A Rational Design of Pseudomonas putida KT2440 capable of Anaerobic Respiration

Abstract

Pseudomonas putida KT2440 is a metabolically versatile, HV1-certified, genetically accessible, and thus interesting microbial chassis for biotechnological applications. However, its obligate aerobic nature hampers production of oxygen sensitive products and drives up costs in large scale fermentation. The inability to perform anaerobic fermentation has been attributed to insufficient ATP production and an inability to produce pyrimidines under these conditions. Addressing these bottlenecks enabled growth under micro-oxic conditions, but does not lead to growth or survival under anoxic conditions.Here, a data-driven approach was used to develop a rational design for a P. putida KT2440 derivative strain capable of anaerobic respiration. To come to the design, data derived from a genome comparison of 1628 Pseudomonas strains was combined with genome-scale metabolic modelling simulations and a transcriptome dataset of 47 samples representing 14 environmental conditions from the facultative anaerobe Pseudomonas aeruginosa.The results indicate that the implementation of anaerobic respiration in P. putida KT2440 would require at least 61 additional genes of known function, at least 8 genes encoding proteins of unknown function, and 3 externally added vitamins.

The anoxic electrode‐driven fructose catabolism of Pseudomonas putida KT2440

“…Additional mutation of negative regulators, such as PtxS, which regulates 2KGA metabolism (del Castillo et al, 2008;Daddaoua et al, 2010;Udaondo et al, 2018), or Cra, might also add to the alleviation of limited intracellular carbon flux of KT2440 in the case of anoxic catabolism of glucose and fructose, respectively. Nevertheless, improving the carbon flux would only be a part of the puzzle: an in-silico approach by Kampers et al (2021) revealed that genetic and metabolic engineering to enable a facultatively anaerobic lifestyle in KT2440 was a much more elaborate process than previously thought. Thus, substitution of oxygendependent reactions to acclimate the cell to anoxic environment of the BES should also be taken into consideration.…”