Binding characteristics of a bacterial expansin (BsEXLX1) for various types of pretreated lignocellulose

BsEXLX1 from Bacillus subtilis is the first discovered bacterial expansin as a structural homolog of a plant expansin, and it exhibited synergism with cellulase on the cellulose hydrolysis in a previous study. In this study, binding characteristics of BsEXLX1 were investigated using pretreated and untreated Miscanthus x giganteus in comparison with those of CtCBD3, a cellulose-binding domain from Clostridium thermocellum. The amounts of BsEXLX1 bound to cellulose-rich substrates were significantly lower than those of CtCBD3. However, the amounts of BsEXLX1 bound to lignin-rich substrates were much higher than those of CtCBD3. A binding competition assay between BsEXLX1 and CtCBD3 revealed that binding of BsEXLX1 to alkali lignin was not affected by the presence of CtCBD3. This preferential binding of BsEXLX1 to lignin could be related to root colonization in plants by bacteria, and the bacterial expansin could be used as a lignin blocker in the enzymatic hydrolysis of lignocellulose.     

Developing Bacillus spp. as a cell factory for production of microbial enzymes and industrially important biochemicals in the context of systems and synthetic biology

Increasing concerns over limited petroleum resources and associated environmental problems are motivating the development of efficient cell factories to produce chemicals, fuels, and materials from renewable resources in an environmentally sustainable economical manner. Bacillus spp., the best characterized Gram-positive bacteria, possesses unique advantages as a host for producing microbial enzymes and industrially important biochemicals. With appropriate modifications to heterologous protein expression and metabolic engineering, Bacillus species are favorable industrial candidates for efficiently converting renewable resources to microbial enzymes, fine chemicals, bulk chemicals, and fuels. Here, we summarize the recent advances in developing Bacillus spp. as a cell factory. We review the available genetic tools, engineering strategies, genome sequence, genome-scale structure models, proteome, and secretion pathways, and we list successful examples of enzymes and industrially important biochemicals produced by Bacillus spp. Furthermore, we highlight the limitations and challenges in developing Bacillus spp. as a robust and efficient production host, and we discuss in the context of systems and synthetic biology the emerging opportunities and future research prospects in developing Bacillus spp. as a microbial cell factory.     

Targeted nitric oxide delivery preferentially induces glioma cell chemosensitivity via altered p53 and O6‐Methylguanine‐DNA Methyltransferase activity

Glioblastoma multiforme is the most common malignant central nervous system tumor, and also among the most difficult to treat due to a lack of response to chemotherapeutics. New methods of countering the mechanisms that confer chemoresistance to malignant gliomas could lead to significant advances in the quest to identify novel drug combinations or targeted drug delivery systems for cancer therapy. In this study, we investigate the use of a targeted nitric oxide (NO) donor as a pretreatment to sensitize glioma cells to chemotherapy. The protein chlorotoxin (CTX) has been shown to preferentially target glioma cells, and we have developed CTX–NO, a glioma‐specific, NO‐donating CTX derivative. Pretreatment of cells with CTX–NO followed by 48‐h exposure to either carmustine (BCNU) or temozolomide (TMZ), both common chemotherapeutics used in glioma treatment, resulted in increased efficacy of both therapeutics. After CTX–NO exposure, both T98G and U‐87MG human malignant glioma cells show increased sensitivity to BCNU and TMZ. Further investigation revealed that the consequences of this combination therapy was a reduction in active levels of the cytoprotective enzyme MGMT and altered p53 activity, both of which are essential in DNA repair and tumor cell resistance to chemotherapy. The combination of CTX–NO and chemotherapeutics also led to decreased cell invasion. These studies indicate that this targeted NO donor could be an invaluable tool in the development of novel approaches to treat cancer. Biotechnol. Bioeng. 2013; 110: 1211–1220. © 2012 Wiley Periodicals, Inc.     

Dongia rigui sp. nov., isolated from freshwater of a large wetland in Korea

A motile, curved to twisted rod-shaped aerobic bacterium, designated strain 04SU4-P^T, was isolated from freshwater collected from the Woopo wetland (Republic of Korea). Cells were observed to be Gram-stain negative, catalase negative and oxidase positive. The major fatty acids (>10 % of the total) were identified as C_19:0 ω8c cyclo (24.6 %), C_16:0 (24.3 %) and C_18:1 ω7c (13.1 %). The DNA G+C content was determined to be 71.5 mol%. The major polar lipids were identified as phosphatidylglycerol, phosphatidylethanolamine, one unknown phospholipid and one unknown aminolipid. The major ubiquinone was determined to be Q-10. A phylogenetic tree based on 16S rRNA gene sequences showed that strain 04SU4-P^T forms an evolutionary lineage within the genus Dongia and its nearest neighbour is Dongia mobilis LM22^T (98.0 % sequence similarity). Genomic DNA–DNA hybridization of stain 04SU4-P^T with D. mobilis LM22^T showed relatedness of only 34.2 %. The phenotypic characteristics indicate the strain 04SU4-P^T can be distinguished from the sole member of the genus Dongia. On the basis of the data presented in this study, strain 04SU4-P^T represents a novel species, for which the name Dongia rigui is proposed. The type strain is 04SU4-P^T (KCTC 23341^T = JCM 17521^T).     

