Genome-wide survey of tissue-specific microRNA and transcription factor regulatory networks in 12 tissues.

Tissue-specific miRNAs (TS miRNA) specifically expressed in particular tissues play an important role in tissue identity, differentiation and function. However, transcription factor (TF) and TS miRNA regulatory networks across multiple tissues have not been systematically studied. Here, we manually extracted 116 TS miRNAs and systematically investigated the regulatory network of TF-TS miRNA in 12 human tissues. We identified 2,347 TF-TS miRNA regulatory relations and revealed that most TF binding sites tend to enrich close to the transcription start site of TS miRNAs. Furthermore, we found TS miRNAs were regulated widely by non-tissue specific TFs and the tissue-specific expression level of TF have a close relationship with TF-genes regulation. Finally, we describe TSmiR (http://bioeng.swjtu.edu.cn/TSmiR), a novel and web-searchable database that houses interaction maps of TF-TS miRNA in 12 tissues. Taken together, these observations provide a new suggestion to better understand the regulatory network and mechanisms of TF-TS miRNAs underlying different tissues.

Bacterial consortia constructed for the decomposition of Agave biomass.

Research has shown that a greater variety of enzymes, as well as variety of microorganisms producing enzymes, can have an overall synergistic effect on the decomposition of lignocellulosic biomass for the production of value-added bio-products. Here, 8 cellulase-degrading bacterial isolates were selected to develop co-, tri-, and tetra-cultures for the decomposition of lignocellulosic biomass. Glucose and xylose equivalents released from imitation biomass media containing 0.5% (w/v) beechwood xylan and 0.5% (w/v) Avicel was measured using di-nitrosalicylic acid for all consortia, along with cell growth and survival. Thereafter, 6 co- and 2 tri-cultures with greatest decomposition were examined for ability to degrade Agave americana fiber. Interestingly, when strains were paired up in co-culture, four pairs: G+5, G+A, C+A1, and G+A1 produced high reducing sugars in 24 h: 6 µM, 8 µM, 8 µM, and finally, 6 µM, respectively. From 4 co-cultures with highest reducing sugar equivalents, tri- and tetra-cultures were produced. The bacterial consortia which had the highest reducing sugars detected were 2 tri-cultures: G + A1 + A4 and G + A1 + 5, displaying levels as high as 9 µM and 5 µM in day 1, respectively. All co- and tri-cultures maintained high cell survival for 14 days with 0.5 g ground Agave. Upon evaluating Agave dry weight after treatment, it was evident that almost half the biomass could be decomposed in 14 days. Scanning electron microscopy of treated Agave supported decomposition when compared with the control. These bacterial consortia have potential for further study of value-added by-product production during metabolism of lignocellulosic biomasses.

Increased expression of ß-glucosidase A in Clostridium thermocellum 27405 significantly increases cellulase activity.

ß-glucosidase A (bglA) in Clostridium thermocellum 27405 was increased by expression from shuttle vector pIBglA in attempts to increase cellulase activity and ethanol titer by lowering the end product inhibition of cellulase. Through a modified electrotransformation protocol C. thermocellum transformant (+MCbglA) harbouring pIBglA was produced. The ß-glucosidase activity of +MCbglA was 2.3- and 1.6-fold greater than wild-type (WT) during late log and stationary phases of growth. Similarly, total cellulase activity of +MCbglA was shown to be 1.7-, 2.3- and 1.6-fold greater than WT during, log, late log and stationary phases of growth. However, there was no significant correlation found between increased cellulase activity and increased ethanol titers for +MCbglA compared with the WT. C. thermocellum has industrial potential for consolidated bioprocessing (CBP) to make a more cost effective production of biofuels; however, the hydrolysis rate of the strain is still hindered by end product inhibition. We successfully increased total cellulase activity by increased expression of bglA and thereby increased the productivity of C. thermocellum during the hydrolysis stage in CBP. Our work also lends insights into the complex metabolism of C. thermocellum for future improvement of this strain.

Newly isolated and characterized bacteria with great application potential for decomposition of lignocellulosic biomass.

This study focuses on the isolation and characterization of bacteria from municipal waste and peat to determine those bacteria with good potential for modification and decomposition of lignocellulosic biomass for industrial application. Twenty cellulase-producing bacteria belonging to four major phyla - Firmicutes, Actinobacteria, Proteobacteria and Bacteroidetes - were found when screened on carboxymethyl cellulose-containing agar. Six isolates also exhibited activities towards filter paper as the sole carbon source in salt media, while 12 exhibited activities towards xylan when screened on xylan-containing plates. Moreover, 5 isolates survived in and increased the absorbance of 1% black liquor in salt media by an average of 2.07-fold after 21 days of incubation. Similarly, these 5 isolates increased the absorbance of 0.1% pure lignin at 280 nm in salt media, indicating modification of lignin. Additionally, the Fourier transform infrared spectroscopy analysis of 1% barley straw treated for 21 days with these 5 strains showed a preference for consumption of hemicelluloses over lignin; however, a change in lignin was observed. A Bacillus strain (55S5) and a Pseudomonas strain (AS1) displayed the greatest potential for lignocellulose decomposition due to a variety of cellulase activities, as well as xylanase activity and modification of lignin. Several of these isolates have good potential for industrial use in the degradation of lignocellulosic biomass.

