A systems biology approach to investigate the effect of pH-induced gene regulation on solvent production by Clostridium acetobutylicum in continuous culture

<p>Abstract</p> <p>Background</p> <p><it>Clostridium acetobutylicum </it>is an anaerobic bacterium which is known for its solvent-producing capabilities, namely regarding the bulk chemicals acetone and butanol, the latter being a highly efficient biofuel. For butanol production by <it>C. acetobutylicum </it>to be optimized and exploited on an industrial scale, the effect of pH-induced gene regulation on solvent production by <it>C. acetobutylicum </it>in continuous culture must be understood as fully as possible.</p> <p>Results</p> <p>We present an ordinary differential equation model combining the metabolic network governing solvent production with regulation at the genetic level of the enzymes required for this process. Parameterizing the model with experimental data from continuous culture, we demonstrate the influence of pH upon fermentation products: at high pH (pH 5.7) acids are the dominant product while at low pH (pH 4.5) this switches to solvents. Through steady-state analyses of the model we focus our investigations on how alteration in gene expression of <it>C. acetobutylicum </it>could be exploited to increase butanol yield in a continuous culture fermentation.</p> <p>Conclusions</p> <p>Incorporating gene regulation into the model of solvent production by <it>C. acetobutylicum </it>enables an accurate representation of the pH-induced switch to solvent production to be obtained and theoretical investigations of possible synthetic-biology approaches to be pursued. Steady-state analyses suggest that, to increase butanol yield, alterations in the expression of single solvent-associated genes are insufficient; a more complex approach targeting two or more genes is required.</p

Coenzyme A-transferase-independent butyrate re-assimilation in Clostridium acetobutylicum - evidence from a mathematical model

The hetero-dimeric CoA-transferase CtfA/B is believed to be crucial for the metabolic transition from acidogenesis to solventogenesis in Clostridium acetobutylicum as part of the industrial-relevant acetone-butanol-ethanol (ABE) fermentation. Here, the enzyme is assumed to mediate re-assimilation of acetate and butyrate during a pH-induced metabolic shift and to faciliate the first step of acetone formation from acetoacetyl-CoA. However, recent investigations using phosphate-limited continuous cultures have questioned this common dogma. To address the emerging experimental discrepancies, we investigated the mutant strain Cac-ctfA398s::CT using chemostat cultures. As a consequence of this mutation, the cells are unable to express functional ctfA and are thus lacking CoA-transferase activity. A mathematical model of the pH-induced metabolic shift, which was recently developed for the wild type, is used to analyse the observed behaviour of the mutant strain with a focus on re-assimilation activities for the two produced acids. Our theoretical analysis reveals that the ctfA mutant still re-assimilates butyrate, but not acetate. Based upon this finding, we conclude that C. acetobutylicum possesses a CoA-tranferase-independent butyrate uptake mechanism that is activated by decreasing pH levels. Furthermore, we observe that butanol formation is not inhibited under our experimental conditions, as suggested by previous batch culture experiments. In concordance with recent batch experiments, acetone formation is abolished in chemostat cultures using the ctfa mutant

Mathematical modelling of clostridial acetone-butanol-ethanol fermentation

Clostridial acetone-butanol-ethanol (ABE) fermentation features a remarkable shift in the cellular metabolic activity from acid formation, acidogenesis, to the production of industrial-relevant solvents, solventogensis. In recent decades, mathematical models have been employed to elucidate the complex interlinked regulation and conditions that determine these two distinct metabolic states and govern the transition between them. In this review, we discuss these models with a focus on the mechanisms controlling intra- and extracellular changes between acidogenesis and solventogenesis. In particular, we critically evaluate underlying model assumptions and predictions in the light of current experimental knowledge. Towards this end, we briefly introduce key ideas and assumptions applied in the discussed modelling approaches, but waive a comprehensive mathematical presentation. We distinguish between structural and dynamical models, which will be discussed in their chronological order to illustrate how new biological information facilitates the ‘evolution’ of mathematical models. Mathematical models and their analysis have significantly contributed to our knowledge of ABE fermentation and the underlying regulatory network which spans all levels of biological organization. However, the ties between the different levels of cellular regulation are not well understood. Furthermore, contradictory experimental and theoretical results challenge our current notion of ABE metabolic network structure. Thus, clostridial ABE fermentation still poses theoretical as well as experimental challenges which are best approached in close collaboration between modellers and experimentalists

