Dear Editor:Clostridium acetobutylicum, a gram-positive, anaerobic, spore-forming bacterium, is capable of using a wide variety of carbon sources to produce acetone, butanol and ethanol. To improve solvent productivity of C. acetobutylicum, metabolic engineering is considered as a useful tool in developing strains with industrially desirable characteristics. However, to date, there are few useful methods for genetic manipulation of C. acetobutylicum, especially for gene disruption. To our knowledge, two types of vectors, including non-replicative and replicative integrative plasmids, have been developed for gene-inactivation in C. acetobutylicum. By using non-replicative integrative plasmids, buk and solR genes of C. acetobutylicum were inactivated [1,2]. However, due to their low frequencies of transformation and recombination, the non-replicative integrative plasmids are usually transformed at less than 1 integrative transformant per mg plasmid DNA. To obtain the integrative mutant, it may require higher transformation frequencies up to 10 5 , but the typical transformation frequencies were reported at 10 3 [3]. Harris et al. described the construction of a replicative integrative plasmid pETSPO and its application in the disruption of gene spo0A which could not be inactivated by using the non-replicative integrative plasmid [4]. With the functional replication origin in C. acetobutylicum, pETSPO increases opportunity for homologous recombination, but it is still time-consuming to screen for double crossover integration. Therefore, a more efficient tool for targeted gene inactivation in the C. acetobutylicum is much needed.Recently, a new strategy was developed to construct gene inactivation mutants by using group II intron-based Targetron technology. The mobile group II intron, originating from the Lactococcus lactis L1.LtrB intron, has been successfully used in a wide range of bacteria including Clostridium perfringens [5]. Without a proper replicon and/or promoter, the targetron plasmid pJIR750ai for C. perfringens from Sigma Aldrich was not applicable for gene disruption in the C. acetobutylicum directly (data not shown). Therefore, a modified targetron plasmid pSY6 was created by cloning the L1.LtrB group II intron fragment into the pIMP1-ptb, which was an E. coli-C. acetobutylicum shuttle vector containing a ptb (phosphotransbutyrylase) promoter [6].The gene buk, encoding the butyrate kinase, catalyzes the production of butyrate, and the gene solR located on the megaplasmid of the strain, encodes a putative repressor of solvent formation genes [7,8]. pSY6-buk and pSY6-solR vectors, constructed based on pSY6 ( Figure 1A and Supplementary information, Figure S1), were electroporated into C. acetobutylicum ATCC 824, respectively. Then, the cells were incubated overnight to induce the intron invasion (See Supplementary information, Materials and Methods). The overnight cultures were spread onto CGM medium (25 μg/ml erythromycin) and the transformants were analyzed

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BackgroundClostridium acetobutylicum, a gram-positive and spore-forming anaerobe, is a major strain for the fermentative production of acetone, butanol and ethanol. But a previously isolated hyper-butanol producing strain C. acetobutylicum EA 2018 does not produce spores and has greater capability of solvent production, especially for butanol, than the type strain C. acetobutylicum ATCC 824.ResultsComplete genome of C. acetobutylicum EA 2018 was sequenced using Roche 454 pyrosequencing. Genomic comparison with ATCC 824 identified many variations which may contribute to the hyper-butanol producing characteristics in the EA 2018 strain, including a total of 46 deletion sites and 26 insertion sites. In addition, transcriptomic profiling of gene expression in EA 2018 relative to that of ATCC824 revealed expression-level changes of several key genes related to solvent formation. For example, spo0A and adhEII have higher expression level, and most of the acid formation related genes have lower expression level in EA 2018. Interestingly, the results also showed that the variation in CEA_G2622 (CAC2613 in ATCC 824), a putative transcriptional regulator involved in xylose utilization, might accelerate utilization of substrate xylose.ConclusionsComparative analysis of C. acetobutylicum hyper-butanol producing strain EA 2018 and type strain ATCC 824 at both genomic and transcriptomic levels, for the first time, provides molecular-level understanding of non-sporulation, higher solvent production and enhanced xylose utilization in the mutant EA 2018. The information could be valuable for further genetic modification of C. acetobutylicum for more effective butanol production.

