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Objectives: Korean developmental dyslexic upper grade children’s text comprehension abilities were investigated considering the mode of texts (reading vs listening) as well as the type of texts (narrative vs expository).Methods: Sixteen 5th to 6th graders with developmental dyslexia (DD) and grade and cognition-matched typically developing children (TD) participated in 4 text comprehension tasks. Each child responded to 32 questions, 8 in each text, tapping comprehension of texts counterbalancing the effect of mode and type of texts.Results: First, children with DD performed lower than TD children in text comprehension, reflecting developmental dyslexic Korean children’s performance cross linguistically even with the high orthographic transparency of Hangeul. Second, children with DD performed better in the mode of reading compared to the mode of listening, which was the same as the TD children. Third, the effect of type of text was meaningful to only children with DD, while TD children’s performance between narrative and expository text was not different.Conclusion: Korean upper grade children with DD seemed to rely heavily on the mode of reading in comprehending texts similarly to their grade-matched children, while children with DD had greater difficulties in comprehending the expository texts both in reading and listening modes compared to the narrative texts. Each child with DD’s developmental level of the type and mode of texts needs to be considered to support his/her text comprehension abilities. Further studies need to be extended to the Korean language considering the type of texts with the DIER model.

Hydrogen has been receiving considerable attention as a clean energy carrier. Thermococcus onnurineus NA1, a hyperthermophilic archaeon, is capable of producing hydrogen using carbon monoxide (CO) in a growth-associated manner, providing a promising platform to upcycle industrial waste gases. In order to improve cost-effectiveness of the biological hydrogen production from carbon monoxide (CO), we developed formulation of a low-cost medium for T. onnurineus 156T by substituting fishmeal and sea salt for yeast extract (YE) and reagentgrade salts, respectively. In serum bottle cultures, hydrogen production by the 156T strain was less than 30% in fishmeal-containing media when compared to that of the YE-containing MM1 medium. However, hydrogen production was increased substantially by adaptive evolution in the fishmeal-sea salt (FMSS) medium. In bioreactor experiments, the resulting strain 156T-FA showed shorter lag phase and 70% higher maximum hydrogen production rate in FMSS medium than the parent strain 156T. Our results showed that the H2 production cost was reduced by ca. 95%, when YE and chemical salts were replaced by fishmeal and sea salt, respectively.

Constant (C)-region switching of heavy (H) and/or light (L) chains in antibodies (Abs) can affect their affinity and specificity, as demonstrated using mouse, human, and chimeric mouse-human (MH) Abs. However, the consequences of C-region switching between evolutionarily distinct mammalian and avian Abs remain unknown. To explore C-region switching in mouse-chicken (MC) Abs, we investigated antigen-binding parameters and thermal stability of chimeric MC-6C407 and MC-3D8 IgY Abs compared with parental mouse IgGs and chimeric MH Abs (MH-6C407 IgG and MH-3D8 IgG) bearing identical corresponding variable (V) regions. The two MC-IgYs exhibited differences in antigen-binding parameters and thermal stability from their parental mouse Abs. However, changes were similar to or less than those between chimeric MH Abs and their parental mouse Abs. The results demonstrate that mammalian and avian Abs share compatible V-C region interfaces, which may be conducive for the design and utilization of mammalian-avian chimeric Abs.

We introduce a disposable non-enzymatic glucose sensor that uses nanoporous Pt instead of enzymes since nanoporous Pt is an ideal electrode material for the electrochemical sensing of glucose. In this work, a nanoporous Pt electrode composed of Pt nanopore-containing particles is formed by simply dispensing a mixture of nanoporous Pt particles and a binding polymer onto a conducting circuit screen-printed on polyimide film; the nanoporous Pt particles were prepared by chemically reducing a Pt precursor in a reverse-micellar phase. This dispensing method is more cost-effective and scalable for mass production in comparison to electroplating. Electrodes based on nanoporous Pt particles dispensed in the manner were tested as non-enzymatic disposable glucose sensors; they exhibited good linearities and acceptable performance in phosphate-buffered saline (PBS) solution at pH 7.4. In human whole blood, the sensors also properly responded to glucose levels. Error grid analysis reveals that most of the data are distributed regions A and B (A: 64%, A + B: 98%).

