Fructooligosaccharides production by Schedonorus arundinaceus sucrose:sucrose 1-fructosyltransferase constitutively expressed to high levels in Pichia pastoris

Abstract

The non-saccharolytic yeast Pichia pastoris was engineered to express constitutively the mature region of sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99) from Tall fescue (Schedonorus arundinaceus). The increase of the transgene dosage from one to nine copies enhanced 7.9-fold the recombinant enzyme (Sa1-SSTrec) yield without causing cell toxicity. Secretion driven by the Saccharomyces cerevisiae α-factor signal peptide resulted in periplasmic retention (38%) and extracellular release (62%) of Sa1-SSTrec to an overall activity of 102.1 U/ml when biomass reached (106 g/l, dry weight) in fed-batch fermentation using cane sugar for cell growth. The volumetric productivity of the nine-copy clone PGFT6x-308 at the end of fermentation (72 h) was 1422.2 U/l/h. Sa1-SSTrec purified from the culture supernatant was a monomeric glycoprotein optimally active at pH 5.0-6.0 and 45-50 °C. The removal of N-linked oligosaccharides by Endo Hf treatment decreased the enzyme stability but had no effect on the substrate and product specificities. Sa1-SSTrec converted sucrose (600 g/l) into 1-kestose (GF₂) and nystose (GF₃) in a ratio 9:1 with their sum representing 55-60% (w/w) of the total carbohydrates in the reaction mixture. Variations in the sucrose (100-800 g/l) or enzyme (1.5-15 units per gram of substrate) concentrations kept unaltered the product profile. Sa1-SSTrec is an attractive candidate enzyme for the industrial production of short-chain fructooligosaccharides, most particularly 1-kestose.

Keywords: 1-kestose; FOS; Fructooligosaccharides; Fructosyltransferase; GAP promoter; Pichia pastoris.