Fermentation of hexoses and pentoses from sugarcane bagasse hydrolysates into ethanol by Spathaspora hagerdaliae

The present study evaluated 13 strains of yeast for ethanol and xylitol production from xylose. Among them, Spathaspora hagerdaliae UFMG-CM-Y303 produced ethanol yields (Y_P/S) of 0.25 g g^− 1 and 0.39 g g^− 1 under aerobic and microaerophilic conditions, respectively, from a mixture of glucose and xylose in flasks. A pH of 5.0 and an inoculum of 3.0 × 10⁸ cells mL^− 1r resulted in the highest ethanol yields. These conditions were tested in a bioreactor for fermenting a medium containing an enzymatic hydrolysate of sugarcane bagasse with 15.5 g L^− 1 of glucose and 3 g L^− 1 of xylose, and achieved a Y_P/S of 0.47 g g^− 1, in relation to total available sugar. These results suggest that S. hagerdaliae UFMG-CM-Y303 has potential for use in second-generation ethanol studies.

     

Spidroins from the Brazilian spider Nephilengys cruentata (Araneae: Nephilidae)

Spiders produce up to six different kinds of silk, each one for a specific biological function. Spider silks are also known for their unique mechanical properties. The possibility of producing new materials with similar properties motivated research on these silk proteins (spidroins). Using expression sequence tags, we identified four spidroins produced by major ampullate, minor ampullate, flagelliform and tubuliform silk glands from the Brazilian spider Nephilengys cruentata (Araneae: Nephilidae). The new protein sequences showed substantial similarity to other spidroins previously described, with high content of alanine and glycine due to the presence of the highly repetitive motifs (polyAla, (GA)_n, (GGX)_n, (GPGGX)_n). Similarities among sequences were also observed between the different spidroins with the exception of tubuliform spidroin, which presents a unique complex amino acid sequence with high amounts of serine and low amounts of glycine.     

Effectiveness of liposomes to transfect livestock fibroblasts

The development of an efficient transfection system in livestock cells is an important step towards investigating gene transfer and the functioning and production of transgenic animals. Important factors involved in cationic liposome mediated gene transfer were evaluated through in vitro transfection of bovine, caprine and ovine fibroblast cells. Transfection of plasmid DNA complexes of different commercially available liposomes (Lipofectamine, Lipofectin, Cellfectin and DMRIE-C; Gibco-BRL, USA) was evaluated utilizing the following parameters: DNA/liposome ratio, cell density, DNA conformation, and the effect of transfection time on the efficiency of bovine fibroblasts to express a reporter gene. The effects and concentrations of liposomes were also evaluated in caprine and ovine fibroblasts. Lipofectamine alone and Lipofectamine with Plus reagent induced high-frequency expression of beta-galactosidase and neo genes in all cells evaluated (47 and 88.3%, respectively). Regarding phenotype, chromosomal stability was similar in transfected and non-transfected cells. The parameters set in this study will establish a foundation for utilizing transfected fibroblast cells to generate transgenic animals through nuclear transfer technology and gene function studies.     

Application of electrochemically produced aluminum hydroxide gel for prepurification of recombinant synthetic green fluorescent protein produced in tobacco leaves

The use of recombinant proteins has increased greatly in recent years, as also have increased the number of techniques and materials used for their production and purification. Among the different types of bioreactors being studied, there is a general consensus among scientists that production in green plant tissues such as leaves is more feasible. However, the presence of chlorophyll and phenolic compounds in plant extracts, which can precipitate and denature the proteins besides damaging separation membranes and gels, makes this technology impracticable on a commercial scale. In the present work, the adsorption to electrochemically produced aluminum hydroxide gel was applied as a prepurification step for recombinant synthetic green fluorescent protein (sGFP), also referred to as enhanced green fluorescent protein, produced in Nicotiana benthamiana leaves. Removal efficiencies of 99.7% of chlorophyll, 88.5% of phenolic compounds, and 38.5% of native proteins from the N. benthamiana extracts were achieved without removing sGFP from the extracts. As electrochemical preparation of aluminum hydroxide gel is a cost‐effective technique, its use can substantially contribute to the development of future production platforms for recombinant proteins produced in green plant tissues of pharmaceutical and industrial interest. © 2011 American Institute of Chemical Engineers Biotechnol. Prog.,, 2011     

Microparticle bombardment of Stylosanthes guianensis: transformation parameters and expression of a methionine-rich 2S albumin gene

An effective protocol to generate stable transformants of the tropical forage legume Stylosanthes guianensis (Aubl.) Sw. in a selection-free system was developed. Based on transient reporter gene expression, we have obtained transformation rates of 3.47% using 30-day-old calli as target, 1300 psi helium pressure, 12.5 cm microprojectile flight distance, 10–20 mm distance between macrocarrier membrane and stopping screen and 10–20 mm gap distance between the shock wave generator and the macrocarrier. These parameters were utilized to produce transgenic S. guianensis plants expressing Be2S1 from Bertholletia excelsa that codes for a methionine-rich storage protein driven by a green-tissue specific promoter, Ats1 from Arabidopsis thaliana. Transgenic plants were identified by a PCR-based high-throughput screen in a selective agent-free system, employing pools of 20–50 regenerating shoots. The integration of the exogenous gene in the host genome was confirmed by Southern blot analysis of PCR-positive plants. The expression of the introduced gene was confirmed in leaf tissue of transgenic plants by Northern and Western blot analyses. Immunoblots of cellular fractions showed that BE2S1 expressed in Stylosanthes is mainly targeted to the vacuoles.     

Radiation as a tool to remove selective marker genes from transgenic soybean plants

The present study evaluated the use of γ-radiation to physically remove selective marker genes previously introduced into the soybean genome. Homozygous seeds from a transgenic soybean line carrying the gus and ahas transgenes were irradiated with γ-rays. Six plants presenting a deleted gus gene were analyzed by Southern blot to confirm removal of both ahas and gus genes. Line 1A presented an absence of the gus gene cassette and presence of the ahas gene cassette.