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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.

Succinic acid is a four-carbon dicarboxylic acid produced as one of the fermentation products of anaerobic metabolism. Based on the complete genome sequence of a capnophilic succinic acid-producing rumen bacterium, Mannheimia succiniciproducens, gene knockout studies were carried out to understand its anaerobic fermentative metabolism and consequently to develop a metabolically engineered strain capable of producing succinic acid without by-product formation. Among three different CO 2 -fixing metabolic reactions catalyzed by phosphoenolpyruvate (PEP) carboxykinase, PEP carboxylase, and malic enzyme, PEP carboxykinase was the most important for the anaerobic growth of M. succiniciproducens and succinic acid production. Oxaloacetate formed by carboxylation of PEP was found to be converted to succinic acid by three sequential reactions catalyzed by malate dehydrogenase, fumarase, and fumarate reductase. Major metabolic pathways leading to by-product formation were successfully removed by disrupting the ldhA, pflB, pta, and ackA genes. This metabolically engineered LPK7 strain was able to produce 13.4 g/liter of succinic acid from 20 g/liter glucose with little or no formation of acetic, formic, and lactic acids, resulting in a succinic acid yield of 0.97 mol succinic acid per mol glucose. Fed-batch culture of M. succiniciproducens LPK7 with intermittent glucose feeding allowed the production of 52.4 g/liter of succinic acid, with a succinic acid yield of 1.16 mol succinic acid per mol glucose and a succinic acid productivity of 1.8 g/liter/h, which should be useful for industrial production of succinic acid.Succinic acid, also known as butanedioic or amber acid, is a four-carbon dicarboxylic acid synthesized as an intermediate of the tricarboxylic acid cycle and also as one of the mixed-acid fermentation products. There is growing interest in the production of succinic acid from renewable resources by microbial fermentation because succinic acid can be used in numerous applications (36,37). Some anaerobic bacteria, such as Anaerobiospirillum succiniciproducens (6) and Actinobacillus succinogenes (10), produce succinic acid as a major fermentation product of their metabolism. During anaerobic fermentation, the greenhouse gas CO 2 is fixed by carboxylation reactions, converting C 3 to C 4 metabolites. Although A. succiniciproducens and A. succinogenes are able to produce relatively more succinic acid than other microorganisms, concomitant production of metabolic by-products, such as acetic, formic, and lactic acids, is problematic because it reduces the succinic acid yield and makes the purification process difficult and costly.With an aim to reduce by-product formation, several different approaches have been taken. In one example, Guettler et al. (9) developed a monofluoroacetate-resistant mutant strain of A. succinogenes that produced less acetic and formic acids than the parent strain. In other studies, Lee et al. used glycerol as a carbon source (22) and supplied hydrogen gas (21) during fermentation to reduce b...

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.

An anaerobic landfill leachate bioreactor was operated with crystalline cellulose and sterile landfill leachate until a steady state was reached. Cellulose hydrolysis, acidogenesis, and methanogenesis were measured. Microorganisms attached to the cellulose surfaces were hypothesized to be the cellulose hydrolyzers. 16S rRNA gene clone libraries were prepared from this attached fraction and also from the mixed fraction (biomass associated with cellulose particles and in the planktonic phase). Both clone libraries were dominated by Firmicutes phylum sequences (100% of the attached library and 90% of the mixed library), and the majority fell into one of five lineages of the clostridia. Clone group 1 (most closely related to Clostridium stercorarium), clone group 2 (most closely related to Clostridium thermocellum), and clone group 5 (most closely related to Bacteroides cellulosolvens) comprised sequences in Clostridium group III. Clone group 3 sequences were in Clostridium group XIVa (most closely related to Clostridium sp. strain XB90). Clone group 4 sequences were affiliated with a deeply branching clostridial lineage peripherally associated with Clostridium group VI. This monophyletic group comprises a new Clostridium cluster, designated cluster VIa. Specific fluorescence in situ hybridization (FISH) probes for the five groups were designed and synthesized, and it was demonstrated in FISH experiments that bacteria targeted by the probes for clone groups 1, 2, 4, and 5 were very abundant on the surfaces of the cellulose particles and likely the key cellulolytic microorganisms in the landfill bioreactor. The FISH probe for clone group 3 targeted cells in the planktonic phase, and these organisms were hypothesized to be glucose fermenters.Landfilling is still one of the most common forms of disposal of organic solid waste worldwide. However, it is becoming increasingly obvious that current waste disposal practices are not sustainable. The development of anaerobic digestion technologies to efficiently convert organic solid wastes, like municipal solid waste (MSW) and agricultural waste, to methane is driven by the need for alternative sources of fuels and the need to mitigate the environmental impacts of landfills, such as uncontrolled greenhouse gas emissions and leachate production (9, 36).The composition of MSW tends to vary depending on climatic, seasonal, and cultural factors, but it is commonly rich in biodegradable material. In general, MSW contains between 40 and 70% cellulosic waste, depending on the factors mentioned above and the level of processing to which the waste is subjected (9, 19, 25). The conversion of cellulosic material to methane is mediated by four microbial populations, including cellulolytic microbes, noncellulolytic saccharolytic microbes, syntrophic hydrogen-producing bacteria, and methanogenic Archaea (4, 9).It is generally accepted that hydrolysis is the slowest and therefore the rate-limiting step in biomethanogenesis of cellulosic material (4,19,25). Therefore, an increase in the rate...

IMPORTANCEThe fear conditioning and extinction neurocircuitry has been extensively studied in healthy and clinical populations, with a particular focus on posttraumatic stress disorder. Despite significant overlap of symptoms between posttraumatic stress disorder and anxiety disorders, the latter has received less attention. Given that dysregulated fear levels characterize anxiety disorders, examining the neural correlates of fear and extinction learning may shed light on the pathogenesis of underlying anxiety disorders.OBJECTIVES To investigate the psychophysiological and neural correlates of fear conditioning and extinction recall in anxiety disorders and to document how these features differ as a function of multiple diagnoses or anxiety severity. DESIGN, SETTING, AND PARTICIPANTSThis investigation was a cross-sectional, case-control, functional magnetic resonance imaging study at an academic medical center. Participants were healthy controls and individuals with at least 1 of the following anxiety disorders: generalized anxiety disorder, social anxiety disorder, specific phobia, and panic disorder. The study dates were between March 2013 and May 2015.EXPOSURES Two-day fear conditioning and extinction paradigm. CONCLUSIONS AND RELEVANCE Despite no skin conductance response group differences during extinction recall, brain activation patterns between anxious and healthy individuals differed. These findings encourage future studies to examine the conditions longitudinally and in the context of treatment trials to improve and guide therapeutics via advanced neurobiological understanding of each disorder.

2,3-Butanediol (2,3-BD) has great potential for diverse industries, including chemical, cosmetics, agriculture, and pharmaceutical areas. However, its industrial production and usage are limited by the fairly high cost of its petro-based production. Several bio-based 2,3-BD production processes have been developed and their economic advantages over petro-based production process have been reported. In particular, many 2,3-BD-producing microorganisms including bacteria and yeast have been isolated and metabolically engineered for efficient production of 2,3-BD. In addition, several fermentation processes have been tested using feedstocks such as starch, sugar, glycerol, and even lignocellulose as raw materials. Since separation and purification of 2,3-BD from fermentation broth account for the majority of its production cost, cost-effective processes have been simultaneously developed. The construction of a demonstration plant that can annually produce around 300 tons of 2,3-BD is scheduled to be mechanically completed in Korea in 2019. In this paper, core technologies for bio-based 2,3-BD production are reviewed and their potentials for use in the commercial sector are discussed.

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