High-level constitutive expression of leech hyaluronidase with combined strategies in recombinant Pichia pastoris
Research article published in Appl Microbiol Biotechnol (2020)
Abstract
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 (PAOX1) 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 PGAP, PGAP(m), and PTEF1 and signal peptides α-factor, nsB, and sp23, the enzyme activity of extracellular LHyal reached 1.38 × 105 U/mL in shake flasks. N-terminal engineering with neutral polar amino acids further increased LHyal activity to 2.06 × 105 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 × 105 U/mL. Finally, LHyal activity of 2.12 × 106 U/mL was achieved in a 3-L fermenter, with a high productivity of 1.96 × 104 U/mL/h. The engineered LHyal-producing Pichia pastoris strains will be more attractive for production of hyaluronidase on industrial scale.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Summary
Hyaluronidases that break down hyaluronan are widely used for preparation of low molecular weight hyaluronan.
Why This Matters for Hirudotherapy
This study engineered recombinant Pichia pastoris strains for constitutive, high-level expression of leech hyaluronidase (LHyal), eliminating the need for toxic methanol induction by combining optimized promoters (PGAP, PGAP(m), PTEF1), signal peptides, N-terminal engineering, and transcription factor co-expression. The investigators achieved LHyal activity of 2.12 × 10⁶ U/mL in a 3-L fermenter with productivity of 1.96 × 10⁴ U/mL/h. This work is relevant to ASH's domain because hyaluronidase is a component of the leech secretome that degrades hyaluronan, and scalable production could support research into leech-derived enzymatic tools and potential applications. The caveat is that this is a bioprocess engineering study focused on industrial enzyme production; it does not address hirudotherapy, clinical use, or in-vivo activity of LHyal, and no therapeutic claims are made.
Citation
High-level constitutive expression of leech hyaluronidase with combined strategies in recombinant Pichia pastoris.
Huang H et al. · Appl Microbiol Biotechnol, 2020
Added to ASH library: May 27, 2026 · Site last updated: June 18, 2026