Pyrrole and furan oligoglycosides from the starfish Asterina batheri and their inhibitory effect on the production of pro-inflammatory cytokines in lipopolysaccharide-stimulated bone marrow-derived dendritic cells

Three new pyrrole oligoglycosides, astebatheriosides A–C (1–3), and a new furan oligoglycoside, astebatherioside D (4), were isolated from the starfish Asterina batheri by various chromatographic methods. Their structures were elucidated by spectroscopic and chemical methods. Compounds 2, 3, and 4 moderately inhibited IL-12 p40 production in lipopolysaccharide (LPS)-stimulated bone marrow-derived dendritic cells (BMDCs) with IC₅₀ values of 36.4, 31.6, and 22.8 μM, respectively.     

Oxazolopyridines and thiazolopyridines as monoamine oxidase B inhibitors for the treatment of Parkinson’s disease

In Parkinson’s disease, the motor impairments are mainly caused by the death of dopaminergic neurons. Among the enzymes which are involved in the biosynthesis and catabolism of dopamine, monoamine oxidase B (MAO-B) has been a therapeutic target of Parkinson’s disease. However, due to the undesirable adverse effects, development of alternative MAO-B inhibitors with greater optimal therapeutic potential towards Parkinson’s disease is urgently required. In this study, we designed and synthesized the oxazolopyridine and thiazolopyridine derivatives, and biologically evaluated their inhibitory activities against MAO-B. Structure–activity relationship study revealed that the piperidino group was the best choice for the R¹ amino substituent to the oxazolopyridine core structure and the activities of the oxazolopyridines with various phenyl rings were between 267.1 and 889.5 nM in IC₅₀ values. Interestingly, by replacement of the core structure from oxazolopyrine to thiazolopyridine, the activities were significantly improved and the compound 1n with the thiazolopyridine core structure showed the most potent activity with the IC₅₀ value of 26.5 nM. Molecular docking study showed that van der Waals interaction in the human MAO-B active site could explain the enhanced inhibitory activities of thiazolopyridine derivatives.     

Discovery of aryl-biphenyl-2-ylmethylpiperazines as novel scaffolds for 5-HT7 ligands and role of the aromatic substituents in binding to the target receptor

It has been reported that 5-HT₇ receptors are promising targets of depression and neuropathic pain. 5-HT₇ receptor antagonists have exhibited antidepressant-like profiles, while agonists have represented potential therapeutics for pain. In the course of our ongoing efforts to discover novel 5-HT₇ modulators, we designed an arylpiperazine scaffold with a substituted biphenyl-2-ylmethyl group. A series of biphenyl-2-yl-arylpiperazinylmethanes were then prepared, which showed a broad spectrum of binding affinities to the 5-HT₇ receptor depending upon the substituents attached to the biphenyl and aryl functionalities. Among those synthesized compounds, the compounds 1–24 and 1–26 showed the best binding affinities to the 5-HT₇ receptor with K_i values of 43.0 and 46.0 nM, respectively. Structure–activity relationship study in conjunction with molecular docking study proposed that the 5-HT₇ receptor might have two distinctive hydrophobic binding sites, one specific for aromatic 2-OCH₃ substituents within the arylpiperazine and the other for biphenyl methoxy group.     

Structure–activity relationship of human glutaminyl cyclase inhibitors having an N-(5-methyl-1H-imidazol-1-yl)propyl thiourea template

In an effort to design inhibitors of human glutaminyl cyclase (QC), we have synthesized a library of N-aryl N-(5-methyl-1H-imidazol-1-yl)propyl thioureas and investigated the contribution of the aryl region of these compounds to their structure–activity relationships as cyclase inhibitors. Our design was guided by the proposed binding mode of the preferred substrate for the cyclase. In this series, compound 52 was identified as the most potent QC inhibitor with an IC₅₀ value of 58 nM, which was two-fold more potent than the previously reported lead 2. Compound 52 is a most promising candidate for future evaluation to monitor its ability to reduce the formation of pGlu-Aβ and Aβ plaques in cells and transgenic animals.     

Isolation of a thermophilic bacterium capable of low-molecular-weight polyethylene degradation

A thermophilic bacterium capable of low-molecular-weight polyethylene (LMWPE) degradation was isolated from a compost sample, and was identified as Chelatococcus sp. E1, through sequencing of the 16S rRNA gene. LMWPE was prepared by thermal degradation of commercial PE in a strict nitrogen atmosphere. LMWPE with a weight-average-molecular-weight (Mw) in the range of 1,700–23,700 was noticeably mineralized into CO₂ by the bacterium. The biodegradability of LMWPE decreased as the Mw increased. The low molecular weight fraction of LMWPE decreased significantly as a result of the degradation process, and thereby both the number-average-molecular-weight and Mw increased after biodegradation. The polydispersity of LMWPE was either narrowed or widened, depending on the initial Mw of LMWPE, due to the preferential elimination of the low molecular weight fraction, in comparison to the high molecular weight portion. LMWPE free from an extremely low molecular weight fraction was also mineralized by the strain at a remarkable rate, and FTIR peaks assignable to C–O stretching appeared as a result of microbial action. The FTIR peaks corresponding to alkenes also became more intense, indicating that dehydrogenations occurred concomitantly with microbial induced oxidation.