Robustness of Pseudomonas 1 putida KT2440 as a host for ethanol 2 biosynthesis

Expansion of the burgeoning biofuels agenda involves not only the design of suitable genetic and metabolic devices but also their deployment into suitable hosts that can endure the stress brought about by the products themselves. The microorganisms easiest to genetically manipulate for these endeavours (e.g., Escherichia coli) are often afflicted by an undesirable sensitivity to the very product that they are engineered to synthesize. In this context, we have examined the resistance to the stress arising from ethanol synthesis and/or its addition to cultures of recombinant Pseudomonas putida, using as a benchmark the same trait in an E. coli strain. To this end, ethanologenic strains of these two species were constructed by functionally expressing pdc (pyruvate decarboxylase) and adhB (alcohol dehydrogenase) from Zymomonas mobilis. Recombinants were compared under anoxic conditions as ethanol producers, and cell survival, stress resistance, and phenotypic stability were quantified in each case. P. putida consistently outperformed E. coli in every ethanol tolerance test conducted – whether the alcohol was produced endogenously or added exogenously. These results highlight the value of such bacterium as a microbial cell factory for the production of biofuels owing to its naturally preevolved ability to withstand different kinds of chemical stresses.This study was supported by the BIO Program of the Spanish Ministry of Economy and Competitiveness, the ST-FLOW and ARIAYS Contracts of the EU, and the PROMT Project of the CAM.Peer reviewe