Metabolic Engineering of Corynebacterium glutamicum for Trehalose Overproduction: Role of the TreYZ Trehalose Biosynthetic Pathway

Trehalose has many potential applications in biotechnology and the food industry due to its protective effect against environmental stress. Our work explores microbiological production methods based on the capacity of Corynebacterium glutamicum to excrete trehalose. We address here raising trehalose productivity through homologous overexpression of maltooligosyltrehalose synthase and the maltooligosyltrehalose trehalohydrolase genes. In addition, heterologous expression of the UDP-glucose pyrophosphorylase gene from Escherichia coli improved the supply of glycogen. Gene expression effects were tested on enzymatic activities and intracellular glycogen content, as well as on accumulated and excreted trehalose. Overexpression of the treY gene and the treY/treZ synthetic operon significantly increased maltooligosyltrehalose synthase activity, the rate-limiting step, and improved the specific productivity and the final titer of trehalose. Furthermore, a strong decrease was noted in glycogen accumulation. Expression of galU/treY and galU/treYZ synthetic operons showed a partial recovery in the intracellular glycogen levels and a significant improvement in both intra- and extracellular trehalose content.     

Genetic Characterization of the Phenylacetyl-Coenzyme A Oxygenase from the Aerobic Phenylacetic Acid Degradation Pathway of Escherichia coli

We show here that the paaABCDE genes of the paa cluster responsible for phenylacetate degradation in Escherichia coli W encode a five-component oxygenase that hydroxylates phenylacetyl-coenzyme A (CoA), the first intermediate of the pathway. The primary structure of the subunits of bacterial phenylacetyl-CoA oxygenases revealed that these enzymes constitute the prototype of a new and distinct group of the large bacterial diiron multicomponent oxygenase family.     

Study of the effect of the peptide loading and solvent system in SPPS by HRMAS-NMR.

The SPPS methodology has continuously been investigated as a valuable model to monitor the solvation properties of polymeric materials. In this connection, the present work applied HRMAS-NMR spectroscopy to examine the dynamics of an aggregating peptide sequence attached to a resin core with varying peptide loading (up to 80%) and solvent system. Low and high substituted BHAR were used for assembling the VQAAIDYING sequence and some of its minor fragments. The HRMAS-NMR results were in agreement with the swelling of each resin, i.e. there was an improved resolution of resonance peaks in the better solvated conditions. Moreover, the peptide loading and the attached peptide sequence also affected the spectra. Strong peptide chain aggregation was observed mainly in highly peptide loaded resins when solvated in CDCl3. Conversely, due to the better swelling of these highly loaded resins in DMSO, improved NMR spectra were acquired in this polar aprotic solvent, thus enabling the detection of relevant sequence-dependent conformational alterations. The more prominent aggregation was displayed by the VQAAIDYING segment and not by any of its intermediary fragments and these findings were also corroborated by EPR studies of these peptide-resins labelled properly with an amino acid-type spin probe.     

Size-Related Differential Seed Predation in a Heavily Defaunated Neotropical Rain Forest

Hunting and habitat loss represent an increasingly common anthropogenic impact. Available evidence suggests that defaunation is typically differential: medium/large mammals are most affected while small rodents are less affected, or even favored. In heavily impacted areas, such as Los Tuxtlas, the residual mammalian fauna is dominated by small rodents. We tested the expectation that if small rodents will preferentially attack small-seeded species, large-seeded species may escape predation in the absence of medium/large seed predators and that this may affect germination and, possibly, recruitment. Experiments with caged rodents ( Heteromys desmarestianus ) under laboratory conditions showed a preference for smaller seeds. A field experiment involving seeds of contrasting size showed that small, unprotected seeds experienced a predation risk 30-times larger than protected seeds, while the effect of protection was not significant for large seeds. These patterns of predation led to significant differences in germination: protected small-seeded species had a fourfold greater germination than unprotected small-seeded species, while germination was not significantly different between exposed and protected large seeds. The observed contrasts in germination suggest that under heavy defaunation, small-seeded species are likely to be penalized by the overabundance of small rodents, while large-seeded species escape predation. Our results are consistent with the observation of dense seedling carpets dominated by large-seeded species in the understory of Los Tuxtlas. We speculate that the patterns we uncovered may explain, at least partly, the impoverished diversity of the understory vegetation that characterizes heavily defaunated understories and that this has the potential to affect forest regeneration.     