Characterization and Metabolic Engineering of Transcription Factors and Redox Dynamics in Candidate Consolidated Bioprocessing Biocatalysts

This thesis studies the metabolic engineering of candidate consolidated bioprocessing biocatalyst microorganisms through targeting regulatory genes, with an emphasis on redox metabolism. Consolidated bioprocessing is the single-step hydrolysis and conversion of lignocellulosic material to biofuels. The biocatalysts considered are Clostridium thermocellum and Caldicellulosiruptor bescii, and the primary product of interest is ethanol. Both organisms are thermophilic anaerobic bacteria which encode and express genes that facilitate the deconstruction and solubilization of lignocellulose into fermentable carbohydrates. Furthermore, these organisms ferment these carbohydrates into ethanol, organic acids, as well as other fermentation products. We seek to improve redox metabolism and osmotolerance in these organsisms toward a biorefining objective goal of engineering a biocatalyst capable of facilitating economically viable consolidated bioprocessing.Expression profiling, transcription factor regulon mapping, genetic engineering, and analytical fermentation were approaches employed to assay and understand which specific traits can be beneficially altered. The traits sought to be altered are characteristically complex, co-opting many cellular sub-processes to enable a molecular mechanism resulting in an observable trait. Such traits are notoriously difficult not only to understand, but to alter through classical metabolic engineering. Instead, the possibility of making system-wide changes through a minimal number of genetic alterations to methodically selected and/or screened regulatory genes was investigated.Active redox-dependent systems were characterized in both bacteria, many of which are controlled by the global redox-state sensing transcription factor Rex. Eliminating Rex control over gene expression in C. bescii resulted in a more reduced intracellular redox state, and ultimately drives increased ethanol synthesis. A method for quantifying important redox metabolites intracellularly is also adopted and validated for use with C. thermocellum. This approach was extended to less characterized gene targets and, arguably, even more complex traits. Screening of single-gene deletion mutants identified two strains of C. bescii showing phenotypic growth differences in elevated osmolarity conditions. One strain housed a deletion of the fapR gene, while the other a deletion of the fruR/cra gene. Characterizing these transcription factors and their regulons elucidates mechanisms which this organism uses to facilitate survival at elevated osmolarities. We are also able to construct genetic variants in C. bescii which are substantially more osmotolerant than native strains, highlighting the usefulness of these genes as targets and the applicability, and important considerations, of our metabolic engineering approach

Metabolic engineering for butanol yield enhancement in Clostridium acetobutylicum

Clostridium acetobutylicum is the model solventogenic saccharolytic Clostridium spp. representing a group of bacteria which exclusively produce acetone and n-butanol along with the common solvent, ethanol; known as the ABE pathway. There is broad utility for n-butanol, particularly as a transport fuel but also as an industrial solvent and as a platform chemical. Hydrogen is also a major product of this organism by way of reduction of protons via ferredoxin coupled hydrogenase activity, where electron flux to this product is mediated by the oxidation of organic metabolic intermediates by the enzymes pyruvate ferredoxin oxidoreductase (PFOR) and the electron bifurcating activity of butyryl-CoA dehydrogenase (BCD). The role of BCD was explored utilising homologous recombination in-frame deletion methods, however, the apparent essentiality of the gene resulted in maintenance of the vector and the target gene in the genome, likely as a result of a random vector integration event. Replacing BCD with trans-2-enoyl-CoA reductase (TER) presents a metabolic engineering opportunity by subversion of electron flux to ferredoxin, and ultimately hydrogen gas production, furthermore, it allows us to investigate the importance of the bifurcating role of BCD. Hypothetically, successful replacement of BCD with TER should result in an alcohologenic fermentation, as the cells attempt to maintain redox cofactor homeostasis. The expression of TER resulted in a significant improvement in solvent productivity. Nevertheless, the electron bifurcating activity of BCD appears to be an essential metabolic function for C. acetobutylicum, and DNA-seq data from a mutant strain obtained from a third party suggests that this is due to the role of hydrogenase in maintaining the proton motive force - in which case a complementary mutation interrupting the function of the proton powered flagella will ultimately facilitate the replacement of BCD with TER. A prototypic lactose inducible orthogonal expression system was applied in order to maximise the flux to butanol in the TER expressing parent strain. A control study using a strain expressing the lactose binding transcriptional activator and the TcdR sigma factor produced an altered phenotype where enhanced solvent production was observed and a computational approach was used to try to identify TcdR promotor binding sites in the C. acetobutylicum genome offering some insight as to the cause of the adjusted phenotype and a new regulator of solventogenesis is proposed

