Efficient endo-β-1,3-glucanase expression in Pichia pastoris for co-culture with Agrobacterium sp. for direct curdlan oligosaccharide production

Abstract

The production of curdlan oligosaccharides, a multifunctional and valuable carbohydrate, by hydrolyzing polysaccharides is of great interest. The endo-β-1,3-glucanase derived from Trichoderma harzianum was expressed in Pichia pastoris with three commonly used promoters (AOX1, GAP and FLD1). The purified recombinant endo-β-1,3-glucanase expressed by Pichia pastoris with GAP promoter displayed high specific activity at pH 5.5 and 50 °C. Thereafter, a co-culture system of Pichia pastoris GS115 (GAP promoter) and Agrobacterium sp. was constructed in which Agrobacterium sp.-metabolized curdlan can be directly hydrolyzed by Pichia pastoris-secreted endo-β-1,3-glucanase to produce functional curdlan oligosaccharides. The co-culture conditions were optimized and the process was carried out in a 7-L bioreactor. The maximum yield of curdlan oligosaccharides reached 18.77 g/L with 3-10 degrees of polymerization. This study presents a novel and easy curdlan oligosaccharide production strategy that can replace traditional sophisticated production procedures and could potentially be implemented for production of other oligosaccharides.

Keywords: Agrobacterium sp.; Co-culture; Curdlan oligosaccharides; Pichia pastoris; endo-β-1,3-glucanase.