Inactivation of isocitrate lyase leads to increased production of medium-chain-length poly(3-hydroxyalkanoates) in Pseudomonas putida

Medium-chain-length (mcl) poly(3-hydroxyalkanoates) (PHAs) are storage poly mers that are produced from various substrates and accumulate in Pseudomona s strains belonging to rRNA homology group I. In experiments aimed at incre asing PHA production in Pseudomonas strains, we generated an mel PHA-overpr oducing mutant of Pseudomonas putida KT2442 by transposon mutagenesis, in w hich the aceA gene was knocked out. This mutation inactivated the glyoxylat e shunt and reduced the in vitro activity of isocitrate dehydrogenase, a ra te-limiting enzyme of the citric acid cycle. The genotype of the mutant was confirmed by DNA sequencing, and the phenotype was confirmed by biochemica l experiments, The aceA mutant was not able to grow on acetate as a sole ca rbon source due to disruption of the glyoxylate bypass and exhibited two- t o fivefold lower isocitrate dehydrogenase activity than the wild type. Duri ng growth on gluconate, the difference between the mean PHA accumulation in the mutant and the mean PHA accumulation in the wild-type strain was 52%, which resulted in a significant increase in the amount of mel PHA at the en d of the exponential phase in the mutant P, putida KT217, On the basis of a stoichiometric flux analysis we predicted that knockout of the glyoxylate pathway in addition to reduced flux through isocitrate dehydrogenase should lead to increased flux into the fatty acid synthesis pathway. Therefore, e nhanced carbon flow towards the fatty acid synthesis pathway increased the amount of mel PHA that could be accumulated by the mutant.