Crystal structure of CelM2, a bifunctional glucanase-xylanase protein from a metagenome library

Degradation of polysaccharides by cellulases and xylanases plays an important role in the carbon cycle, but only occurs in plant cell walls, a few bacteria and some animals. This process is also critical in processes such as biomass degradation and fuel production in the conversion cycles of cellulosic biomass. The enzyme CelM2 is bifunctional, because it is able to effectively hydrolyze barley glucan and xylan. Here, we show the crystal structure of the bifunctional enzyme CelM2, isolated from a metagenome library, and describe the sequence information and structure of its two domains. We believe that CelM2 is attractive as an industrial enzyme and that the structural results presented herein provide insights that are relevant to the genetic engineering of multifunctional enzymes.     

Overexpression of ATP Sulfurylase in Indian Mustard Leads to Increased Selenate Uptake, Reduction, and Tolerance

In earlier studies, the assimilation of selenate by plants appeared to be limited by its reduction, a step that is thought to be mediated by ATP sulfurylase. Here, the Arabidopsis APS1 gene, encoding a plastidic ATP sulfurylase, was constitutively overexpressed in Indian mustard (Brassica juncea). Compared with that in untransformed plants, the ATP sulfurylase activity was 2- to 2.5-fold higher in shoots and roots of transgenic seedlings, and 1.5- to 2-fold higher in shoots but not roots of selenate-supplied mature ATP-sulfurylase-overexpressing (APS) plants. The APS plants showed increased selenate reduction: x-ray absorption spectroscopy showed that root and shoot tissues of mature APS plants contained mostly organic Se (possibly selenomethionine), whereas wild-type plants accumulated selenate. The APS plants were not able to reduce selenate when shoots were removed immediately before selenate was supplied. In addition, Se accumulation in APS plants was 2- to 3-fold higher in shoots and 1.5-fold higher in roots compared with wild-type plants, and Se tolerance was higher in both seedlings and mature APS plants. These studies show that ATP sulfurylase not only mediates selenate reduction in plants, but is also rate limiting for selenate uptake and assimilation.     

Decolorization of azo dye using PVA-immobilized microorganisms

A microbial consortium having a high capacity for rapid decolorization of azo dye (RED RBN) was immobilized by a phosphorylated polyvinyl alcohol (PVA) gel. The immobilized-cell beads exhibited a color removal capability of 75%, even at a high concentration of RED RBN (500 mg l(-1)) within 12 h using flask culture. The continuous operation was conducted at a hydraulic retention time (HRT) of 5-20 h in which the dye loading rate ranged from 240 to 60 mg dye h(-1). A removal efficiency exceeding 90% was obtained at the HRT higher than 10 h. No recognizable destruction of bead appearance was observed in the 6-month operation. Examination of the mechanism of the decolorization process by cell beads indicated that it proceeded primarily by biological decolorization associated with partial adsorption of the dye onto the entrapped cells and gel matrix. Microscopic observation revealed that the microbial consortium contained in the gel beads was at least made up of three kinds of bacterial species. From the economical viewpoint, alternative cheaper nitrogen sources such as fish meal, soybean meal, pharmamedia and vita yeast powder were examined.     

Anaerobic bio-hydrogen production from ethanol fermentation: the role of pH

Hydrogen was produced by an ethanol-acetate fermentation at pH of 5.0 +/- 0.2 and HRT of 3 days. The yield of hydrogen was 100-200 ml g Glu(-1) with a hydrogen content of 25-40%. This fluctuation in the hydrogen yield was attributed to the formation of propionate and the activity of hydrogen utilizing methanogens. The change in the operational pH for the inhibition of this methanogenic activity induced a change in the main fermentation pathway. In this study, the main products were butyrate, ethanol and propionate, in the pH ranges 4.0-4.5, 4.5-5.0 and 5.0-6.0, respectively. However, the activity of all the microorganisms was inhibited below pH 4.0. Therefore, pH 4.0 was regarded as the operational limit for the anaerobic bio-hydrogen production process. These results indicate that the pH plays an important role in determining the type of anaerobic fermentation pathway in anaerobic bio-hydrogen processes.     

Disentangling the effects of isolation-by-distance and isolation-by-environment on genetic differentiation among <Emphasis Type="Italic">Rhododendron</Emphasis> lineages in the subgenus <Emphasis Type="Italic">Tsutsusi</Emphasis>