σ54 (σL) plays a central role in carbon metabolism in the industrially relevant Clostridium beijerinckii

International audiencethe solventogenic C. beijerinckii DSM 6423, a microorganism that naturally produces isopropanol and butanol, was previously modified by random mutagenesis. In this work, one of the resulting mutants was characterized. this strain, selected with allyl alcohol and designated as the AA mutant, shows a dominant production of acids, a severely diminished butanol synthesis capacity, and produces acetone instead of isopropanol. Interestingly, this solvent-deficient strain was also found to have a limited consumption of two carbohydrates and to be still able to form spores, highlighting its particular phenotype. sequencing of the AA mutant revealed point mutations in several genes including CIBE_0767 (sigL), which encodes the σ 54 sigma factor. Complementation with wild-type sigL fully restored solvent production and sugar assimilation and Rt-qpCR analyses revealed its transcriptional control of several genes related to solventogensis, demonstrating the central role of σ 54 in C. beijerinckii DSM 6423. Comparative genomics analysis suggested that this function is conserved at the species level, and this hypothesis was further confirmed through the deletion of sigL in the model strain C. beijerinckii NCIMB 8052. In the context of worldwide energy transition, research for alternatives to fossil fuels has become a priority. In particular, the replacement of petrochemistry by a low carbon emission industry has been a major challenge as our global consumption of petrochemicals keeps on increasing 1. The valorization of plant biomass to synthesize ethanol by microbial fermentation has already been pioneered for biofuel production 2 and could therefore be applied to bio-based chemistry 3. A few strains from the Clostridium genus are naturally able to produce isopropanol and butanol 4,5 , two compounds that could be used as biochemical and biofuel, respectively. However, those organisms are not producing these metabolites in quantities compatible with an economically viable industrial process 6. However, with the increasing availability of efficient genetic tools in Clostridia 7 , metabolic engineering approaches could be undertaken to enhance solvent productivity. Clostridium beijerinckii DSM 6423 (NRRL B-593) is the only natural isopropanol-butanol producing strain whose genome and transcriptome have been investigated 8. It may therefore be the best candidate for genetic engineering, although its particular physiology is still poorly understood. For this purpose, gaining additional knowledge on metabolism regulation in this strain would greatly benefit future optimization efforts. In particular, identifying the molecular effectors controlling solvent production may provide valuable insights to define adequate genetic engineering strategies. As no genetic toolbox was available for this particular strain, Máté de Gerando and coworkers performed random mutagenesis coupled with genome shuffling to increase isopropanol productivity by selecting isopropanol-tolerant strains 9. In this work, random mutagenesis followed by allyl alcohol selection also generated an interesting mutant, further referred to as AA mutant. This strain mainly produces acids, shows no isopropanol production and a strongly attenuated butanol synthesis capacity. These results are consistent with those obtained in Clostridium acetobutylicum DSM 1792, in which mutants obtained in the presence of allyl alcohol-precursor of the highly toxic acrolein molecule in the reaction catalyzed by alcohol dehydrogenases-permitted the selection of butanol-deficient strains 10. Nevertheless, in both cases the key mutated genes causing these phenotypes have not been clearly identified

Nitrogen metabolism and butanol production by South African clostridium beijerinckii and clostridium saccharobutylicum strains

