Efficient Expression of Human Lysozyme Through the Increased Gene Dosage and Co-expression of Transcription Factor Hac1p in Pichia pastoris

  • Jun Liu
  • Qin Han
  • Qikun Cheng
  • Yuanyuan Chen
  • Ruxin Wang
  • Xin Li
  • Yulan Liu
  • Dazhong YanEmail author


In this work, the high-level expression of the human lysozyme (HLY) was achieved by both optimization of the gene copy number and co-expression of the transcription factor Hac1p for the unfolded protein response (UPR) in the host strain Pichia pastoris KM71H. A series of recombinant constructs with various numbers of HLY expression cassettes was generated for the production of recombinant strains integrated with different copies of the HLY gene. The copy number of the HLY gene was determined by real-time quantitative polymerase chain reaction, and the recombinant strains of P. pastoris carrying one, two, three, four, or six copies of the HLY gene were obtained. Maximum extracellular protein and lysozyme enzyme activity reached 436.99 ± 26.08 μg/mL and 61,900 ± 2036.47 U/mL, respectively, in the recombinant strain HLYH4-3 with the four copies of the HLY gene after shaking flask fermentation. Moreover, the co-expression of the transcription factor Hac1p in the recombinant strains further enhanced the HLY yields. Extracellular protein and lysozyme enzyme activity, respectively, reached 517.82 ± 4.19 μg/mL and 78,600 ± 1134.95 U/mL by using the Hac1p co-expression strain HLYH4-3/Hac1p. These values are the highest recorded level of human lysozyme expressed by P. pastoris in shaking flask fermentation so far.



This study was financially supported by the Open Project of Hubei Key Laboratory of Animal Nutrition and Feed Science (Grant Number 201809).


This study was financially supported by the Open Project of Hubei Key Laboratory of Animal Nutrition and Feed Science (Grant Number 201809).

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conficts of interest.


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Jun Liu
    • 1
    • 2
  • Qin Han
    • 2
  • Qikun Cheng
    • 2
  • Yuanyuan Chen
    • 2
  • Ruxin Wang
    • 2
  • Xin Li
    • 2
  • Yulan Liu
    • 1
  • Dazhong Yan
    • 1
    • 2
    Email author
  1. 1.Hubei Key Laboratory of Animal Nutrition and Feed ScienceWuhan Polytechnic UniversityWuhanChina
  2. 2.School of Biology and Pharmaceutical EngineeringWuhan Polytechnic UniversityWuhanChina

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