Combined strategies to improve the expression of acidic mammalian chitinase in Pichia pastoris for the production of N, N′-diacetylchitobiose

Acidic mammalian chitinase (AMCase) could hydrolyze chitin into (GlcNAc)(2) at pH 2.0. Therefore, it was essential to develop an engineered strain with a high-yield AMCase. Herein, multiple strategies including codon optimization, multiple expression cassettes insertion, co-expression of helper proteins, were developed to construct a recombinant Pichia pastoris with the high-level secretion of AMCase. The recombinant strain harboring four copies of codon-optimized AMCase-encoding gene ChiD and co-expression of HACl presented the highest chitinase activity of 570 U/L in high density fermentation, which was 934 folds higher than that in E. coli reported before. Then, the recombinant AMCase was applied to hydrolyze colloidal chitin with a conversion rate of 90 % at pH 2.0, which could save half of the water consumption during the preparation of colloidal chitin. Finally, we also demonstrated that the hydrolysate of AMCase could promote seed germination of wheat and rice, improving the growth of roots and seedling.

Automated summary

Through multiple engineering strategies, a recombinant Pichia pastoris with high-level secretion of AMCase was successfully constructed, showing significantly improved chitinase activity under high-density fermentation conditions. The hydrolysis of colloidal chitin by the recombinant AMCase not only could reduce water consumption significantly, but also promote seed germination and seedling growth of wheat and rice.