Reducing Biomass Recalcitrance by Heterologous Expression of a Bacterial Peroxidase in Tobacco (\u3cem\u3eNicotiana benthamiana\u3c/em\u3e)

Commercial scale production of biofuels from lignocellulosic feed stocks has been hampered by the resistance of plant cell walls to enzymatic conversion, primarily owing to lignin. This study investigated whether DypB, the lignin-degrading peroxidase from Rodococcus jostii, depolymerizes lignin and reduces recalcitrance in transgenic tobacco (Nicotiana benthamiana). The protein was targeted to the cytosol or the ER using ER-targeting and retention signal peptides. For each construct, five independent transgenic lines were characterized phenotypically and genotypically. Our findings reveal that expression of DypB in the cytosol and ER does not affect plant development. ER-targeting increased protein accumulation, and extracts from transgenic leaves showed higher activity on classic peroxidase substrates than the control. Intriguingly, in situ DypB activation and subsequent saccharification released nearly 200% more fermentable sugars from transgenic lines than controls, which were not explained by variation in initial structural and non-structural carbohydrates and lignin content. Pyrolysis-GC-MS analysis showed more reduction in the level of lignin associated pyrolysates in the transgenic lines than the control primarily when the enzyme is activated prior to pyrolysis, consistent with increased lignin degradation and improved saccharification. The findings reveal for the first time that accumulation and in situ activation of a peroxidase improves biomass digestibility

Elucidation of gene involvement in the siderophores biosynthesis by targeted mutagenesis in Mycobacterium species

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Microbial Cellulose Synthesis

Cellulose, the most abundant biopolymer on earth, has served mankind in countless applications from crude building resources to feedstock for advanced synthetic materials. As interest grows in alternative fuel resources, cellulose is among the primary contenders as a feedstock for generating these fuels. In recent years, interest in plant-derived, photosynthetically fixed cellulose has intensified, as this remains the major untapped natural source of stored solar energy in the form of carbon-carbon bonds. Although not usually considered in the biofuel sector, the ubiquity of microbial cellulose and the engineering of photosynthetic microbes for cellulose production make microbial cellulose a viable candidate. Not only does the remarkable versatility of microbial cellulose make its study worthwhile, but the microbes responsible for its synthesis serve as a simpler model for understanding the complex biological processes underlying plant cellulose synthesis

Isolation and characterization of two cellulose morphology mutants of Gluconacetobacter hansenii ATCC23769 producing cellulose with lower crystallinity.

Gluconacetobacter hansenii, a Gram-negative bacterium, produces and secrets highly crystalline cellulose into growth medium, and has long been used as a model system for studying cellulose synthesis in higher plants. Cellulose synthesis involves the formation of β-1,4 glucan chains via the polymerization of glucose units by a multi-enzyme cellulose synthase complex (CSC). These glucan chains assemble into ordered structures including crystalline microfibrils. AcsA is the catalytic subunit of the cellulose synthase enzymes in the CSC, and AcsC is required for the secretion of cellulose. However, little is known about other proteins required for the assembly of crystalline cellulose. To address this question, we visually examined cellulose pellicles formed in growth media of 763 individual colonies of G. hansenii generated via Tn5 transposon insertion mutagenesis, and identified 85 that produced cellulose with altered morphologies. X-ray diffraction analysis of these 85 mutants identified two that produced cellulose with significantly lower crystallinity than wild type. The gene disrupted in one of these two mutants encoded a lysine decarboxylase and that in the other encoded an alanine racemase. Solid-state NMR analysis revealed that cellulose produced by these two mutants contained increased amounts of non-crystalline cellulose and monosaccharides associated with non-cellulosic polysaccharides as compared to the wild type. Monosaccharide analysis detected higher percentages of galactose and mannose in cellulose produced by both mutants. Field emission scanning electron microscopy showed that cellulose produced by the mutants was unevenly distributed, with some regions appearing to contain deposition of non-cellulosic polysaccharides; however, the width of the ribbon was comparable to that of normal cellulose. As both lysine decarboxylase and alanine racemase are required for the integrity of peptidoglycan, we propose a model for the role of peptidoglycan in the assembly of crystalline cellulose

