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

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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.

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Funding

This work was financially supported by a grant from the Key Technologies R&D Program of Jiangsu Province (BE2019630), the National Natural Science Foundation of China (31670092), the Fundamental Research Funds for the Central Universities (JUSRP51707A), and the National First-class Discipline Program of Light Industry Technology and Engineering (LITE2018-16).

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Authors and Affiliations

  1. The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China

    Hao Huang, Qixing Liang, Yang Wang, Jian Chen & Zhen Kang

  2. The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China

    Hao Huang, Qixing Liang, Yang Wang, Jian Chen & Zhen Kang

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  1. Hao Huang
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  2. Qixing Liang
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  3. Yang Wang
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Correspondence to Jian Chen or Zhen Kang.

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Huang, H., Liang, Q., Wang, Y. et al. High-level constitutive expression of leech hyaluronidase with combined strategies in recombinant Pichia pastoris. Appl Microbiol Biotechnol 104, 1621–1632 (2020). https://doi.org/10.1007/s00253-019-10282-7

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