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

Abstract:

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 α 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 extra-cellular protein l-1 (enzyme activity 4000 u l-1). Incubation of the recombinant enzyme in sucrose (500 g l-1) yielded approximately 40 % (w/v) of total sugars as 1-kestose. Yeast fermentation in the presence of sucrose (50 g l-1) increased biomass from 60 g l-1 to 90 g l-1 dry weight and led to the direct formation and accumulation of fructans of different degree of polymerisation.