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Zusammenfassung:
BackgroundPseudomonas putida is a metabolically versatile, genetically
accessible, and stress-robust species with outstanding potential to be
used as a workhorse for industrial applications. While industry
recognises the importance of robustness under micro-oxic conditions for
a stable production process, the obligate aerobic nature of P. putida,
attributed to its inability to produce sufficient ATP and maintain its
redox balance without molecular oxygen, severely limits its use for
biotechnology applications.ResultsHere, a combination of genome-scale
metabolic modelling and comparative genomics is used to pinpoint
essential O2-dependent processes. These explain the inability of the
strain to grow under anoxic conditions: a deficient ATP generation and
an inability to synthesize essential metabolites. Based on this, several
P. putida recombinant strains were constructed harbouring acetate kinase
from Escherichia coli for ATP production, and a class I dihydroorotate
dehydrogenase and a class III anaerobic ribonucleotide triphosphate
reductase from Lactobacillus lactis for the synthesis of essential
metabolites. Initial computational designs were fine-tuned by means of
adaptive laboratory evolution.ConclusionsWe demonstrated the value of
combining in silico approaches, experimental validation and adaptive
laboratory evolution for microbial design by making the strictly aerobic
Pseudomonas putida able to grow under micro-oxic conditions.
