experimental characterization of AraRmediated regulation in Clostridium acetobutylicum. Based on the reconstructed AraR regulons, a novel ribulokinase, AraK, present in all analyzed Clostridium species was identified, which was a nonorthologous replacement of previously characterized ribulokinases. The predicted function of the araK gene was confirmed by inactivation of the araK gene in C. acetobutylicum and biochemical assays using purified recombinant AraK. In addition to the genes involved in arabinose utilization and arabinoside degradation, extension of the AraR regulon to the pentose phosphate pathway genes in several Clostridium species was revealed. The predicted AraR-binding sites in the C. acetobutylicum genome and the negative effect of L-arabinose on DNA-regulator complex formation were verified by in vitro binding assays. The predicted AraR-controlled genes in C. acetobutylicum were experimentally validated by testing gene expression patterns in both wild-type and araR-inactivated mutant strains during growth in the absence or presence of L-arabinose.

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“…The metabolic network of C. autoethanogenum was also compared with GEMs of other Clostridial species (i.e. Clostridium ljungdahlii (Nagarajan et al, 2013), Clostridium beijerinckii (Milne et al, 2011), Clostridium acetobutylicum (Lee et al, 2008;Senger and Papoutsakis, 2008) and Clostridium sticklandii (Fonknechten et al, 2010) to compare genome annotations and assist in gap-filling. The final curated model was tested for the ability of cellular growth in both autotrophic and heterotrophic conditions.…”

Section: Metabolic Reconstruction Processmentioning

confidence: 99%

“…acetobutylicum (Lee et al, 2008;Senger and Papoutsakis, 2008) and C. ljungdahlii (Nagarajan et al, 2013) (Table S1). …”

Section: Biomass Compositionmentioning

confidence: 99%

Arginine deiminase pathway provides ATP and boosts growth of the gas-fermenting acetogen Clostridium autoethanogenum

Valgepea

Loi

Behrendorff

et al. 2017

Metabolic Engineering

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Arginine deiminase pathway provides ATP and boosts growth of the gas-fermenting acetogen Clostridium autoethanogenum Metabolic Engineering, http://dx.doi.org/10.1016Engineering, http://dx.doi.org/10. /j.ymben.2017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. AbstractAcetogens are attractive organisms for the production of chemicals and fuels from inexpensive and non-food feedstocks such as syngas (CO, CO 2 and H 2 ). Expanding their product spectrum beyond native compounds is dictated by energetics, particularly ATP availability. Acetogens have evolved sophisticated strategies to conserve energy from reduction potential differences between major redox couples, however, this coupling is sensitive to small changes in thermodynamic equilibria. To accelerate the development of strains for energy-intensive products from gases, we used a genome-scale metabolic model (GEM) to explore alternative ATP-generating pathways in the gas-fermenting acetogen 1 Present address: Department of Plant and Environmental Science, University of Copenhagen, Copenhagen, Denmark Clostridium autoethanogenum. Shadow price analysis revealed a preference of C.autoethanogenum for nine amino acids. This prediction was experimentally confirmed under heterotrophic conditions. Subsequent in silico simulations identified arginine (ARG) as a key enhancer for growth. Predictions were experimentally validated, and faster growth was measured in media containing ARG (t D~4 h) compared to growth on yeast extract (t D~9 h). The growth-boosting effect of ARG was confirmed during autotrophic growth.Metabolic modelling and experiments showed that acetate production is nearly abolished and fast growth is realised by a three-fold increase in ATP production through the arginine deiminase (ADI) pathway. The involvement of the ADI pathway was confirmed by metabolomics and RNA-sequencing which revealed a ~500-fold up-regulation of the ADI pathway with an unexpected down-regulation of the Wood-Ljungdahl pathway. The data presented here offer a potential route for supplying cells with ATP, while demonstrating the usefulness of metabolic modelling for the discovery of native pathways for stimulating growth or enhancing energy availability.

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Genome‐scale model for Clostridium acetobutylicum: Part II. Development of specific proton flux states and numerically determined sub‐systems

Cited by 72 publications

References 63 publications

Development of a Gene Knockout System Using Mobile Group II Introns (Targetron) and Genetic Disruption of Acid Production Pathways in Clostridium beijerinckii

Development of a Gene Knockout System Using Mobile Group II Introns (Targetron) and Genetic Disruption of Acid Production Pathways in Clostridium beijerinckii

Ribulokinase and Transcriptional Regulation of Arabinose Metabolism in Clostridium acetobutylicum

Arginine deiminase pathway provides ATP and boosts growth of the gas-fermenting acetogen Clostridium autoethanogenum

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