Ecological speciation has long been noted as a central topic in the field of evolutionary biology, and investigation into the relative importance of ecological and geographical factors is becoming increasingly emphasized. We surveyed genetic variation of 277 samples from 25 populations of nine Rhododendron species within Tsutsusi subgenus in Taiwan using simple sequence repeats of expressed sequence tags. Bayesian clustering revealed four genetic lineages: (1) the Rhododendron simsii, Rhododendron kanehirai, and Rhododendron nakaharae lineage (lineage 1); (2) the Rhododendron longiperulatum, Rhododendron breviperulatum, and Rhododendron noriakianum lineage (lineage 2); (3) the Rhododendron rubropilosum lineage (lineage 3); and (4) the Rhododendron oldhamii lineage (lineage 4). Asymmetric introgressions were found from lineage 3 into lineages 1 and 2 (introgressed lineages). Genetic admixture of non-R. oldhamii species was also revealed by a neighbor-joining tree. Variation partitioning showed that environment explained much larger portions of genetic variation than geography between non-introgressed lineages (i.e., between R. oldhamii and other lineages). However, the Mantel and partial Mantel tests and the multiple matrix regression with randomization found that isolation-by-distance played a more important role than isolation-by-environment (IBE) in contributing to genetic variation in most between lineage comparisons. Nevertheless, strong IBE was found when compared between non-introgressed lineages of R. oldhamii and R. rubropilosum, suggesting post-speciation ecological divergence. Several environmental variables, including annual mean temperature, aspect, isothermality, seasonal precipitation, slope, and soil pH, could be important ecological drivers involved in reproductive isolation between R. oldhamii and non-R. oldhamii species within the Tsutsusi subgenus.     

Engineering surface hydrophobicity improves activity of Bacillus thermocatenulatus lipase 2 enzyme

Bacillus thermocatenulatus lipase 2 (BTL2) is a promising industrial enzyme used in biodiesel production. Although BTL2 has high thermostability and good resistance to organic solvents, the activity of BTL2 is suboptimal for industrial processes. To improve BTL2 activity, we engineered BTL2 lipase by modulating hydrophobicity of its lid domain. Through site‐directed mutagenesis, we constructed three mutants, namely Y225F+S232A, S232A+T236V and Q185L, to cover all uncharged hydrophilic amino acids within the lid domain. Activities of these mutants were characterized. Our findings suggest that one mutant (Y225F+S232A) showed ～35% activity increase in catalyzing heterogeneous hydrolytic reactions relevant for industrial applications. A mathematical framework was established to account for different molecular events that contribute to the observed apparent catalytic activities. Increases in hydrophobicity of lid domains were associated with increased interfacial adsorption of lipases and lower molecular enzymatic activities. The measured apparent activities of lipases include contributions from both events. Lid hydrophobicity can thus result in different changes in lipase activities depending on the mutation site. Our work demonstrates the feasibility of increasing BTL2 activity by modulating the hydrophobicity of lid domains and provides some guidelines for further improving BTL2 activity.     

Taurine resumed neuronal differentiation in arsenite-treated N2a cells through reducing oxidative stress,endoplasmic reticulum stress,and mitochondrial dysfunction

The goal of the study is to investigate the preventive effect of taurine against arsenite-induced arrest of neuronal differentiation in N2a cells. Our results revealed that taurine reinstated the neurite outgrowth in arsenite-treated N2a cells. Meanwhile, arsenite-induced oxidative stress and mitochondrial dysfunction as well as degradation of mitochondria DNA (mtDNA) were also inhibited by co-treatment of taurine. Since oxidative stress and mitochondrial dysfunction is closely associated with endoplasmic reticulum (ER) stress, we further examined indicators of ER stress, 78 kDa glucose-regulated protein (GRP78), and C/EBP-homologous protein (CHOP) protein expression. The results demonstrated that taurine significantly reduced arsenite-induced ER stress in N2a cells. In the parallel experiment, arsenite-induced disruption of intracellular calcium homeostasis was also ameliorated by taurine. The proven bio-function of taurine preserved a preventive effect against deleteriously cross-talking between oxidative stress, mitochondria, and ER. Overall, the results of the study suggested that taurine reinstated neuronal differentiation by inhibiting oxidative stress, ER stress, and mitochondrial dysfunction in arsenite-treated N2a cells.     

Expression of the IL-2 gene in the potato (Solanum tuberosum cv. Superior)

We intended to examine the expression of the T-cell growth factor (human interleukin-2) so that a binary vector, pSSK-1, carrying the IL-2 gene was constructed and transferred intoA. tumefaciens for the purpose of the transformation of the potato (Solanum tuberosum cv. Superior). All of theAgrobacterium-infected potato explants were regenerated to shoots in modified MS medium and 81% of them rooted on the medium containing kanamycin (200 mg/L). Southern and Northern analysis were performed to verify the transformation events. EL-ISA test indicated that IL-2 protein was produced from IL-2-transformed potatoes. These results suggested expression and production of the IL-2 protein from the transgenic potato.     

Reconstruction of methanol and formate metabolic pathway in non-native host for biosynthesis of chemicals and biofuels

One-carbon feedstock such as methanol and formate has attracted much attention as carbon substrate of industrial biotechnology for production of value-added chemicals and biofuels. Productivity improvement of natural one-carbon metabolic pathways in native hosts such as methanotrophs is somewhat difficult due to inefficient genetic tools and low specific growth rate. As an alternative, metabolic engineering can create new and efficient metabolic pathways of one-carbon substrate that can be readily transferred to non-native hosts. In this paper, recent progresses in protein and metabolic engineering for creation of methanol and formate-utilizing synthetic pathways based on RuMP cycle and formolase are reviewed. Perspectives on one-carbon metabolic pathway engineering in non-native host are also discussed.