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Background   Pseudomnas putida  is a natural producer of medium chain length polyhydroxyalkanoates (mcl-PHA), a polymeric precursor of bioplastics. A two-fold increase of mcl-PHA production via inactivation of the glucose dehydrogenase gene  gcd , limiting the metabolic flux towards side products like gluconate was achieved before. Here, we investigated the overproduction of enzymes catalyzing limiting steps of mcl-PHA precursor formation.    Results  A genome-based  in silico  model for  P. putida  KT2440 metabolism was employed to identify potential genetic targets to be engineered for the improvement of mcl-PHA production using glucose as sole carbon source. Here, overproduction of pyruvate dehydrogenase subunit AcoA in the  P. putida  KT2440 wild type and the Δ gcd  mutant strains led to an increase of PHA production. In controlled bioreactor batch fermentations PHA production was increased by 33% in the  acoA  overexpressing wild type and 121% in the  acoA  overexpressing Δ gcd  strain in comparison to  P. putida  KT2440. Overexpression of  pgl- encoding 6-phosphoglucolactonase did not influence PHA production. Transcriptome analyses of engineered PHA producing  P. putida  in comparison to its parental strains revealed the induction of genes encoding glucose 6-phosphate dehydrogenase and pyruvate dehydrogenase. In addition, NADPH seems to be quantitatively consumed for efficient PHA synthesis, since a direct relationship between low levels of NADPH and high concentrations of the biopolymer were observed. In contrast, intracellular levels of NADH were found increased in PHA producing organisms.    Conclusion  Production of mcl-PHAs was enhanced in  P. putida  when grown on glucose via overproduction of a pyruvate dehydrogenase subunit (AcoA) in combination with a deletion of the glucose dehydrogenase ( gcd ) gene as predicted by  in silico  elementary flux mode analysis.

Production of medium chain length polyhydroxyalkanoate in metabolic flux optimized Pseudomonas putida