Includes bibliographical references.The acetone- butanol-ethanol (ABE) fermentation was one of the first fermentation processes to be industrialized on a large scale, and the dominant product, butanol is particularly significant due to its potential as a modern day fuel additive or fuel extender in the petrochemical industry. A collection of 19 solventogenic Clostridium beijerinckii and 11 Clostridium saccharobutylicum strains isolated from the National Chemical Products (NCP) ABE fermentation plant in Germiston, South Africa, were classed according to species by a quick species-specific colony PCR and by rifampicin screening methods respectively. The speciesspecific PCR aims to provide a rapid means of assessing any contamination of an ABE batch fermentation by differentiating between C. saccharobutylicum and C. beijerinckii species. Random Amplification of Polymorphic DNA (RAPD) analysis generated four C. beijerinckii and two C. saccharobutylicum strain groups respectively. Multilocus Sequence Typing (MLST) was developed for a smaller selection of strains and showed a further two strain groups within the NCP C. beijerinckii strains and three groups within the C. saccharobutylicum strains

Characterization of a transposon-induced pleiotropic metronidazole resistant mutant of Clostridium acetobutylicum P262

Bibliography: leaves [168]-207.Metronidazole is a pro-drug which must be reduced to elicit a bactericidal effect. In the clostridia, some of the electron transport proteins that provide the source of electrons for the reductive activation of metronidazole play a key role in electron distribution, which in turn regulates the direction of carbon flow in the cell. The aim of this research project was to isolate electron transport gene(s) from the solvent-producing Clostridium acetobutylicum strain P262, using transposon-induced metronidazole resistance as a selection system. In the process, the feasibility of transposon mutagenesis in this strain, which lacks conventional systems for DNA delivery, was assessed, and the nature of metronidazole susceptibility in the C. acetobutylicum wild type was investigated. The metronidazole resistant transconjugant of interest, referred to as mutant 3R, was shown to harbour a single insertion of the Tn925: :Tn917 transposon cointegrate within a structural gene, designated sum (susceptibility to metronidazole)

Studies on the glucose family phosphotransferases of Clostridium beijerinckii

Revival of the ABE fermentation will be enhanced by the ability of bacterial strains to utilise cheap, renewable substrates containing a range of fermentable carbohydrates. Development of an effective process will, however, depend on a detailed understanding of the mechanisms of uptake and metabolism of the available sugars. The predominant mechanism for uptake of sugars and sugar derivatives in the clostridia is the phosphoenolpyruvate (PEP) - dependent phosphotransferase system (PTS), which not only catalyses the concurrent uptake and phosphorylation of its substrate but also plays a central role in regulation of carbohydrate metabolism. Complete characterization of the PTS in the solventogenic clostridia will therefore be instrumental in developing strategies for constructing effective fermentation strains. The Clostridium beijerinckii 8052 genome encodes 43 complete phosphotransferase systems, including sixteen belonging to the glucose-glucoside family. Three of the PTSs are members of the glucose subgroup in a phylogenetic branch, and might therefore transport glucose. Since glucose has been shown to repress utilization of other sugars by Clostridium beijerinckii, these systems could also potentially be involved in glucose sensing and carbon catabolite repression (CCR). The cbei 0751 gene encoding a IICBA PTS permease was amplified by PCR, and cloned into Escherichia coli ZSC113, a mutant which cannot take up and phosphorylate glucose and mannose. Transformants showed a positive fermentation phenotype for glucose and mannose. Extracts showed glucose PTS activity, and cbei 0751 was therefore shown to be a functional glucose PTS. The activity was inhibited by mannose confirming that the system also recognises mannose as a substrate. The expression of this gene appeared to be constitutive although quantitative expression was not performed. Similar experiments were used to investigate the function of a second system encoded by cbei 4983 (IICB) and cbei 4982 (IIA). Although these genes were successfully cloned, their function could not be identified. Since the cbei 4984 gene encodes a putative glycoside hydrolase, this suggests that the primary function of this PTS may be to transport and phosphorylate a disaccharide, but further experimental analysis is required to identify the substrate of this system. Attempts to inactivate the two phosphotransferases to examine the effect on the cells were not successful