Constitutive expression of enzymatically active Gluconacetobacter diazotrophicus levansucrase in the methylothrophic yeast Pichia pastoris

Gluconacetobacter diazotrophicus levansucrase (LsdA) was constitutively expressed in P. pastoris under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP) without being toxic to the host yeast. Fusion of the mature part of LsdA to the N-terminal signal sequence of Saccharomyces cerevisiae a factor allowed efficient secretion of the enzyme. After fermentation of a recombinant Pichia pastoris strain harbouring one copy of the IsdA expression cassette integrated in the genome, LsdA activity was totally detected in the bioreactor culture supernatant yielding 0.2 g of total extracellular protein I - 1 (enzyme activity 4000 u r - 1 ). Incubation of the recombinant enzyme in sucrose (500 g I - 1 ) yielded approximately 40 % (w/v) of total sugars as 1-kestose. Yeast fermentation in the presence of sucrose (50 g I - 1 ) increased biomass from 60 g I - 1 to 90 g r - 1 dry weight and led to the direct formation and accumulation of fructans of different degree of polymerisation.