RESUMO
Production of biofuels such as ethanol from non-grain crops may contribute to alleviating the global energy crisis and reducing the potential threat to food security. Tobacco (Nicotiana tabacum) is a commercial crop with high biomass yield. Breeding of starch-rich tobacco plants may provide alternative raw materials for the production of fuel ethanol. We cloned the small subunit gene NtSSU of ADP-glucose pyrophosphorylase (NtAGPase), which controls starch biosynthesis in tobacco, and constructed a plant expression vector pCAMBIA1303-NtSSU. The NtSSU gene was overexpressed in tobacco upon Agrobacterium-mediated leaf disc transformation. Phenotypic analysis showed that overexpression of NtSSU gene promoted the accumulation of starch in tobacco leaves, and the content of starch in tobacco leaves increased from 17.5% to 41.7%. The growth rate and biomass yield of the transgenic tobacco with NtSSU gene were also significantly increased. The results revealed that overexpression of NtSSU gene could effectively redirect more photosynthesis carbon flux into starch biosynthesis pathway, which led to an increased biomass yield but did not generate negative effects on other agronomic traits. Therefore, NtSSU gene can be used as an excellent target gene in plant breeding to enrich starch accumulation in vegetative organs to develop new germplasm dedicated to fuel ethanol production.
Assuntos
Biomassa , Regulação da Expressão Gênica de Plantas , Melhoramento Vegetal , Folhas de Planta/genética , Plantas Geneticamente Modificadas/metabolismo , Amido , Nicotiana/metabolismoRESUMO
With the discovery of the significant medicinal value of alginate oligosaccharides and bioethanol produced by microalgae, alginate lyase has been the focus of research in all fields. Five alginate lyase genes in cluster from Vibrio alginolyticus were cloned and expressed in Escherichia coli. SDS-PAGE and enzyme activity showed that four of the five genes have the activity to degrade alginate. Optimization of the induction conditions, protein purification and enzyme properties of rAlgV3 with the highest enzyme activity were studied. The results showed that the enzyme activity of recombinant enzyme rAlgV3 increased from 2.34×10⁴ U/L to 1.68×10⁵ U/L, which was 7.3 times higher than before. The optimal reaction temperature was 40 °C, and the enzyme was relatively stable between 4 °C and 20 °C. The enzyme had a higher activity between pH 6.5 and 9.0, with the optimum pH 8.0. It showed a wide range of pH that the alginate lyase can exist stably between pH 4.5 and 9.5. Appropriate concentrations of NaCl and Fe²⁺, Fe³⁺ ions promoted enzyme activity. SDS and Cu²⁺ ions inhibited the enzyme activity. The enzyme degraded Poly-M fragments and Poly-G fragments, with a wide range of substrate properties. The degraded product of sodium alginate of rAlgV3 analyzed by ESI-MS mainly was oligosaccharides with a polymerization degree of 2 to 3, which means that rAlgV3 was an endo-type alginate lyase. This enzyme has the potential in the development of third-generation bioethanol and the production of alginate oligosaccharides.
RESUMO
During the dilute acid pretreatment of lignocellulosic materials such as corn stover, hemicellulose is hydrolyzed into monosaccharides, and meanwhile, toxic by-products are simultaneously generated, which may influence ethanol fermentation thereafter. Studies on the inhibitory effects of the by-products on ethanol fermentation are of practical use for further improvement of ethanol yield from lignocellulosic materials. Five by-products, including acetic acid, formic acid, vanillin, furfural and 5-hydroxymethylfurfural, were identified to be the main components in the hydrolysate of dilute acid pretreatment of local corn stover, which were added into the medium at different concentrations to study their impacts on the growth and ethanol fermentation of a recombinant xylose-utilizing yeast strain, S. cerevisiae 6508-127. The ethanol production was inhibited by formic acid and acetic acid to a lesser extent than that to the growth, and formic acid was shown to be much more toxic than acetic acid, showing severe inhibitory effects at the concentration of 1g/L, half of the concentration for acetic acid which showed remarkably negative effects on ethanol fermentation. Vanillin caused a much longer lag-phase in growth when the concentration was 2g/L, and the lag-phase was not obvious at lower concentrations. At the concentration of 6g/L, vanillin completely inhibited the fermentation as well as the cell growth. 5-Hydroxymethylfurfural was showed to remarkably inhibit ethanol production, but the biomass yield was higher by exogenous addition of 5-Hydroxymethylfurfural than control. Furfural at 0.5~1.5g/L inhibited the cell growth, but the ethanol yield was higher than that of the control experiment. It was also found that vanillin, furfural and 5-hydroxymethylfurfural could be assimilated and metabolized by S. cerevisiae 6508-127 under the experimental conditions.
RESUMO
The stress resistance of Saccharomyces cerevisiae industrial strains for molasses to high-concentration ethanol,high temperature,high osmotic pressure,furfural toxicity,phenol toxicity,acetic acid toxicity and G418 toxicity were analyzed by the spot dilution growth assays in this paper. The results showed that the stress resistances among these industrial strains were obviously different. The strains AS2.1189 and AS2.1190 are more resistant to the tested stress factors than any others .The strain 396 is the most resistant to the acetic acid toxicity and G418 toxicity,and the strain 2610 is the most resistant to the high temperature.