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Auto-induction-based Rapid Evaluation of Extracellular Enzyme Expression from Lac Operator-involved Recombinant Escherichia coli

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Abstract

Degeneration of engineered strains and decreased production of target gene products were often observed during recombinant bioprocess. Although several strategies have been developed to ensure high levels of target gene products, these methods seemed to be complex and laborious. By investigating possible factors contributing to the decreased yield, degeneration of host cells was identified as the main cause. Based on the principle of auto-induction and the fact that the interaction between colored substrates and gene products could present visible changes, a convenient and accurate screening method was developed to evaluate the production performance of engineered strains. Due to the typicality of pullulanase from genus Klebsiella, which has been a model for the research of secretion mechanism in gram-negative bacteria, an engineered E. coli producing extracellular pullulanase was employed to illustrate the method. Consequently, according to the capability to form a colorless halo, colonies could be divided into two groups, one surrounded by clear zone and the other unable to present transparent haloes. Furthermore, the high capability of these colonies was confirmed by performing pullulanase production in liquid medium. Compared with other methods for evaluating engineered strains, the visible screening technique was suggested to have the advantages of effectiveness and accuracy.

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Acknowledgments

Financial supports from the National Hi-Tech Research and Development Program of China (863 Program) (2012AA022207), the National Key Basic Research and Development Program of China (973 Program) (2011CB710800), the National Natural Science Foundation of China (NSFC) (21376107), the Program of Introducing Talents of Discipline to Universities (111 Project) (111-2-06), the High-end Foreign Experts Recruitment Program (GDW20133200113), the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, and the Jiangsu province “Collaborative Innovation Center for Advanced Industrial Fermentation” industry development program are gratefully appreciated.

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Authors and Affiliations

  1. Key Laboratory of Industrial Biotechnology, Ministry of Education and School of Biotechnology, Jiangnan University, Wuxi, 214122, People’s Republic of China

    Wen-Bo Chen, Yao Nie, Xiao-Qing Mu, Wei Yan & Yan Xu

  2. Institute of Agro-Products Processing Science and Technology, Chinese Academy of Agricultural Science, Beijing, 100193, China

    Wen-Bo Chen

  3. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu Province, China

    Yan Xu

  4. Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ, 08854, USA

    Rong Xiao

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  1. Wen-Bo Chen
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  2. Yao Nie
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  3. Xiao-Qing Mu
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Correspondence to Yao Nie or Yan Xu.

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Chen, WB., Nie, Y., Mu, XQ. et al. Auto-induction-based Rapid Evaluation of Extracellular Enzyme Expression from Lac Operator-involved Recombinant Escherichia coli . Appl Biochem Biotechnol 174, 2516–2526 (2014). https://doi.org/10.1007/s12010-014-1201-y

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