Abstract

Pseudomonas sp. LFM046 was selected as the best medium-chain-length polyhydroxyalkanoate (PHAMCL) producer from glucose and fructose, presenting a better performance than Pseudomonas putida KT2440, the most studied bacterial strain for the production of this kind of polymer. A core metabolic network consisting mainly of Entner-Doudoroff (ED) pathway (linear or cyclic), pentose phosphate pathway (PPP), tricarboxylic acid cycle (TCA), glyoxylic acid cycle (GAC) and de novo fatty acids biosynthesis (FAB) was reconstructed as the supplier of monomers for PHA biosynthesis . The reconstruction was based on sequenced genomes available from different Pseudomonas species. Shaken flask cultivations revealed two clearly distinguished phases in the culture: cell multiplication and PHA accumulation. PHAMCL produced by Pseudomonas sp. LFM046 from glucose and fructose contained 3 mol% 3-hydroxyhexanoate, 29 mol% of 3-hydroxyoctanoate, 65 mol% of 3-hydroxydecanoate and 3 mol% of 3-hydroxydodecanoate. During accumulation phase, PHA yield from carbohydrates reached 0.2531 g/g. Elementary mode analysis revealed that the best performance in converting glucose into PHAMCL should be achieved with an almost equimolar distribution of the glucose flux in the ED and PPP, as well as no flux in the TCA and GCA pathways. This metabolic flux allows to obtain a PHA yield from carbohydrates of 0.3307 g/g. Therefore, the actual performance of Pseudomonas sp. LFM046 corresponds to about 77% of the maximum theoretical yield. Since data from cultures using 13C glucose indicated a flux in PPP twice higher than in ED, it was suggested that increasing carbon flux in ED would improve the performance of Pseudomonas sp. LFM046 to convert hexoses into PHA. The Pseudomonas genome is being sequenced to confirm the reconstructed metabolic network and to improve flux distribution analysis.