Molecular cloning and characterization of a high DP fructan: fructan 1‐fructosyl transferase from Viguiera discolor (Asteraceae) and its heterologous expression in Pichia pastoris

Abstract

Inulin‐type fructans are stored in the tuberous roots of the Brazilian cerrado plant Viguiera discolor Baker (Asteraceae). In Cynara scolymus (artichoke) and Echinops ritro (globe thistle), the fructans have a considerably higher degree of polymerization (DP) than in Cichorium intybus (chicory) and Helianthus tuberosus (Jerusalem artichoke). It was shown before that the higher DP in some species can be attributed to the properties of their fructan: fructan 1‐fructosyl transferases (1‐FFTs; EC 2.4.1.100), enzymes responsible for chain elongation. Here, we describe the cloning of a high DP (hDP) 1‐FFT cDNA from V. discolor and its heterologous expression in Pichia pastoris. Starting from 1‐kestose and Neosugar P (a mixture of oligo‐inulins from microbial origin) as substrates, the recombinant enzyme produces a typical hDP inulin profile in vitro, closely resembling the one observed in vivo. The enzyme shows no invertase activity and sucrose: sucrose 1‐fructosyl transferase (1‐SST; EC 2.4.1.99) activity in vitro. Pattern evolution during incubation suggests that inulins with DP ≥ 6 are much better substrates than sucrose or lower DP oligo‐fructans. Because hDP inulin‐type fructans show superior properties for specific food and non‐food applications, the hDP 1‐FFT gene from V. discolor has potential for the production of hDP inulin in vitro or in transgenic crops.