Characterization and analysis of nifH genes from Paenibacillus sabinae T27.

Paenibacillus sabinae T27 (CCBAU 10202=DSM 17841) is a gram-positive, spore-forming diazotroph with high nitrogenase activities. Three nifH clusters were cloned from P. sabinae T27. Phylogenetic analysis revealed that NifH1, NifH2 and NifH3 cluster with Cyanobacterium. Each of the coding regions of nifH1, nifH2 and nifH3 from P. sabinae T27 under the control of the nifH promoter of Klebsiella pneumoniae could partially restore nitrogenase activity of K. pneumoniae nifH(-) mutant strain 1795, which has no nitrogenase activity. This suggests that the three nifH genes from P. sabinae T27 have some function in nitrogen fixation. RT-PCR showed that all three nifH genes were expressed under nitrogen-fixing growth conditions. Using promoter vectors which have promoterless lacZ gene, three putative promoter regions of nifH genes were identified.

Characterization of some efficient cellulase producing bacteria isolated from paper mill sludges and organic fertilizers.

The wide variety of bacteria in the environment permits screening for more efficient cellulases to help overcome current challenges in biofuel production. This study focuses on the isolation of efficient cellulase producing bacteria found in organic fertilizers and paper mill sludges which can be considered for use in large scale biorefining. Pure isolate cultures were screened for cellulase activity. Six isolates: S1, S2, S3, S4, E2, and E4, produced halos greater in diameter than the positive control (Cellulomonas xylanilytica), suggesting high cellulase activities. A portion of the 16S rDNA genes of cellulase positive isolates were amplified and sequenced, then BLASTed to determine likely genera. Phylogenetic analysis revealed genera belonging to two major Phyla of Gram positive bacteria: Firmicutes and Actinobacteria. All isolates were tested for the visible degradation of filter paper; only isolates E2 and E4 (Paenibacillus species) were observed to completely break down filter paper within 72 and 96 h incubation, respectively, under limited oxygen condition. Thus E2 and E4 were selected for the FP assay for quantification of total cellulase activities. It was shown that 1% (w/v) CMC could induce total cellulase activities of 1652.2±61.5 and 1456.5±30.7 µM of glucose equivalents for E2 and E4, respectively. CMC could induce cellulase activities 8 and 5.6X greater than FP, therefore CMC represented a good inducing substrate for cellulase production. The genus Paenibacillus are known to contain some excellent cellulase producing strains, E2 and E4 displayed superior cellulase activities and represent excellent candidates for further cellulase analysis and characterization.

Cellulase activities in biomass conversion: measurement methods and comparison.

Cellulose, the major constituent of all plant materials and the most abundant organic molecule on the Earth, is a linear biopolymer of glucose molecules, connected by ß-1,4-glycosidic bonds. Enzymatic hydrolysis of cellulose requires mixtures of hydrolytic enzymes including endoglucanases, exoglucanases (cellobiohydrolases), and ß-glucosidases acting in a synergistic manner. In biopolymer hydrolysis studies, enzyme assay is an indispensable part. The most commonly used assays for the individual enzymes as well as total cellulase activity measurements, including their advantages and limitations, are summarized in this review article. In addition, some novel approaches recently used for enzyme assays are summarized.

The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass.

Lignocellulosic biomass is a renewable and abundant resource with great potential for bioconversion to value-added bioproducts. However, the biorefining process remains economically unfeasible due to a lack of biocatalysts that can overcome costly hurdles such as cooling from high temperature, pumping of oxygen/stirring, and, neutralization from acidic or basic pH. The extreme environmental resistance of bacteria permits screening and isolation of novel cellulases to help overcome these challenges. Rapid, efficient cellulase screening techniques, using cellulase assays and metagenomic libraries, are a must. Rare cellulases with activities on soluble and crystalline cellulose have been isolated from strains of Paenibacillus and Bacillus and shown to have high thermostability and/or activity over a wide pH spectrum. While novel cellulases from strains like Cellulomonas flavigena and Terendinibacter turnerae, produce multifunctional cellulases with broader substrate utilization. These enzymes offer a framework for enhancement of cellulases including: specific activity, thermalstability, or end-product inhibition. In addition, anaerobic bacteria like the clostridia offer potential due to species capable of producing compound multienzyme complexes called cellulosomes. Cellulosomes provide synergy and close proximity of enzymes to substrate, increasing activity towards crystalline cellulose. This has lead to the construction of designer cellulosomes enhanced for specific substrate activity. Furthermore, cellulosome-producing Clostridium thermocellum and its ability to ferment sugars to ethanol; its amenability to co-culture and, recent advances in genetic engineering, offer a promising future in biofuels. The exploitation of bacteria in the search for improved enzymes or strategies provides a means to upgrade feasibility for lignocellulosic biomass conversion, ultimately providing means to a 'greener' technology.