The Entner–Doudoroff pathway empowers Pseudomonas putida KT2440 with a high tolerance to oxidative stress

Authors

  • Max Chavarría,

    1. Systems and Synthetic Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), Madrid, Spain
    2. Escuela de Química, Universidad de Costa Rica, San José, Costa Rica
    Search for more papers by this author
    • Ex aequo contribution.
  • Pablo I. Nikel,

    1. Systems and Synthetic Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), Madrid, Spain
    Search for more papers by this author
    • Ex aequo contribution.
  • Danilo Pérez-Pantoja,

    1. Systems and Synthetic Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), Madrid, Spain
    Search for more papers by this author
  • Víctor de Lorenzo

    Corresponding author
    • Systems and Synthetic Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), Madrid, Spain
    Search for more papers by this author

For correspondence. E-mail vdlorenzo@cnb.csic.es; Tel. (+34) 91 585 4536; Fax (+34) 91 585 4506.

Summary

Glucose catabolism of Pseudomonas putida is carried out exclusively through the Entner–Doudoroff (ED) pathway due to the absence of 6-phosphofructokinase. In order to activate the Embden–Meyerhof–Parnas (EMP) route we transferred the pfkA gene from Escherichia coli to a P. putida wild-type strain as well as to an eda mutant, i.e. lacking 2-keto-3-deoxy-6-phosphogluconate aldolase. PfkAE. coli failed to redirect the carbon flow from the ED route towards the EMP pathway, suggesting that ED was essential for sugar catabolism. The presence of PfkAE. coli was detrimental for growth, which could be traced to the reduction of ATP and NAD(P)H pools along with alteration of the NAD(P)H/NADP+ ratio. Pseudomonas putida cells carrying PfkAE. coli became highly sensitive to diamide and hydrogen peroxide, the response to which is very demanding of NADPH. The inhibitory effect of PfkAE. coli could in part be relieved by methionine, the synthesis of which relies much on NADPH. These results expose the role of the ED pathway for generating the redox currency (NADPH) that is required for counteracting oxidative stress. It is thus likely that environmental bacteria that favour the ED pathway over the EMP pathway do so in order to gear their aerobic metabolism to endure oxidative-related insults.

Ancillary