The antigen-binding properties of single chain Fv antibodies (scFvs) can vary depending on the position and type of fusion tag used, as well as the host cells used for expression. The issue is even more complicated with a catalytic scFv antibody that binds and hydrolyses a specific antigen. Herein, we investigated the antigen-binding and -hydrolysing activities of the catalytic anti-nucleic acid antibody 3D8 scFv expressed in Escherichia coli or HEK293f cells with or without additional amino acid residues at the N- and C-termini. DNA-binding activity was retained in all recombinant forms. However, the DNA-hydrolysing activity varied drastically between forms. The DNA-hydrolysing activity of E. coli-derived 3D8 scFvs was not affected by the presence of a C-terminal human influenza haemagglutinin (HA) or His tag. By contrast, the activity of HEK293f-derived 3D8 scFvs was completely lost when additional residues were included at the N-terminus and/or when a His tag was incorporated at the C-terminus, whereas a HA tag at the C-terminus did not diminish activity. Thus, we demonstrate that the antigen-binding and catalytic activities of a catalytic antibody can be separately affected by the presence of additional residues at the N- and C-termini, and by the host cell type.

BackgroundGas-fermenting acetogens have received a great deal of attention for their ability to grow on various syngas and waste gas containing carbon monoxide (CO), producing acetate as the primary metabolite. Among them, someClostridiumspecies, such asC. ljungdahliiandC. autoethanogenum, are of particular interest as they produce fuel alcohols as well. Despite recent efforts, alcohol production by these species is still unsatisfactory due to their low productivity and acetate accumulation, necessitating the isolation of strains with better phenotypes.ResultsIn this study, a novel alcohol-producing acetogen (Clostridiumsp. AWRP) was isolated, and its complete genome was sequenced. This bacterium belongs the same phylogenetic group asC. ljungdahlii,C. autoethanogenum,C. ragsdalei, andC. coskatiibased on 16S rRNA homology; however, the levels of genome-wide average nucleotide identity (gANI) for strain AWRP compared with these strains range between 95 and 96%, suggesting that this strain can be classified as a novel species. In addition, strain AWRP produced a substantial amount of ethanol (70–90 mM) from syngas in batch serum bottle cultures, which was comparable to or even exceeded the typical values obtained using its close relatives cultivated under similar conditions. In a batch bioreactor, strain AWRP produced 119 and 12 mM of ethanol and 2,3-butanediol, respectively, while yielding only 1.4 mM of residual acetate. Interestingly, the alcohologenesis of this strain was strongly affected by oxidoreduction potential (ORP), which has not been reported with other gas-fermenting clostridia.ConclusionConsidering its ethanol production under low oxidoreduction potential (ORP) conditions,Clostridiumsp. AWRP will be an interesting host for biochemical studies to understand the physiology of alcohol-producing acetogens, which will contribute to metabolic engineering of those strains for the production of alcohols and other value-added compounds from syngas.

Empathy toward the distress of others is thought to motivate helping behaviour, in the form of voluntary action to eliminate that distress. Neuropeptide oxytocin is associated with various social cognitive abilities, including empathy and prosocial behaviour. The anterior cingulate cortex is known to be one of the brain regions underlying empathy, and one in which oxytocin receptors are expressed. However, the relationship between helping behaviour and oxytocin in the anterior cingulate cortex is still unclear. The present study investigated whether oxytocin in the anterior cingulate cortex is involved in rats' helping behaviour. In Experiment 1, we examined the influence of blockading the oxytocin receptors in the anterior cingulate cortex on helping behaviour. Impeding oxytocin in the anterior cingulate cortex delayed learning of the helping behaviour. In Experiment 2, we examined immunofluorescent colocalization of oxytocin receptors and c-fos proteins in the anterior cingulate cortex, the anterior insular cortex, and the amygdala in rats that acquired helping behaviour. We found increased c-fos expression in oxytocin receptor-containing neurons in the anterior cingulate cortex and amygdala when the rats acquired helping behaviour. In addition, the change in neural activation was found in the late phase of the learning. These results suggest that the oxytocin in the cingulate-amygdala pathways may play an important role in helping behaviour.