Environmental Control of Early Succession on a Large Landslide in a Tropical Dry Ecosystem (Casita Volcano, Nicaragua)

We described the plant communities on a large landslide in a human-dominated area of tropical dry forest landscape (Casita Volcano, Nicaragua) and in the adjacent forest, 3 yr after landslide occurrence. At both sites, we determined the relationships between spatial changes in environmental factors and the spatial distribution of species and plant traits. Subsequently, we tested the hypothesis that the compositional similarity between the landslide and the forest increased with a decrease in the distance from the forest edge and the width of the landslide. In the forest, the spatial distribution of species and plant traits was determined mainly by an elevational gradient that was associated with the amount of bare soil, whereas, on the landslide, there was no such gradient but species distributions were influenced mostly by the presence of residual agricultural and forest soils and human disturbance. We did not find an increase in compositional similarity between the landslide and the forest at the edge or in the narrow zones of the landslide. Compared to other landslides, the recovery process was strongly influenced by the extreme abiotic heterogeneity, climate seasonality, and human use in the area. The study of succession in tropical dry landslides located in densely populated zones should focus on understanding the response of regional ecosystems to a complex disturbance regime in which human-induced disturbances play a major role.     

Immunogenicity and safety assessment of the Cuban recombinant hepatitis B vaccine in healthy adults.

Manufactures of biotechnological/biological products (including vaccines) frequently make changes to manufacturing processes of products both during development and after approval. In our case, a non-inferiority bridging study was carried out to demonstrate that changes in the production plant facilities of Cuban recombinant hepatitis B vaccine, Heberbiovac HB, did not affect the safety and immunogenicity of the vaccine. This controlled, randomized, doubled-blinded trial included 501 volunteers, aged between 20 and 64, who were given three doses of vaccine (20 microg HBsAg/mL) at month 0, 1, and 2. Four lots were evaluated (three corresponding to the new production facilities and a control one produced in the older facilities). One month after the third dose, were observed protective levels of anti-HBsAg in 97% of the subjects that concluded the study with a geometric mean antibody titer (GMT) of 931.18 IU/L. Normal values of body mass index (BMI), the younger ages, and being a female, were significantly related to a good antibody response. The vaccine was well tolerated. Pain at the injection site was the most commonly reported symptom. We conclude that Heberbiovac HB vaccine maintains its characteristics after the modifications carried out in the production plant facilities and both, lot obtained in previous facilities and in the new ones, are comparable in terms of safety and immunogenicity.     

Cytoskeletal rearrangement and cell death induced by Bothrops alternatus snake venom in cultured Madin-Darby canine kidney cells.

Bothrops snake venoms cause renal damage, with renal failure being the main cause of death in humans bitten by these snakes. In this work, we investigated the cytoskeletal rearrangement and cytotoxicity caused by Bothrops alternatus venom in cultured Madin-Darby canine kidney (MDCK) cells. Incubation with venom (10 and 100 microg/mL) significantly (p <0.05) decreased the cellular uptake of neutral red dye after 1 and 3 h. Venom (100 microg/mL) also markedly decreased the transepithelial electrical resistance (RT) across MDCK monolayers. Staining with rhodamine-conjugated phalloidin revealed disarray of the cytoskeleton that involved the stress fibers at the basal cell surface and focal adhesion-associated F-actin in the cell-matrix contact region. Feulgen staining showed a significant decrease in the number of cells undergoing mitosis and an increase in the frequency of altered nuclei. Scanning electron microscopy revealed a decrease in the number of microvilli and the presence of cells with a fusiform format. Flow cytometry with annexin V and propidium iodide showed that cell death occurred by necrosis, with little apoptosis, a conclusion supported by the lack of DNA fragmentation characteristic of apoptosis. Pretreating the cells with catalase significantly attenuated the venom-induced loss of viability, indicating a possible involvement of H2O2 in the cellular damage; less protection was observed with superoxide dismutase or N omega-nitro-L-arginine methyl ester. These results indicate that Bothrops alternatus venom is cytotoxic to cultured MDCK cells, possibly via the action of reactive oxygen species. This cytotoxicity could contribute to nephrotoxicity after envenoming by this species.