194 Plant Genetic Engineering: Towards the Third Millennium A.D. Arencibia (Editors) 2000 Elsevier Science B.V. Strategies for Fructan Production in Transgenic Sugarcane (Saccharmu spp L.) and Sweet Potato (/pomoea batata L.) Plants Expressing the Acetobacter diazotrophicus levansucrase L E Trujillo*, J G Arrieta, G A Enriquez, F Dafhnis, A D Fuentes, R Garcia, M Soto, D Alfonso, A Banguela, L Hernandez Plant-Microbe Interaction Laboratory. Plant Division, CIGB, P.O. Box 6162, Havana 10600, Cuba. * Author for correspondence Introduction Fructans are polyfructose molecules produced by the action of fructosyltransferases on sucrose. Included in the diet, fructans function as fiber due to their non-digestibility. Those of low polymerization degree have a sweet-taste and can be used as low-calorie edulcorants. In addition, their ingestion promotes growth of beneficial bacteria in the gut improving humans and animals health (Yun, 1996). Other uses such as fat replacement, emulsifier, etc, are possible (Fuchs, 1993). Difficulties in fructans purification from natural sources or their production through biotechnological methods hamper their potential applications. This problem could be overcome by the recombinant production and accumulation of these carbohydrates in economically important crops. The bacterial fructosyltransferase genes from Bacillus subtilis, Streptococcus mutants, Bacillus amyloliquejaciens or Erwinia amylovora have been successfully used to transform tobacco, potato and maize into fructan-producer crops (Ebskamp et al., 1994; Van der Meer et al., 1994; Caimi et al., 1996; R6ber et al., 1996). Expression of the plant 1-sucrose:sucrose fructosyltransferase gene (1-sst) in transgenic potato and sugar beet resulted in the production of fructo-oligosaccharides (FOS) (Hellwege et al., 1997; S6venier et al., 1998). Enhancement of stress resistance is another application that is envisioned for fructan production in plants. These compounds may be involved in resistance to drought and low temperatures (Hendry and Wallace, 1993; Pilon-Smith et al., 1995). Marketed FOS consist essentially of 1-kestose, nystose and fructofuranosyl nystose produced from sucrose by microbial enzymes. Acetobacter diazotrophicus fructosyltransferase (LsdA) yields high levels of 1-kestose (Tfimbara et al., 1999), the commercially most attractive FOS (Yun et al., 1990). Herein, we focus on strategies followed for the recombinant expression of LsdA in two economically important crops: sugarcane (Saccharum spp L.) and sweet potato (Ipomoea batatas L.). Both cultivars show physiological features that could allow successful production and accumulation of fructans. Recombinant LsdA is Produced Biologically Active in Eukariotic Hosts The methylotrophic yeast Pichia pastoris was chosen as a model host to study LsdA expression in eukaryotic cells, before attempting the enzyme expression in the sucrose-rich plants sugarcane and sweet potato. According to this strategy, the product of a truncated fragment of the gene encoding levansucrase (lsdA) from Acetobacter diazotrophicus SRT4 was expressed and secreted in P. pastoris