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High-Level Extracellular Production of Glucose Oxidase by Recombinant Pichia Pastoris Using a Combined Strategy

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Abstract

In this work, a combined strategy was developed to improve the production of glucose oxidase (GOD) (EC 1.1.3.4) in Pichia pastoris. One of the main challenges facing protein production by the high-density fermentation of P. pastoris is the high demand for oxygen. Another challenge is how to balance a reduction in oxygen consumption and its effects on protein production. Herein, a combined strategy involving mannitol co-feeding, two-stage methanol induction, and the co-expression of the transcriptional activator general control non-derepressible 4 (GCN4) from P. pastoris was used. A two-stage, co-feeding strategy, based on a mannitol/methanol mixture in a 3-L fermentor was used to enhance cell viability and protein production. This resulted in an increased GOD yield of 1208.2 U/mL compared with a control strain (427.6 U/mL). An increase in the copy number of the GCN4 gene enhanced the GOD yield (1634.7 U/mL) by 2.8-fold and the protein concentration (19.55 g/L) by 1.58-fold compared with the control (7.59 g/L). This strategy illustrates a way to overcome the high oxygen requirement during high-density fermentation of P. pastoris and balances the reduction of oxygen consumption and protein production. Moreover, the series of strategies presented in this work provide valuable and novel information for the industrial production of GOD.

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Acknowledgments

This project was financially supported by the National High Technology Research and Development Program of China (863 Program, 2011AA100905), the Major State Basic Research Development Program of China (973 Program, 2013CB733902), Synergetic Innovation Center Of Food Safety and Nutrition, the Natural Science Foundation of Jiangsu Province (BK2012553), the China Postdoctoral Science Foundation(2013M540538), and the 111 Project (111-2-06).

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Authors and Affiliations

  1. Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China

    Lei Gu, Juan Zhang & Baihong Liu

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

    Guocheng Du

  3. National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, 214122, China

    Jian Chen

  4. School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, China

    Lei Gu, Juan Zhang, Baihong Liu, Guocheng Du & Jian Chen

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  1. Lei Gu
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  2. Juan Zhang
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  4. Guocheng Du
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  5. Jian Chen
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Correspondence to Juan Zhang or Jian Chen.

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Gu, L., Zhang, J., Liu, B. et al. High-Level Extracellular Production of Glucose Oxidase by Recombinant Pichia Pastoris Using a Combined Strategy. Appl Biochem Biotechnol 175, 1429–1447 (2015). https://doi.org/10.1007/s12010-014-1387-z

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  • DOI: https://doi.org/10.1007/s12010-014-1387-z

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