Combinatorial strategy of sorbitol feeding and low-temperature induction leads to high-level production of alkaline β-mannanase in Pichia pastoris

A process for efficient production of an alkaline β-mannanases from Bacillus sp. N16-5 was established by heterologous expression using Pichia pastoris. A high producing strain was generated by removing the native β-mannanases signal peptide and increasing the copy number of the mature β-mannanases gene. High cell density fermentation of this strain in 1-L bioreactor led to a production level of 4164 U/mL after 96 h of induction. Sorbitol co-feeding and temperature-lowering strategies both increased the β-mannanase production levels. Combined usage of these two strategies achieved the most effective result—the enzyme level reached 6336 U/mL within 84 h, which to our best knowledge is the highest production level reported for the expression of extreme β-mannanase thus far. The strategy described in this work can also be adapted to express other important industrial enzymes with extreme properties.

► Removal of the native signal peptide is a prerequisite to achieve high yield.
► Increasing the gene dosage dramatically increased the mannanase production level.
► The combinatorial strategy clearly led to better performance than either alone.
► The enzyme activity of the expressed alkaline β-mannanase reached 6336 U/ml.
► This is the highest alkaline β-mannanases level produced to date.