Additional file 1: Fig. S1. Product patterns of C. ljungdahlii DSM 13528 grown on various culture media (unbuffered); Table S1. Composition of the AMv2 medium; Table S2. Composition of the modified PETC medium; Table S3. Composition of the RM medium.

3D8 single-chain Fv (scFv) is a catalytic nucleic acid antibody with anti-viral activity against a broad spectrum of viruses. Here we investigated the functional stability of 3D8 scFv to provide a basis for engineering a 3D8 scFv derivative and for developing stable formulations with improved stability and potential use as an anti-viral agent. The stability of 3D8 scFv was assessed by measuring its DNA-hydrolyzing activity under different biochemical and physical conditions using a fluorescence resonance energy transfer (FRET)-based method. In addition, the anti-influenza (H9N2) effect of 3D8 scFv was evaluated in A549 cells. 3D8 scFv was stable at 50°C for 6h at pH 7.2, for 3 days at pH 4–10 at 37°C and 30 days at pH 4–8 at 37°C. The stability was not affected by a reducing condition, freeze–thawing for up to 30 cycles, or lyophilization. Evaluation of the anti-virus effect showed that cells treated with 32–128 units of 3D8 scFv showed a 50% decrease in influenza replication compared to untreated cells. Based on its enzymatic stability in various biochemical and physical environments, 3D8 scFv holds good potential for development as an anti-viral therapeutic.

A novel metabolic pathway was designed for the production of 3-aminopropionic acid (3-AP), an important platform chemical for manufacturing acrylamide and acrylonitrile. Using a fumaric acid producing Escherichia coli strain as a host, the Corynebacterium glutamicum panD gene (encoding L-aspartate-α-decarboxylase) was overexpressed and the native promoter of the aspA gene was replaced with the strong trc promoter, which allowed aspartic acid production through the aspartase-catalyzed reaction. Additional overexpression of aspA and ppc genes, and supplementation of ammonium sulfate in the medium allowed production of 3.49 g/L 3-AP. The 3-AP titer was further increased to 3.94 g/L by optimizing the expression level of PPC using synthetic promoters and RBS sequences. Finally, native promoter of the acs gene was replaced with strong trc promoter to reduce acetic acid accumulation. Fed-batch culture of the final strain allowed production of 32.3 g/L 3-AP in 39 h.

Conventional procedures to assay RNA degradation by a protein with ribonuclease (RNase) activity require a step to isolate intact RNA molecules, which are used as a substrate. Here, we established a novel "In-cell RNA hydrolysis assay" in which RNAs within cells are used as a substrate for the RNA-hydrolyzing protein, thereby avoiding the need to prepare intact RNA molecules. In this method, the degree of RNA degradation is indicated by the fluorescence intensity of RNA molecules released from fixed and permeabilized cells following treatment with the potential RNase. A catalytic 3D8 antibody capable of degrading RNAs and pancreatic RNase A were used as model RNases. Our data demonstrate that the novel In-cell RNA hydrolysis assay is a reliable and sensitive method to analyze the activities of potential RNA-hydrolyzing proteins such as catalytic antibodies.

There have recently been significant advances in bio-based production of chemicals from renewable resources. Microorganisms belonging to the genus Clostridium have been considered as one of the promising hosts for the production of desired chemicals of wide industrial use. Clostridium strains have capability to utilize diverse carbon sources, including C5 and C6 substrates, which thus allows production of chemicals from inexpensive and abundant biomass such as corn stover, straw, and woody waste. In addition, Clostridium strains naturally produce various chemicals, such as acetic acid, butyric acid, ethanol, isopropanol, butanol, 1,3-propanediol, 2,3-butanediol, and acetone. Recently, several important strategies for the metabolic engineering of Clostridium have been developed not only for the enhanced production of these natural products and but also for the production of non-natural isobutanol production. Here, we review the strategies employed for the development of metabolically engineered Clostridium strains for the production of such chemicals and provide future perspectives.

