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Fructans, homopolymers of fructose produced by fructosyltransferases (FTs), are emerging as intriguing components in halophiles since they are thought to be associated with osmotic stress tolerance and overall fitness of microorganisms and plants under high-salinity conditions. Here, we report on the full characterization of the first halophilic FT, a levansucrase from Halomonas smyrnensis AAD6T (HsLsc; EC 2.4.1.10). The encoding gene (lsc) was cloned into a vector with a 6xHis Tag at its C-terminus, then expressed in Escherichia coli. The purified recombinant enzyme (47.3 kDa) produces levan and a wide variety of fructooligosaccharides from sucrose, but only in the presence of high salt concentrations (> 1.5 M NaCl). HsLsc showed Hill kinetics and pH and temperature optima of 5.9 and 37 °C, respectively. Interestingly, HsLsc was still very active at salt concentrations close to saturation (4.5 M NaCl) and was selectively inhibited by divalent cations. The enzyme showed high potential in producing novel saccharides derived from raffinose as both fructosyl donor and acceptor and cellobiose, lactose, galactose, and ʟ-arabinose as fructosyl acceptors. With its unique biochemical characteristics, HsLsc is an important enzyme for future research and potential industrial applications in a world faced with drought and diminishing freshwater supplies. [ABSTRACT FROM AUTHOR]

Levansucrase (EC 2.4.1.10) was identified as a constitutive exoenzyme in 14 Gluconacetobacter diazotrophicus strains recovered from different host plants in diverse geographical regions. The enzyme, consisting of a single 60-kDa polypeptide, hydrolysed sucrose to synthesise oligofructans and levan. Sugar-cane-associated strains of the most abundant genotype (electrophoretic type 1) showed maximal values of levansucrase production. These values were three-fold higher than those of the isolates recovered from coffee plants. Restriction fragment length polymorphism analysis revealed a high degree of conservation of the levansucrase locus (lsdA) among the 14 strains under study, which represented 11 different G. diazotrophicus genotypes. Targeted disruption of the lsdA gene in four representative strains abolished their ability to grow on sucrose, indicating that the endophytic species G. diazotrophicus utilises plant sucrose via levansucrase. [ABSTRACT FROM AUTHOR]

Bacterial levan [β(2,6)-linked polyfructan] has potential applications in the food, bio-energetic, medical, pharmaceutical, and other industries. The lack of technically and economically feasible large-scale production systems limits the commercial exploitation of levan. Gluconacetobacter diazotrophicus secretes a levansucrase (LsdA, EC 2.4.1.10) that synthesizes high levels of levan and fructooligosaccharides from sucrose. This bacterium is not attractive for the cost-effective production of LsdA. In this research, we used Pichia pastoris and Nicotiana tabacum as hosts for LsdA production and direct levan synthesis, respectively. The recombinant yeast constitutively expressing the lsdA gene acquired saccharolytic capacity and secreted LsdA to a yield 9-fold higher than the value reported for the natural host. The occurrence of N-glycosylation in the yeast-produced LsdA did not affect the catalytic efficiency, substrate specificity, or product profile compared to the native non-glycosylated enzyme. This finding prompted us to express the lsdA gene in vacuoles, the most physiologically appropriate compartment to direct levan formation within the plant cell. Constitutive expression of LsdA fused to the vacuolar targeting signal of an onion fructosyltransferase allowed the accumulation of highly polymerized levan (above 104 fructose residues) in mature tobacco leaves where the polymer represented between 10 and 70% (w/w) of total dry weight. The latter value is the highest reported in the literature for a levan-producing transgenic plant grown in soil. No drastic physiological changes were observed in tobacco plants with levan yields up to 30% (w/w) in leaves. The polymer production remained stable in the plant progenies pointing for potential application in biotechnology. [ABSTRACT FROM AUTHOR]

Sugarcane (Saccharum spp. hybrid) emerges as an ideal crop for the cost-effective transgenic production of fructans due to its high efficiency for fixing carbon and storing the substrate sucrose. As other gramineous species, sugarcane is recalcitrant to genetic transformation. In this work, we optimized conditions for the transformation of sugarcane cv. C1051 -73 via microprojectile bombardment of embryogenic calli. The genes encoding the enhanced green-fluorescent protein (eGFP) and the neomycin phosphotransferase (nptII), both under the control of the maize ubiquitin 1 (Ubi-1) promoter, were used for the early detection of transient transformation events and for the selection of stable transfor-mants, respectively. DNA was efficiently delivered into the cell without causing drastic damages in calli bombarded at the distance of 11 cm and the argon pressure of 90 PSI. Non-mosaic transgenic plantlets were recovered by increasing the geneticin concentration from 20 mg/L during callus growth to 25 mg/L for the shooting and rooting steps. Moreover, using the optimized transformation procedure, we recovered twenty transgenic sugarcane lines carrying the Gluconacetobacter diazotrophicus levansucrase gene (lsdA) modified for vacuolar targeting of the enzyme, as a strategy for fructan production. Southern blot and PCRanalysis revealed the stable presence of the chimaeric lsdA in the primary stalk and sprouts of plants grown under field conditions. None of the transgenic lines accumulated levan in mature stems or leaves, although one of them showed evident levansucrase activity in leaf extracts. [ABSTRACT FROM AUTHOR]

Abstract: Bacterial levansucrase (EC 2.4.1.10) converts sucrose into non-linear levan consisting of long β(2,6)-linked fructosyl chains with β(2,1) branches. Bacterial levan has wide food and non-food applications, but its production in industrial reactors is costly and low yielding. Here, we report the constitutive expression of Gluconacetobacter diazotrophicus levansucrase (LsdA) fused to the vacuolar targeting pre-pro-peptide of onion sucrose:sucrose 1-fructosyltransferase (1-SST) in tobacco, a crop that does not naturally produce fructans. In the transgenic plants, levan with degree of polymerization above 104 fructosyl units was detected in leaves, stem, root, and flowers, but not in seeds. High levan accumulation in leaves led to gradual phenotypic alterations that increased with plant age through the flowering stage. In the transgenic lines, the fructan content in mature leaves varied from 10 to 70% of total dry weight. No oligofructans were stored in the plant organs, although the in vitro reaction of transgenic LsdA with sucrose yielded β(2,1)-linked FOS and levan. Transgenic lines with levan representing up to 30mgg−1 of fresh leaf weight produced viable seeds and the polymer accumulation remained stable in the tested T1 and T2 progenies. The lsdA-expressing tobacco represents an alternative source of highly polymerized levan. [Copyright &y& Elsevier]