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pH-stat fed-batch process to enhance the production of cis, cis-muconate from benzoate by Pseudomonas putida KT2440-JD1

Authors

  • Jozef B. J. H. van Duuren,

    Corresponding author
    1. Wageningen UR Food & Biobased Research, P.O. Box 17, 6700AA Wageningen, The Netherlands
    2. Helmholtz Centre for Infection Research, Synthetic and Systems Biology, Inhoffenstraße 7, D-38124 Braunschweig, Germany
    • Wageningen UR Food & Biobased Research, P.O. Box 17, 6700AA Wageningen, The Netherlands
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  • Dorien Wijte,

    1. TNO Defence, Security and Safety, P.O. Box 45, 2289AA Rijswijk, The Netherlands
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  • Bianka Karge,

    1. Helmholtz Centre for Infection Research, Synthetic and Systems Biology, Inhoffenstraße 7, D-38124 Braunschweig, Germany
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  • Vítor A.P. Martins dos Santos,

    1. Helmholtz Centre for Infection Research, Synthetic and Systems Biology, Inhoffenstraße 7, D-38124 Braunschweig, Germany
    Current affiliation:
    1. Wageningen University, Laboratory of Systems and Synthetic Biology, Dreijenplein 10, Building number 316, 6703 HB Wageningen, The Netherlands.
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  • Yu Yang,

    1. Wageningen UR Food & Biobased Research, P.O. Box 17, 6700AA Wageningen, The Netherlands
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  • Astrid E. Mars,

    1. Wageningen UR Food & Biobased Research, P.O. Box 17, 6700AA Wageningen, The Netherlands
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  • Gerrit Eggink

    1. Wageningen UR Food & Biobased Research, P.O. Box 17, 6700AA Wageningen, The Netherlands
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Abstract

Pseudomonas putida KT2440-JD1 is able to cometabolize benzoate to cis, cis-muconate in the presence of glucose as growth substrate. P. putida KT2440-JD1 was unable to grow in the presence of concentrations above 50 mM benzoate or 600 mM cis, cis-muconate. The inhibitory effects of both compounds were cumulative. The maximum specific uptake rate of benzoate was higher than the specific production rate of cis, cis-muconate during growth on glucose in the presence of benzoate, indicating that a benzoate derivative accumulated in the cells, which is likely to be catechol. Catechol was shown to reduce the expression level of the ben operon, which encodes the conversion of benzoate to cis, cis-muconate. To prevent overdoses of benzoate, a pH-stat fed-batch process for the production of cis, cis-muconate from benzoate was developed, in which the addition of benzoate was coupled to the acidification of the medium. The maximum specific production rate during the pH-stat fed-batch process was 0.6 g (4.3 mmol) g dry cell weight−1 h−1, whereas 18.5 g L−1 cis, cis-muconate accumulated in the culture medium with a molar product yield of close to 100%. Proteome analysis revealed that the outer membrane protein H1 was upregulated during the pH-stat fed-batch process, whereas the expression of 10 other proteins was reduced. The identified proteins are involved in energy household, transport, translation of RNA, and motility. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2012

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