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Genetic engineering of Pseudomonas putida KT2440 for rapid and high-yield production of vanillin from ferulic acid

Applied Microbiology and Biotechnology volume 98, pages 137–149 (2014)Cite this article

Abstract

Vanillin is one of the most important flavoring agents used today. That is why many efforts have been made on biotechnological production from natural abundant substrates. In this work, the nonpathogenic Pseudomonas putida strain KT2440 was genetically optimized to convert ferulic acid to vanillin. Deletion of the vanillin dehydrogenase gene (vdh) was not sufficiant to prevent vanillin degradation. Additional inactivation of a molybdate transporter, identified by transposon mutagenesis, led to a strain incapable to grow on vanillin as sole carbon source. The bioconversion was optimized by enhanced chromosomal expression of the structural genes for feruloyl-CoA synthetase (fcs) and enoyl-CoA hydratase/aldolase (ech) by introduction of the strong tac promoter system. Further genetic engineering led to high initial conversion rates and molar vanillin yields up to 86 % within just 3 h accompanied with very low by-product levels. To our knowledge, this represents the highest productivity and molar vanillin yield gained with a Pseudomonas strain so far. Together with its high tolerance for ferulic acid, the developed, plasmid-free P. putida strain represents a promising candidate for the biotechnological production of vanillin.

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Acknowledgments

The authors would like to thank the Federal Ministry of Science and Education (BMBF), Germany, for funding this project (Systembiologie in Pseudomonas, FZK 315406). We also thank Armin Huber, Jens Pfannstiel, and Oliver Simon from the Proteomics Core Facility of Life Science Center, University of Hohenheim, Germany, for providing the proteomics data. We also thank Georg Sprenger for helpful discussions.

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  1. Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, 70569, Stuttgart, Germany

    Nadja Graf & Josef Altenbuchner

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  1. Nadja Graf
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  2. Josef Altenbuchner
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Correspondence to Josef Altenbuchner.

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Graf, N., Altenbuchner, J. Genetic engineering of Pseudomonas putida KT2440 for rapid and high-yield production of vanillin from ferulic acid. Appl Microbiol Biotechnol 98, 137–149 (2014). https://doi.org/10.1007/s00253-013-5303-1

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  • DOI: https://doi.org/10.1007/s00253-013-5303-1

Keywords

  • Bioconversion
  • Pseudomonas putida
  • Vanillin
  • Ferulic acid
  • Genetic engineering
  • Plasmid-free