A subset of monoclonal anti-DNA autoantibodies enters a variety of living cells. Here, we aimed to identify the endocytic receptors recognized by an internalizing anti-nucleic acid autoantibody, the 3D8 single-chain variable fragment (scFv). We found that cell surface binding and internalization of 3D8 scFv were inhibited markedly in soluble heparan sulfate (HS)/chondroitin sulfate (CS)-deficient or -removed cells and in the presence of soluble HS and CS. 3D8 scFv colocalized intracellularly with either HS proteoglycans (HSPGs) or CSPGs in HeLa cells. 3D8 scFv was co-endocytosed and co-precipitated with representative individual HSPG and CSPG molecules: syndecan-2 (a transmembrane HSPG), glypican-3 (a glycosylphosphatidylinositol (GPI)-anchored HSPG); CD44 (a transmembrane CSPG); and brevican (a GPI-anchored CSPG). Collected data indicate that 3D8 scFv binds to the negatively charged sugar chains of both HSPGs and CSPGs and is then internalized along with these molecules, irrespective of how these proteoglycans are associated with the cell membrane. This is the first study to show that anti-DNA antibodies enter cells via both HSPGs and CSPGs simultaneously. The data may aid understanding of endocytic receptors that bind anti-DNA autoantibodies. The study also provides insight into potential cell membrane targets for macromolecular delivery.

L-ornithine is a non-essential amino acid for various industrial applications in food industry. In this study, Corynebacterium glutamicum ATCC 13032 was metabolically engineered for the production of L-ornithine. First, the proB and argF genes were deleted to block the competitive branch pathway and to block the conversion of L-ornithine to citrulline, respectively. In addition, the argR gene encoding the regulatory repressor of the L-arginine operon was also deleted. The resulting strain produced 230 mg/L of L-ornithine from glucose in flask culture. This base strain was further engineered by the plasmid-based overexpression of the argCJBD genes from C. glutamicum ATCC 21831, which resulted in the production of 7.19 g/L of L-ornithine. To enrich the NADPH pool, the carbon flux was redirected towards the pentose phosphate pathway by changing the start codons of the pgi and zwf genes and replacing the native promoter of the tkt operon with the strong sod promoter. Fed-batch cultivation of this final strain YW06 (pSY223) allowed production of 51.5 g/L of L-ornithine in 40 h with the overall productivity of 1.29 g/L/h. The results obtained in this study demonstrate the possibility of efficiently producing L-ornithine by metabolically engineered C. glutamicum. Biotechnol. Bioeng. 2015;112: 416–421. © 2014 Wiley Periodicals, Inc.

In recent years, a number of techniques and tools have been developed for genome engineering and gene expression control to achieve desired phenotypes of various bacteria. Here we review and discuss the recent advances in bacterial genome manipulation and gene expression control techniques, and their actual uses with accompanying examples. Genome engineering has been commonly performed based on homologous recombination. During such genome manipulation, the counterselection systems employing SacB or nucleases have mainly been used for the efficient selection of desired engineered strains. The recombineering technology enables simple and more rapid manipulation of the bacterial genome. The group II intron-mediated genome engineering technology is another option for some bacteria that are difficult to be engineered by homologous recombination. Due to the increasing demands on high-throughput screening of bacterial strains having the desired phenotypes, several multiplex genome engineering techniques have recently been developed and validated in some bacteria. Another approach to achieve desired bacterial phenotypes is the repression of target gene expression without the modification of genome sequences. This can be performed by expressing antisense RNA, small regulatory RNA, or CRISPR RNA to repress target gene expression at the transcriptional or translational level. All of these techniques allow efficient and rapid development and screening of bacterial strains having desired phenotypes, and more advanced techniques are expected to be seen.