N-Acetyl-D: -neuraminic acid (Neu5Ac) can be produced from N-acetyl-D: -glucosamine (GlcNAc) and pyruvate by a chemoenzymatic process in which an alkaline-catalyzed epimerization transforms GlcNAc to N-acetyl-D: -manosamine (ManNAc). ManNAc is then condensed biocatalytically with pyruvate in the presence of N-acetyl-D: -neuraminic acid lyase (NAL) or by a two-step, fully enzymatic process involving bioconversions of GlcNAc to ManNAc and ManNAc to Neu5Ac using N-acetyl-D: -glucosamine 2-epimerase (AGE) and NAL. There are some drawbacks to this technique, such as lengthy reaction time, and the low conversion rate when the soluble forms of the enzymes are used in the two-step enzymatic process. In this study, the Escherichia coli-expressed AGE and NAL in the supernatant were purified by FP-based affinity chromatography and then immobilized on Amberzyme oxirane resin. These two immobilized enzymes, with a specific activity of 78.18 U/g for AGE and 69.30 U/g for NAL, were coupled to convert GlcNAc to Neu5Ac directly in one reactor. The conversion rate of the two-step reactions from GlcNAc to Neu5Ac was approximately 73% within 24 h. Furthermore, the immobilized AGE and NAL could both be used up to five reaction cycles without loss of activity or significant decrease of the conversion rate.

Cassava, due to its high starch content and low cost, is a promising candidate substrate for large-scale fermentation processes aimed at producing the solvents acetone, butanol and ethanol (ABE). However, the solvent yield from the fermentation of cassava reaches only 60% of that achieved by fermenting corn. We have found that the addition of ammonium acetate (CH(3)COONH(4)) to the cassava medium significantly promotes solvent production from cassava fermented by Clostridium acetobutylicum EA 2018, a mutant with a high butanol ratio. When cassava medium was supplemented with 30 mM ammonium acetate, the acetone, butanol and total solvent production reached 5.0, 13.0 and 19.4 g/l, respectively, after 48 h of fermentation. This level of solvent production is comparable to that obtained from corn medium. Both ammonium (NH(4) (+)) and acetate (CH(3)COO(-)) were required for increased solvent synthesis. We also demonstrated substantially increased acetic and butyric acid accumulation during the acidogenesis phase as well as greater acid re-assimilation during the solventogenesis period in ammonium acetate-supplemented cassava medium. Reverse transcription-polymerase chain reaction analysis indicated that the transcription of several genes encoding enzymes related to acidogenesis and solventogenesis in C. acetobutylicum EA 2018 were enhanced by the addition of ammonium acetate to the cassava medium.

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The veracity of land evaluation is tightly related to the reasonable weights of land evaluation factors. By mapping qualitative linguistic words into a fine-changeable cloud drops and translating the uncertain factor conditions into quantitative values with the uncertain illation based on cloud model, and then, integrating correlation analysis, a new way of figuring out the weight of land evaluation factors is proposed. It may solve the limitations of the conventional ways.

Due to the rapid installation of a massive number of fixed and mobile sensors, monitoring machines are intentionally or unintentionally involved in the production of a large amount of geospatial data. Environmental sensors and related software applications are rapidly altering human lifestyles and even impacting ecological and human health. However, there are rarely specific geospatial sensor web (GSW) applications for certain ecological public health questions. In this paper, we propose an ontology-driven approach for integrating intelligence to manage human and ecological health risks in the GSW. We design a Human and Ecological health Risks Ontology (HERO) based on a semantic sensor network ontology template. We also illustrate a web-based prototype, the Human and Ecological Health Risk Management System (HaEHMS), which helps health experts and decision makers to estimate human and ecological health risks. We demonstrate this intelligent system through a case study of automatic prediction of air quality and related health risk.