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Enhanced human lysozyme production by Pichia pastoris via periodic glycerol and dissolved oxygen concentrations control

  • Biotechnological products and process engineering
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Abstract

In human lysozyme (hLYZ) production by Pichia pastoris, the glycerol fed-batch phase was generally implemented under the environment of “oxygen sufficient-glycerol limited” to achieve high cell-density cultivation during the cell growth phase. However, the structural and functional components in P. pastoris cells were irreversible damaged with more and more reactive oxygen species (ROS) accumulation when cells were exposed to the oxygen sufficient environments for long time, leading to a failure of hLYZ expression. In this study, a novel periodic glycerol and dissolved oxygen concentration (DO) control strategy was proposed to solve these problems. This strategy periodically switched the cultivation environments from “oxygen sufficient-glycerol limited” to “oxygen limited-glycerol sufficient” for 5 cycles. When using this strategy: (1) the highest dry cell weight (DCW) of 143.02 g-DCW/L and the lowest distribution of glycerol towards to cell maintenance (0.0400 1/h) were achieved during the glycerol feeding phase by maintaining ROS levels below 48.39 Fluorescence intensity/g-DCW; (2) the adaption time of P. pastoris cells to methanol induction environments was shortened for about 50%; (3) P. pastoris cell metabolic activities reflected by the activities of alcohol oxidase, formaldehyde dehydrogenase, formate dehydrogenase, and methanol consumption rate, etc., in the successive induction phase were largely enhanced; (4) hLYZ activity reached the highest level of 2.45 × 105 IU/mL, which was about 2-fold than that obtained with the strategy of “oxygen sufficient-glycerol limited,” when the same methanol induction strategy was adopted.

Key points

• A novel periodic glycerol feeding strategy proposed/used for P. pastoris cell growth.

• Higher cell density was obtained by controlling ROS at low level via this strategy.

• The highest hLYZ activity was achieved when initiating induction at higher cell density.

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Data availability

All data generated or analyzed during this study are included in this published article (and its supplementary information files).

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Funding

This work was supported by the Natural Science Foundation of Jiangsu Province (BK20180922, BK20190890), the Jiangsu University Students Practice Innovation Training Program (201911117090Y), and the Key Research & Development Program of Dantu District (SH2018001).

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Authors and Affiliations

  1. School of Food Science and Technology, Yangzhou University, Yangzhou, 225127, China

    Luqiang Jia, Teng Li, Yixuan Wu, Chunsen Wu, Huaxiang Li & Agen Huang

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

    Luqiang Jia

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  1. Luqiang Jia
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  2. Teng Li
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  3. Yixuan Wu
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  4. Chunsen Wu
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  5. Huaxiang Li
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  6. Agen Huang
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Contributions

LJ: Supervision, conceptualization, methodology, writing—review and editing, funding acquisition. TL: Investigation, writing—original draft. YW: Data curation. CW: Visualization, funding acquisition. HL: Supervision, funding acquisition. AH: Supervision, writing—review and editing.

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Correspondence to Luqiang Jia or Agen Huang.

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Jia, L., Li, T., Wu, Y. et al. Enhanced human lysozyme production by Pichia pastoris via periodic glycerol and dissolved oxygen concentrations control. Appl Microbiol Biotechnol 105, 1041–1050 (2021). https://doi.org/10.1007/s00253-021-11100-9

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