Clostridium acetobutylicum is an industrially important organism that produces acetone-butanol-ethanol (ABE). The main objective of this study was to characterize the effects of increased cell density on the production of ABE during the phase transition from acidogenesis to solventogenesis in C. acetobutylicum. The increased ABE productivity of C. acetobutylicum was obtained by increasing the cell density using a newly designed medium (designated C. a cetobutylicum medium 1; CAM1). The maximum OD(600) value of C. acetobutylicum ATCC 824 strain obtained with CAM1 was 19.7, which is 1.8 times higher than that obtained with clostridial growth medium (CGM). The overall ABE productivity obtained in the CAM1-fermetation of the ATCC 824 strain was 0.83 g/L/h, which is 1.5 times higher than that (0.55 g/L/h) obtained with CGM. However, the increased productivity obtained with CAM1 did not result in an increase in the final ABE titer, because phase transition occurred at a high titer of acids.

Gene overexpression is one of the most basic strategies in metabolic engineering, but the factors determining gene expression levels have been poorly studied in Clostridium species. In this study, we found that a short single-stranded 5' untranslated region (UTR) sequence led to decreased gene expression in Clostridium acetobutylicum. Using an in vitro enzyme assay and reverse transcription-quantitative PCR, we found that addition of a small stem-loop at the 5' end of mRNA increased mRNA levels and thereby protein expression levels up to 4.6-fold, possibly protecting mRNA from exonuclease attack. Gene-expression levels were apparently independent of the stability of the added stem-loop; the existence of a stem-loop itself appears to be more important. Our results indicate that efficient expression cassettes can be designed by taking the 5' UTR into consideration, as the expression levels can vary even though the same promoter and RBS are used. These findings will be useful for developing a more reliable gene expression system for metabolic engineering of Clostridium strains.

Growing concerns over limited fossil resources and associated environmental problems are motivating the development of sustainable processes for the production of chemicals, fuels and materials from renewable resources. Metabolic engineering is a key enabling technology for transforming microorganisms into efficient cell factories for these compounds. Systems metabolic engineering, which incorporates the concepts and techniques of systems biology, synthetic biology and evolutionary engineering at the systems level, offers a conceptual and technological framework to speed the creation of new metabolic enzymes and pathways or the modification of existing pathways for the optimal production of desired products. Here we discuss the general strategies of systems metabolic engineering and examples of its application and offer insights as to when and how each of the different strategies should be used. Finally, we highlight the limitations and challenges to be overcome for the systems metabolic engineering of microorganisms at more advanced levels.

To understand the metabolic characteristics of Clostridium acetobutylicum and to examine the potential for enhanced butanol production, we reconstructed the genome-scale metabolic network from its annotated genomic sequence and analyzed strategies to improve its butanol production. The generated reconstructed network consists of 502 reactions and 479 metabolites and was used as the basis for an in silico model that could compute metabolic and growth performance for comparison with fermentation data. The in silico model successfully predicted metabolic fluxes during the acidogenic phase using classical flux balance analysis. Nonlinear programming was used to predict metabolic fluxes during the solventogenic phase. In addition, essential genes were predicted via single gene deletion studies. This genome-scale in silico metabolic model of C. acetobutylicum should be useful for genome-wide metabolic analysis as well as strain development for improving production of biochemicals, including butanol.

Due to the increasing concerns about limited fossil resources and environmental problems, there has been much interest in developing biofuels from renewable biomass. Ethanol is currently used as a major biofuel, as it can be easily produced by existing fermentation technology, but it is not the best biofuel due to its low energy density, high vapor pressure, hygroscopy, and incompatibility with current infrastructure. Higher alcohols, including 1-propanol, 1-butanol, isobutanol, 2-methyl-1-butanol, and 3-methyl-1-butanol, which possess fuel properties more similar to those of petroleum-based fuel, have attracted particular interest as alternatives to ethanol. Since microorganisms isolated from nature do not allow production of these alcohols at high enough efficiencies, metabolic engineering has been employed to enhance their production. Here, we review recent advances in metabolic engineering of microorganisms for the production of higher alcohols.

