Metabolic pathways analysis in PHAs production by Pseudomonas with 13C-labeling experiments

Abstract

Abstract Metabolic flux analysis is a useful tool for metabolism characterization and verification of genetic modification effects; it is a support for decisions on biotechnological process improvement. Commercial production of biodegradable polymers, specifically polyhydroxyalkanoates PHAs, is restricted by production costs, which may be cut by increasing yield from substrate to product, since carbon source for PHAs production accounts up to 50% of the total production costs; additionally, in Pseudomonas sp . LMF046 the experimental yield is between 60-70% of theoretical maximum yield. This work presents metabolic pathways identification, flux quantification and analysis on this strain, employing position isotopomer balancing and measurements of labeling patterns in the polymer by GC-MS. Initial results –using [1- 13 C]glucose-allow to rule out carbohydrate catabolism by EMP pathway, whereas final ones –using a mixture of [U- 13 C]glucose and natural glucose– allow to estimate fraction of glucose metabolized by ED and PP pathways. Metabolic network includes eight intracellular metabolite and 324 isotopomer balances, and it is solved in 1.3 seconds in a Core i5 PC. Sensitivity analysis shows inclusion of carbon natural labeling improves the prediction. The estimated ratio for sugar metabolism into PP and ED pathways, 1.35:0.55, that corresponds to 77% of the theoretical yield, leads to the conclusion that the glucose metabolism in larger proportion by the Pentoses pathway is the main reason for a low yield. The problem has been solved satisfactorily, and a sensitivity analysis shows that it is necessary to reduce uncertainty on labeling measurements.