Increasing gene dosage and chaperones co-expression facilitate the efficient dextranase expression in Pichia pastoris

Abstract

Dextranase (DEX) is an important hydrolase in the food industry, which can degrade the dextran into low-molecular-weight polysaccharides. However, its commercial applications are limited by ineffective heterologous recombination system. Here, we build an efficient Pichia pastoris system for DEX following two different engineering strategies. First, through increasing the gene dosage, the DEX activity increased from 40.13 U/mL to 121.02 U/mL. The up-regulation of the endoplasmic reticulum stress gene ERO1 was detected by the qPCR. Since chaperones are involved in the protein folding and modification, co-expression of chaperones was applied to relieve the endoplasmic reticulum stress and facilitated further increase in DEX activity to 164.78 U/mL. Then, high cell density cultivation of P. pastoris was performed in a 5 L fermenter (cell dry weight reached 105.37 g/L), resulting in the DEX activity to 619.78 U/mL. Finally, under the conditions of 25 °C, pH 5.0, 5% sorbitol as co-substrates, and a feed rate of 7.2 mL/h, a DEX yield of 3257.23 U/mL was obtained through 5-L fed-batch fermentation. DCFH-DA staining showed that the reactive oxygen species level decreased by 68.83% due to sorbitol co-feeding. Our study thus represents an important step for building a robust P. pastoris expression system for recombinant DEX production.