L-ornithine is a non-essential amino acid for various industrial applications in food industry. In this study, Corynebacterium glutamicum ATCC 13032 was metabolically engineered for the production of L-ornithine. First, the proB and argF genes were deleted to block the competitive branch pathway and to block the conversion of L-ornithine to citrulline, respectively. In addition, the argR gene encoding the regulatory repressor of the L-arginine operon was also deleted. The resulting strain produced 230 mg/L of L-ornithine from glucose in flask culture. This base strain was further engineered by the plasmid-based overexpression of the argCJBD genes from C. glutamicum ATCC 21831, which resulted in the production of 7.19 g/L of L-ornithine. To enrich the NADPH pool, the carbon flux was redirected towards the pentose phosphate pathway by changing the start codons of the pgi and zwf genes and replacing the native promoter of the tkt operon with the strong sod promoter. Fed-batch cultivation of this final strain YW06 (pSY223) allowed production of 51.5 g/L of L-ornithine in 40 h with the overall productivity of 1.29 g/L/h. The results obtained in this study demonstrate the possibility of efficiently producing L-ornithine by metabolically engineered C. glutamicum.

The fermentation carried out by the solvent-producing bacterium, Clostridium acetobutylicum, is characterized by two distinct phases: acidogenic and solventogenic phases. Understanding the cellular physiological changes occurring during the phase transition in clostridial fermentation is important for the enhanced production of solvents. To identify protein changes upon entry to stationary phase where solvents are typically produced, we herein analyzed the proteomic profiles of the parental wild type C. acetobutylicum strains, ATCC 824, the non-solventogenic strain, M5 that has lost the solventogenic megaplasmid pSOL1, and the synthetic simplified alcohol forming strain, M5 (pIMP1E1AB) expressing plasmid-based CoA-transferase (CtfAB) and aldehyde/alcohol dehydrogenase (AdhE1). A total of 68 protein spots, corresponding to 56 unique proteins, were unambiguously identified as being differentially present after the phase transitions in the three C. acetobutylicum strains. In addition to changes in proteins known to be involved in solventogenesis (AdhE1 and CtfB), we identified significant alterations in enzymes involved in sugar transport and metabolism, fermentative pathway, heat shock proteins, translation, and amino acid biosynthesis upon entry into the stationary phase. Of these, four increased proteins (AdhE1, CAC0233, CtfB and phosphocarrier protein HPr) and six decreased proteins (butyrate kinase, ferredoxin:pyruvate oxidoreductase, phenylalanyl-tRNA synthetase, adenylosuccinate synthase, pyruvate kinase and valyl-tRNA synthetase) showed similar patterns in the two strains capable of butanol formation. Interestingly, significant changes of several proteins by post-translational modifications were observed in the solventogenic phase. The proteomic data from this study will improve our understanding on how cell physiology is affected through protein levels patterns in clostridia.

Butanol is considered as a superior biofuel, which is conventionally produced by clostridial acetone-butanol-ethanol (ABE) fermentation. Among ABE, only butanol and ethanol can be used as fuel alternatives. Coproduction of acetone thus causes lower yield of fuel alcohols. Thus, this study aimed at developing an improved Clostridium acetobutylicum strain possessing enhanced fuel alcohol production capability. For this, we previously developed a hyper ABE producing BKM19 strain was further engineered to convert acetone into isopropanol. The BKM19 strain was transformed with the plasmid pIPA100 containing the sadh (primary/secondary alcohol dehydrogenase) and hydG (putative electron transfer protein) genes from the Clostridium beijerinckii NRRL B593 cloned under the control of the thiolase promoter. The resulting BKM19 (pIPA100) strain produced 27.9 g/l isopropanol-butanol-ethanol (IBE) as a fuel alcohols with negligible amount of acetone (0.4 g/l) from 97.8 g/l glucose in lab-scale (2 l) batch fermentation. Thus, this metabolically engineered strain was able to produce 99% of total solvent produced as fuel alcohols. The scalability and stability of BKM19 (pIPA100) were evaluated at 200 l pilot-scale fermentation, which showed that the fuel alcohol yield could be improved to 0.37 g/g as compared to 0.29 g/g obtained at lab-scale fermentation, while attaining a similar titer. To the best of our knowledge, this is the highest titer of IBE achieved and the first report on the large scale fermentation of C. acetobutylicum for IBE production.