Pankiller effect of prolonged exposure to menadione on glioma cells: potentiation by vitamin C

Menadione (Vitamin K3) has anti-tumoral effects against a wide range of cancer cells. Its potential toxicity to normal cells and narrow therapeutic range limit its use as single agent but in combination with radiation or other anti-neoplastic agents can be of therapeutic use. In this paper, we first evaluated the early (within 3 h) effect of menadione on ongoing DNA replication. In normal rat cerebral cortex mini-units menadione showed an age dependent anti-proliferative effect. In tissue mini-units prepared from newborn rats, menadione inhibited ongoing DNA replication with an IC (50) of approximately 10 mu M but 50 mu M had no effect on mini-units from prepared adult rat tissue. The effect of short (72 h) and prolonged exposure (1-2 weeks) to menadione alone in the DBTRG.05MG human glioma cells line and in combination with vitamin C was studied. After short period of exposure data show that menadione alone or in combination with vitamin C provided similar concentration-response curves (and IC50 values). Prolonged exposure to these drugs was evaluated by their ability to kill 100% of glioma cells and prevent regrowth when cells are re-incubated in drug-free media. In this long-term assay, menadione:vitamin C at a ratio 1:100 showed higher anti-proliferative activity when compared to each drug alone and allowed to reduce each drug concentration between 2.5 to 5-fold. Similar anti-proliferative effect was demonstrated in 8 patient derived glioblastoma cell cultures. Our data should be able to encourage further advanced studies on animal models to evaluate the potential use of this combination therapy for glioma treatment

Model for the role of the peptidoglycan framework in cellulose synthesis.

<p>(A) The peptidoglycan framework providing guidance of glucan chains (green line) produced by each cellulose synthase complex located in the inner membrane through the periplasm to a pore, located in the outer membrane and in registry, to form a sub-elementary fiber for extrusion. Several sub-elementary fibers produced from more than one extrusion site aggregate to form a 3.5 nm elementary fibril, and adjacent elementary fibrils co-crystallize to form a 6–7 nm microfibril. (B) Effects of disruption of the peptidoglycan framework on assembly of glucan chains. The glucan chains (green line), synthesized in the inner membrane, may not be in perfect registry with the pore, and lack of guidance may result in some glucan chains not participating in the assembly of a sub-elemental fibril. Thus, the number of glucan chains that reach a pore may be reduced, and the less tightly packed glucan chains in the periplasm may allow non-cellulosic polysaccharides (purple curve line) to be incorporated into the sub-elementary fibril.</p

¹³C NMR spectra of cellulose pellicles produced by mutant I-23 (red), mutant #52 (purple), and wild type (black).

<p>(A) Full spectra with carbon-1 to carbon-6 of glucose in crystalline cellulose indicated. (B) The region of the spectra showing the peak area of carbon-4 of glucose in crystalline cellulose (88.9 ppm), indicated by a blue arrow; peak area of carbon-4 of glucose in non-crystalline cellulose (85.0 ppm), indicated by a green arrow; peak areas of 82.0 ppm, indicated by a red arrow, and 83.0 ppm, indicated by a pink arrow, assigned to carbons of monosaccharides in non-cellulosic polysaccharides. (C) The region of the spectra showing the peak areas of 172.8 ppm, indicated by a yellow arrow, and 176.3 ppm peak, indicated by an orange arrow, assigned to ester or acid carboxyl carbons of monosaccharides in non-cellulosic polysaccharides. (D) The region of the spectra showing peaks assigned to methyl groups at ~20 ppm. (E) The region of the spectra showing carbon-1 of monosaccharides in non-cellulosic polysaccharides.</p

XRD diffractograms of cellulose morphology mutants I-23 and #52, their complemented transformants, I-23_CE and #52_CE, and wild type.

<p>For each mutant, complemented transformant and wild type, cellulose pellicles prepared from three biological replicates were used for analysis, and the result of one representative diffractogram is shown.</p