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Efficient expression of the Aspergillus niger acidic protease PrA in Pichia pastoris
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Bingkun LI1, Yiheng ZHENG2, Fei WANG1, *, Lifeng CHENG3, Ding LI4
Acta Microbiologica Sinica | 2024, 64(9) : 3314 - 3329
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Acta Microbiologica Sinica | 2024, 64(9): 3314-3329
• Research Articles •
Efficient expression of the Aspergillus niger acidic protease PrA in Pichia pastoris
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Bingkun LI1, Yiheng ZHENG2, Fei WANG1, *, Lifeng CHENG3, Ding LI4
Affiliations
  • 1 College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang 330045, Jiangxi, China
  • 2 College of Food and Bioengineering, Henan University of Science and Technology, Luoyang 471000, Henan, China
  • 3 Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 410205, Hunan, China
  • 4 Institute of Veterinary Immunology & Engineering, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, Jiangsu, China
Published: 2024-05-13 doi: 10.13343/j.cnki.wsxb.20240116
Outline
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[Objective] To obtain a yeast strain efficiently producing the acidic protease PrA for applications in food processing, feed additives, and other related industries. [Methods] We constructed a recombinant strain of Pichia pastoris expressing PrA by fermentation in shake flasks and measured the enzymatic properties of the expressed PrA. Several strategies, such as signal peptide modification, gene dosage optimization, and co-expression with molecular chaperones, were employed to enhance the production of PrA. Additionally, high-density fermentation was employed to further improve the expression level. [Results] The expressed enzyme PrA showcased the specific activity of 3 974.00 U/mg, with the optimum performance at pH 3.0 and 45 ℃. The production of PrA by the parental strain was 738.03 U/mL. The modification of the MF4I signal peptide increased the production of PrA to 1 206.52 U/mL. Moreover, an increase in the copy number of prA further increased the PrA production to 2 406.47 U/mL. Additionally, co-expression with single or combined molecular chaperones increased the PrA production to 4 091.27 U/mL. After undergoing high-density fermentation, the enzyme activity reached 43 088.00 U/mL within 168 h, representing a 58.4-fold increase compared with the initial production. [Conclusion] High-level expression of PrA was achieved in P. pastoris, which laid a foundation for the future industrial applications. The results provide valuable insights into the research and development of PrA for applications in food processing and feed additives.

Pichia pastoris  /  acidic protease PrA  /  signal peptide  /  gene dosage  /  molecular chaperone  /  high-density fermentation
Bingkun LI, Yiheng ZHENG, Fei WANG, Lifeng CHENG, Ding LI. Efficient expression of the Aspergillus niger acidic protease PrA in Pichia pastoris[J]. Acta Microbiologica Sinica, 2024 , 64 (9) : 3314 -3329 . DOI: 10.13343/j.cnki.wsxb.20240116
Funding
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  • National Natural Science Foundation of China(32002316)
  • Natural Science Foundation of Hunan Province(2023JJ50315)
Appendix
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Year 2024 volume 64 Issue 9
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113
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Article Info
doi: 10.13343/j.cnki.wsxb.20240116
  • Receive Date：2024-02-26
  • Online Date：2026-03-20
  • Published：2024-05-13
Article Data
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History
  • Received：2024-02-26
  • Accepted：2024-05-07
Funding
National Natural Science Foundation of China(32002316)
Natural Science Foundation of Hunan Province(2023JJ50315)
Affiliations
    1 College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang 330045, Jiangxi, China
    2 College of Food and Bioengineering, Henan University of Science and Technology, Luoyang 471000, Henan, China
    3 Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 410205, Hunan, China
    4 Institute of Veterinary Immunology & Engineering, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, Jiangsu, China

Corresponding:

*WANG Fei, E-mail:
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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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