Butanol has been widely used as an important industrial solvent and feedstock for chemical production. Also, its superior fuel properties compared with ethanol make butanol a good substitute for gasoline. Butanol can be efficiently produced by the genus Clostridium through the acetone-butanol-ethanol (ABE) fermentation, one of the oldest industrial fermentation processes. Butanol production via industrial fermentation has recently gained renewed interests as a potential solution to increasing pressure of climate change and environmental problems by moving away from fossil fuel consumption and moving toward renewable raw materials. Great advances over the last 100 years are now reviving interest in bio-based butanol production. However, several challenges to industrial production of butanol still need to be overcome, such as overall cost competitiveness and development of higher performance strains with greater butanol tolerance. This minireview revisits the past 100 years of remarkable achievements made in fermentation technologies, product recovery processes, and strain development in clostridial butanol fermentation through overcoming major technical hurdles.

Butanol is an important industrial solvent and advanced biofuel that can be produced by biphasic fermentation by Clostridium acetobutylicum. It has been known that acetate and butyrate first formed during the acidogenic phase are reassimilated to form acetone-butanol-ethanol (cold channel). Butanol can also be formed directly from acetyl-coenzyme A (CoA) through butyryl-CoA (hot channel). However, little is known about the relative contributions of the two butanol-forming pathways. Here we report that the direct butanol-forming pathway is a better channel to optimize for butanol production through metabolic flux and mass balance analyses. Butanol production through the hot channel was maximized by simultaneous disruption of the pta and buk genes, encoding phosphotransacetylase and butyrate kinase, while the adhE1D485G gene, encoding a mutated aldehyde/alcohol dehydrogenase, was overexpressed. The ratio of butanol produced through the hot channel to that produced through the cold channel increased from 2.0 in the wild type to 18.8 in the engineered BEKW(pPthlAAD**) strain. By reinforcing the direct butanol-forming flux in C. acetobutylicum, 18.9 g/liter of butanol was produced, with a yield of 0.71 mol butanol/mol glucose by batch fermentation, levels which are 160% and 245% higher than those obtained with the wild type. By fed-batch culture of this engineered strain with in situ recovery, 585.3 g of butanol was produced from 1,861.9 g of glucose, with the yield of 0.76 mol butanol/mol glucose and productivity of 1.32 g/liter/h. Studies of two butanol-forming routes and their effects on butanol production in C. acetobutylicum described here will serve as a basis for further metabolic engineering of clostridia aimed toward developing a superior butanol producer., IMPORTANCE Renewable biofuel is one of the answers to solving the energy crisis and climate change problems. Butanol produced naturally by clostridia has superior liquid fuel characteristics and thus has the potential to replace gasoline. Due to the lack of efficient genetic manipulation tools, however, strain improvement has been rather slow. Furthermore, complex metabolic characteristics of acidogenesis followed by solventogenesis in this strain have hampered development of engineered clostridia having highly efficient and selective butanol production capability. Here we report for the first time the results of systems metabolic engineering studies of two butanol-forming routes and their relative importances in butanol production. Based on these findings, a metabolically engineered Clostridium acetobutylicum strain capable of producing butanol to a high titer with high yield and selectivity could be developed by reinforcing the direct butanol-forming flux.

