High-level constitutive expression of leech hyaluronidase with combined strategies in recombinant Pichia pastoris

Hyaluronidases that break down hyaluronan are widely used for preparation of low molecular weight hyaluronan. Leech hyaluronidase (LHyal) is a newly discovered hyaluronidase with outstanding enzymatic properties. The Pichia pastoris expression system of LHyal that depends on AOX1 promoter (P-AOX1) has been constructed. However, the addition of the toxic inducer methanol is a big safety concern. Here, a combinational strategy was adopted for constitutive expression of LHyal to high level in P. pastoris. By optimizing the combination of promoters P-GAP, P-GAP(m), and P-TEF1 and signal peptides alpha-factor, nsB, and sp23, the enzyme activity of extracellular LHyal reached 1.38 x 10(5) U/mL in shake flasks. N-terminal engineering with neutral polar amino acids further increased LHyal activity to 2.06 x 10(5) U/mL. In addition, the impact of overexpressing transcription factors Aft1, Gal4-like, and Yap1 on LHyal production was also investigated. We found the co-expression of Aft1 significantly enhanced the expression of LHyal to 3.03 x 10(5) U/mL. Finally, LHyal activity of 2.12 x 10(6) U/mL was achieved in a 3-L fermenter, with a high productivity of 1.96 x 10(4) U/mL/h. The engineered LHyal-producing Pichia pastoris strains will be more attractive for production of hyaluronidase on industrial scale.