Trehalose Synthase Gene Transfer Mediated by Agrobacterium tumefaciens Enhances Resistance to Osmotic Stress in Sugarcane

Trehalose synthase gene (TSase) from Grifola frondosa was transferred into sugarcane (Sac-charum hybrid L.) using Agrobacterium-mediated method to improve sugarcane drought-tolerance. The results indicated that embryogenic callus of sugarcane was sensitive to A. tumefaciens EHA105 strain in the transformation system employed. The high frequency PPT-resistant plants were obtained from transformated with 3 weeks callus after incubation, which reached 4.5% on average. The transgenic plants were confirmed by PCR and Southern analysis. Some transgenic plants showed multiple phenotypic alterations and some plants demonstrated improvement tolerance to osmotic stress.

Immobilization of Trehalose Synthase on Mesoporous Molecular Sieve

[ Objective] This study aimed to explore the immobitizatian of trehalose synthase on mesoporous molecular sieves. [ Method] With trehalose synthase produced by genetically engineered bacteria as experimental material, rnesoporous molecular sieve MCM-41 was used as a carrier for immobilization of trehalase syn- thase. Properties of trehalose synthase before and after immobilization were compared. [ Result ] Trehalose synthase could be immohihzed on mesoporous molecular sieve MCM-41 by using physical adsorption method. Immobilization of trehalose synthase achieved the best effect at pH 3.0, with enzyme amount of 62.5 mg/g and immobilization duration of 12 h. Compared with the free u, ehalose synthase, immobilized trehalose synthase showed significantly improved pH stability and thermal stability with repeatable operation, which was conducive to the utilization and storage of enzyme. [ Conclusion] This study provided theoretical basis for the devel- opment of new immobilized trehalose synthase.

Whole‑cell catalytic synthesis of trehalose by Corynebacterium glutamicum displaying trehalose synthase on its cell surface

Trehalose is a disaccharide with many applications in cosmetics,refrigeration,and food.Trehalose synthase is a significant enzyme in trehalose production.Escherichia coli is usually used to express this enzyme heterologously.Since E.coli is a pathogenic strain,we chose Corynebacterium glutamicum ATCC13032 as an engineering strain in this study for food safety reasons.Because of its poor permeability,we constructed two recombinant C.glutamicum strains using two anchor proteins,PorH,and short-length NCgl1337,to anchor trehalose synthase from Streptomyces coelicolor on the cell surface and synthesize trehalose directly from maltose.Studies on enzymatic properties indicated that NCgl1337S–ScTreSK246A had better enzyme activity and thermal stability than the free enzyme.After optimizing the whole-cell transformation,the optimal transformation condition was 35°C,pH 7.0,and OD600 of 30.Under this condition,the conversion rate of 300 g/L maltose reached 69.5%in a 5 L fermentor.The relative conversion rate was still above 75%after repeated five times.

Expression of the Grifola frondosa Trehalose Synthase Gene and Improvement of Drought-Tolerance in Sugarcane （Saccharum officinarum L.）

Trehalose Is a nonreduclng dlsaccharlde of glucose that functions as a protectant In the stabilization of blologlcal structures and enhances stress tolerance to abiotic stresses in organisms. We report here the expression of a Grlfola frondosa trehalose synthase （TSase） gene for Improving drought tolerance In sugarcane （Saccharum offlclnarum L.）. The expression of the transgene was under the control of two tandem copies of the CaMV35S promoter and transferred Into sugarcane by Agrobacterium tumefaciens EHA105. The transgenlc plants accumulated high levels of trehalose, up to 8.805-12.863 mg/g fresh weight, whereas It was present at undetectable level in nontransgenlc plants. It has been reported that transgenlc plants transformed with Escherlchla coil TPS （trehalose-6-phosphatesynthase） and/or TPP （trehalose-6-phosphate phosphatase） are severely stunted and have root morphologlc alterations. Interestingly, our transgenlc sugarcane plants had no obvious morphological changes and no growth Inhibition in the field. Trehalose accumulation in 35S-35S：TSase plants resulted In In- creased drought tolerance, as shown by the drought and the drought physiological Indexes, such as the rate of bound water/free water, plasma membrane permeability, malondlaldehyde content, chlorophyll a and b contents, and activity of SOD and POD of the excised leaves. These results suggest that transgenlc plants transformed with the TSase gene can accumulate high levels of trehalose and have enhanced tolerance to drought.

Genetic Transformation of Tobacco with the Trehalose Synthase Gene from Grifola frondosa Fr. Enhances the Resistance to Drought and Salt in Tobacco

Trehalose is a non-reducing disaccharide of glucose that functions as a protectant in the stabilization of biological structures and enhances the tolerance of organisms to abiotic stress. In the present study, we report on the expression of the Grifola frondosa Fr. Trehalose synthase (Tsase) gene for manipulating abiotic stress tolerance in tobacco (Nicotiana tabaccum L.). The expression of the transgene was under the control of two tandem copies of the CaMV3 5 S promoter and was transferred into tobacco by Agrobacterium tumefaciens EHA105. Compared with non-transgenic plants, transgenic plants were able to accumulate high levels of products of trehalose, which were increased up to 2.126–2.556 mg/g FW, although levels were undetectable in non-transgenic plants. This level of trehalose in transgenic plants was 400-fold higher than that of transgenic tobacco plants cotransformed with Escherichia coli TPS and TPP on independent expression cassettes, twofold higher than that of transgenic rice plants transformed with a bi functional fusion gene (TPSP) of the trehalose-6-phosphate (T-6-P) synthase (TPS) and T-6-P phosphatase (TPP) of E. coli, and 12-fold higher than that of transgenic tobacco plants transformed the yeast TPS1 gene. It has been reported that transgenic plants with E. coli TPS and/or TPP were severely stunted and had morphological alterations of their roots. Interestingly, our transgenic plants have obvious morphological changes, including thick and deep-coloured leaves, but show no growth inhibition; moreover, these morphological changes can restore to normal type in T2 progenies. Trehalose accumulation in 35S–35S:Tsase plants resulted in increased tolerance to drought and salt, as shown by the results of tests on drought, salt tolerance, and drought physiological indices, such as water content in excised leaves, malondialdehyde content, chlorophyll a and b contents, and the activity of superoxide dismutase and peroxidase in excised leaves. These results suggest that transgenic plants transformed with the Tsase gene can accumulate high levels of trehalose and have enhanced tolerance to drought and salt.