title: Comprehensive proteome analysis of the response of Pseudomonas putida KT2440 to the flavor compound vanillin.
abstract: Understanding of the molecular response of bacteria to precursors, products and environmental conditions applied in bioconversions is essential for optimizing whole-cell biocatalysis. To investigate the molecular response of the potential biocatalyst Pseudomonas putida KT2440 to the flavor compound vanillin we applied complementary gel- and LC-MS-based quantitative proteomics approaches. Our comprehensive proteomics survey included cytoplasmic and membrane proteins and led to the identification and quantification of 1614 proteins, corresponding to 30% of the total KT2440 proteome. 662 proteins were altered in abundance during growth on vanillin as sole carbon source as compared to growth on glucose. The proteome response entailed an increased abundance of enzymes involved in vanillin degradation, significant changes in central energy metabolism and an activation of solvent tolerance mechanisms. With respect to vanillin metabolism, particularly enzymes belonging to the β-ketoadipate pathway including a transcriptional regulator and porins specific for vanillin uptake increased in abundance. However, catabolism of vanillin was not dependent on vanillin dehydrogenase (Vdh), as shown by quantitative proteome analysis of a Vdh-deficient KT2440 mutant (GN235). Other aldehyde dehydrogenases that were significantly increased in abundance in response to vanillin may replace Vdh and thus may represent interesting targets for improving vanillin production in P. putida KT2440.
  
  
  CONCLUSIONS
  The high demand for the flavor compound vanillin by the food and fragrance industry makes natural vanillin from vanilla pods a scarce and expensive resource rendering its biotechnological production economically attractive. Pseudomonas bacteria are metabolically very versatile and accept a broad range of hydrocarbons as carbon source making them suitable candidates for bioconversion processes. This work describes the impact of vanillin on the metabolism of the reference strain P. putida KT2440 on a proteome wide scale. The high proteome coverage of our proteomics survey allowed us to analyze the regulation of whole protein networks instead of single proteins. We were able to reconstruct the complete degradation pathway of vanillin and to monitor the changes in the energy metabolism of KT2440 induced by vanillin as sole carbon source. Vanillin dehydrogenase (Vdh) was not mandatory for vanillin degradation in KT2440 and may be substituted by other aldehyde dehydrogenases that were up-regulated in a wild-type as well as in a Vdh-deficient strain in the presence of vanillin. Aldehyde dehydrogenases, vanillin specific porins and efflux pump systems identified in study will be interesting targets for optimization of vanillin production in Pseudomonas bacteria. Furthermore, several mechanisms of solvent tolerance were induced by vanillin in KT2440. These include increased abundance of several efflux pump systems, chaperones as well as enzymes involved in cyclopropane fatty acid synthesis and trehalose formation. The present work will deepen the understanding of metabolism of aromatic compounds in P. putida and may lead to a more comprehensive understanding of solvent tolerance mechanisms in Gram-negative bacteria in general. Moreover, it will serve as a basis for further strain developments for a biotechnological production of vanillin in P. putida KT2440 or other Pseudomonas strains, highlighting the role of proteomics surveys as a powerful screening technology.
authors.affiliation: Department of Biosensorics, Institute of Physiology, University of Hohenheim, August von Hartmann-Str. 3, 70599 Stuttgart, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Simon
authors.affiliation: Proteomics Core Facility of the Life Science Center, University of Hohenheim, August von Hartmann-Str. 3, 70599 Stuttgart, Germany.
authors.forename: Iris
authors.initials: I
authors.lastname: Klaiber
authors.affiliation: Department of Biosensorics, Institute of Physiology, University of Hohenheim, August von Hartmann-Str. 3, 70599 Stuttgart, Germany; Proteomics Core Facility of the Life Science Center, University of Hohenheim, August von Hartmann-Str. 3, 70599 Stuttgart, Germany.
authors.forename: Armin
authors.initials: A
authors.lastname: Huber
authors.affiliation: Proteomics Core Facility of the Life Science Center, University of Hohenheim, August von Hartmann-Str. 3, 70599 Stuttgart, Germany. Electronic address: Jens.Pfannstiel@uni-hohenheim.de.
authors.forename: Jens
authors.initials: J
authors.lastname: Pfannstiel
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001547:Benzaldehydes / Q000378:metabolism* / Q000494:pharmacology; D005421:Flavoring Agents / Q000378:metabolism* / Q000494:pharmacology; D009154:Mutation; D020543:Proteome / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics; D016958:Pseudomonas putida / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D001547:Benzaldehydes; D005421:Flavoring Agents; D020543:Proteome; C100058:vanillin
keywords: 2-D DIGE; Biocatalysis; GeLC–MS/MS; Organic solvent tolerance; Pseudomonas putida KT2440; Vanillin