Global energy crisis and limited supply of petroleum fuels have rekindled the worldwide focus towards development of a sustainable technology for alternative fuel production. Utilization of abundant renewable biomass offers an excellent opportunity for the development of an economical biofuel production process at a scale sufficiently large to have an impact on sustainability and security objectives. Additionally, several environmental benefits have also been linked with the utilization of renewable biomass. Butanol is considered to be superior to ethanol due to its higher energy content and less hygroscopy. This has led to an increased research interest in butanol production from renewable biomass in recent years. In this paper, we review the various aspects of utilizing renewable biomass for clostridial butanol production. Focus is given on various alternative substrates that have been used for butanol production and on fermentation strategies recently reported to improve butanol production.

Clostridium acetobutylicum naturally produces acetone as well as butanol and ethanol. Since acetone cannot be used as a biofuel, its production needs to be minimized or suppressed by cell or bioreactor engineering. Thus, there have been attempts to disrupt or inactivate the acetone formation pathway. Here we present another approach, namely, converting acetone to isopropanol by metabolic engineering. Since isopropanol can be used as a fuel additive, the mixture of isopropanol, butanol, and ethanol (IBE) produced by engineered C. acetobutylicum can be directly used as a biofuel. IBE production is achieved by the expression of a primary/secondary alcohol dehydrogenase gene from Clostridium beijerinckii NRRL B-593 (i.e., adh B-593 ) in C. acetobutylicum ATCC 824. To increase the total alcohol titer, a synthetic acetone operon ( act operon; adc-ctfA-ctfB ) was constructed and expressed to increase the flux toward isopropanol formation. When this engineering strategy was applied to the PJC4BK strain lacking in the buk gene (encoding butyrate kinase), a significantly higher titer and yield of IBE could be achieved. The resulting PJC4BK(pIPA3-Cm2) strain produced 20.4 g/liter of total alcohol. Fermentation could be prolonged by in situ removal of solvents by gas stripping, and 35.6 g/liter of the IBE mixture could be produced in 45 h.

Biofuel from renewable biomass is one of the answers to help solve the problems associated with limited fossil resources and climate change. Butanol has superior liquid-fuel characteristics, with similar properties to gasoline, and thus, has the potential to be used as a substitute for gasoline. Clostridia are recognized as a good butanol producers and are employed in the industrial-scale production of solvents. Due to the difficulty of performing genetic manipulations on clostridia, however, strain improvement has been rather slow. Furthermore, complex metabolic characteristics of acidogenesis followed by solventogenesis in this strain have hampered the development of engineered clostridia strains with highly efficient and selective butanol-production capabilities. In recent years, the butanol-producing characteristics in clostridia have been further characterized and alternative pathways discovered. More recently, systems-level metabolic engineering approaches were taken to develop superior strains. Herein, we review recent discoveries of metabolic pathways for butanol production and the metabolic engineering strategies being developed.

To improve butanol selectivity, Clostridium acetobutylicum M5(pIMP1E1AB) was constructed by adhE1-ctfAB complementation of C. acetobutylicum M5, a derivative strain of C. acetobutylicum ATCC 824, which does not produce solvents due to the lack of megaplasmid pSOL1. The gene products of adhE1-ctfAB catalyze the formation of acetoacetate and ethanol/butanol with acid re-assimilation in solventogenesis. Effects of the adhE1-ctfAB complementation of M5 were studied by batch fermentations under various pH and glucose concentrations, and by flux balance analysis using a genome-scale metabolic model for this organism. The metabolically engineered M5(pIMP1E1AB) strain was able to produce 154 mM butanol with 9.9 mM acetone at pH 5.5, resulting in a butanol selectivity (a molar ratio of butanol to total solvents) of 0.84, which is much higher than that (0.57 at pH 5.0 or 0.61 at pH 5.5) of the wild-type strain ATCC 824. Unlike for C. acetobutylicum ATCC 824, a higher level of acetate accumulation was observed during fermentation of the M5 strain complemented with adhE1 and/or ctfAB. A plausible reason for this phenomenon is that the cellular metabolism was shifted towards acetate production to compensate reduced ATP production during the largely growth-associated butanol formation by the M5(pIMP1E1AB) strain.