Cloning and functional analysis of a high DP fructan:fructan 1-fructosyl transferase from Echinops ritro (Asteraceae): comparison of the native and recombinant enzymes

Abstract

Inulin-type fructans are the simplest and most studied fructans and have become increasingly popular as prebiotic health-improving compounds. A natural variation in the degree of polymerization (DP) of inulins is observed within the family of the Asteraceae. Globe thistle (Echinops ritro), artichoke (Cynara scolymus), and Viguiera discolor biosynthesize fructans with a considerably higher DP than Cichorium intybus (chicory), Helianthus tuberosus (Jerusalem artichoke), and Dahlia variabilis. The higher DP in some species can be explained by the presence of special fructan:fructan 1-fructosyl transferases (high DP 1-FFTs), different from the classical low DP 1-FFTs. Here, the RT-PCR-based cloning of a high DP 1-FFT cDNA from Echinops ritro is described, starting from peptide sequence information derived from the purified native high DP 1-FFT enzyme. The cDNA was successfully expressed in Pichia pastoris. A comparison is made between the mass fingerprints of the native, heterodimeric enzyme and its recombinant, monomeric counterpart (mass fingerprints and kinetical analysis) showing that they have very similar properties. The recombinant enzyme is a functional 1-FFT lacking invertase and 1-SST activities, but shows a small intrinsic 1-FEH activity. The enzyme is capable of producing a high DP inulin pattern in vitro, similar to the one observed in vivo. Depending on conditions, the enzyme is able to produce fructo-oligosaccharides (FOS) as well. Therefore, the enzyme might be suitable for both FOS and high DP inulin production in bioreactors. Alternatively, introduction of the high DP 1-FFT gene in chicory, a crop widely used for inulin extraction, could lead to an increase in DP which is useful for a number of specific industrial applications. 1-FFT expression analysis correlates well with high DP fructan accumulation in vivo, suggesting that the enzyme is responsible for high DP fructan formation in planta.

Levans in Excised Leaves ofDactylis glomerata: Effects of Light, Sugars, Temperature and Senescence

“…However, this seems highly unlikely since i. no 6-FFT ~ype enzymes have ever been reported in plants ii. so far fructan biosynthetic enzymes generally show a very low affinity (high Km) for their substrates (Van Laere and Van den Ende, 2002), a noticeable exception being the high DP 1-FFT from Echinops ritro (Van den Ende et al, 2006) showing an apparent donor Km of 15 mM for 1-kestose. Taken togetk, er, these observations suggest that fructan biosynthesis in orchardgrass is independent of 1-SST and FFT-type enzymes but might occur by a combination of 6-SST and 6-SFT enzymes or even by a single 6-SST/ 6-SFT enzyme with characteristics that might resemble those of some bacterial levansucrases.…”

Stem carbohydrate dynamics and expression of genes involved in fructan accumulation and remobilization during grain growth in wheat (Triticum aestivum L.) genotypes with contrasting tolerance to water stress

“…These include 1-fructan exohydrolase (1-FEH) and 6-fructan exohydrolase (6-FEH) [39, 40, 41] and they catalyze the reaction of fructan, which participates in fructan depolymerization, with β (2,1) and β (2,6) linkages, respectively [42]. The β (2,6) linkages are predominant in wheat stems [35, 43]. …”

Heterologous expression and comparative characterization of vacuolar invertases from Cu-tolerant and non-tolerant populations of Elsholtzia haichowensis

“…The methylotrophic yeast P. pastoris has been widely used for expressing invertases from various plants (Huang et al 2003;De Coninck et al 2005;Canam et al 2008;Du et al 2013) and investigating the effects of amino acid substitutions on invertase properties (Fridman et al 2004;Ritsema et al 2005Ritsema et al , 2006Le Roy et al 2007;Schroeven et al 2008;Chen et al 2009). Invertases as well as other enzymes in the GH32 family expressed in P. pastoris showed similar properties to those isolated directly from native tissues (De Coninck et al 2005;Hsieh et al 2006;Van den Ende et al 2006). In our experiment, we successfully expressed the VINs using the wild type P. pastoris X-33 strain.…”