Abstract
BackgroundPseudomonas 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.ResultsHere, 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.ConclusionsThe results indicate that the implementation of anaerobic respiration in P. putida KT2440 would require at least 49 additional genes of known function, at least 8 genes encoding proteins of unknown function, and 3 externally added vitamins.
Highlights
Pseudomonas putida KT2440 is a metabolically versatile, HV1-certified, genetically accessible, and interesting microbial chassis for biotechnological applications
As there is a relatively short evolutionary distance between the strict aerobic P. putida KT2440 and facultative anaerobic Pseudomonas species compared to other anaerobic bacteria [21], it could be reasoned that a minimal set of adaptations would be required to change an aerobic Pseudomonas species into an facultative anaerobic one
Insertion of acetate kinase in P. putida KT2440 Previous designs to obtain P. putida strains surviving anoxic conditions were conceptually based on the hypothesis that survival in anoxic conditions was prevented by a lack of energy conservation and redox balancing [4, 13,14,15,16]
Summary
Pseudomonas putida KT2440 is a metabolically versatile, HV1-certified, genetically accessible, and interesting microbial chassis for biotechnological applications. Using genome metabolic models (GSMs) iJP962 and iJP746 combined with a protein domain comparison (PDC) between six aerobic Pseudomonas putida strains including KT2440 and six facultative anaerobic Pseudomonas strains, three key enzymes were selected and included in the final design: acetate kinase (encoded by ackA), dihydroorotate dehydrogenase (pyrK-pyrD B) and ribonucleotide triphosphate reductase class III (nrdDnrdG). This design was built, and the resulting recombinant strain showed growth under micro-oxic conditions [23]. Earlier work already described an increase in survival rates upon introduction of solely acetate kinase [4, 14], and since the model predictions used in the design only considered full anoxic conditions, survival rates of the recombinant strains under anoxic conditions needed to be tested
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