Metabolic flux analysis of poly(3-hydroxybutyrate-CO-3-hydroxyvalerate) biosynthetic pathways in Ralstonia eutropha NCIMB 11599

Abstract

Studies of application of metabolic flux analysis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3-HV)] biosynthesis in Ralstonia eutropha NCIMB 11599. Improves the production process of P(3HB-co-3HV), a biodegradable plastic, to meet the commercial requirement that the copolymer contains the fraction of 3-hydroxyvalerate (3HV) monomeric unit as much as possible. The research is categorized into four main sections. First section, proposes metabolic network that can describe the metabolic behavior of R. eutropha NCIMB 11599 when it is growing under various conditions, i.e., under different carbon sources and levels of nitrogen limitation. Accordingly, there are four metabolic networks. These pathways are depending upon used substrate and level of nitrogen limitation. Mainly, there are such similarities among them; namely, modified pattern of pentose-phosphate (PP) pathway, acetyl coenzyme A formation, tricarboxylic acid (TCA) cycle, 3-hydroxybutyrate formation route, ammonium assimilation via GS-GOGAT system, residual biomass formation, oxidative phosphorylation and respiratory pathway, and phosphoenolpyruvate (PEP) carboxykinase as the only route of anaplerotic pathway. As for the differences, there are at the catabolism of glucose and propionic acid, 3-hydroxyvalerate formation route, and NADPH-linked malic enzyme as an additional source of NADPH production. Second section, calculation of maximum theoretical 3HV molar fraction in the copolymer is carried out. It can be concluded that the maximum theoretical values (maximum at 75%) are higher than the ones computed from the experimental data (maximum at 53%). This suggests that there is still more room for improvement of 3HV fraction in the copolymer. Interestingly, the improvement can be systematically attained by the application of metabolic engineering. Regarding the third section, identification of principle nodes and possible bottlenecks is accomplished. According to the analysis of fluxsplit ratio, there are two principle nodes in the metabolic network where a mixture of glucose and propionic acid is used as a carbon source. These nodes are acetyl coenzyme A (AcCoA) and propionyl coenzyme A (ProCoA). Providing that the increased molar fraction of 3HV is desired, flux split ratios at these two nodes need to be modified since the fluxes around these two nodes are possible bottlenecks. The modifications can be achieved by; a) increasing the flux of AcCoA that condenses with ProCoA to form more 3HV; b) decreasing the flux of AcCoA that condenses to form less 3HB; c) increasing the flux of ProCoA that condenses with AcCoA to form more 3HV; and d) decreasing the flux of ProCoA that enters MCA cycle. From the above conclusions, the target sites for metabolic engineering are suggested in the last section. as follows; a) engineering the active site of 3-ketothiolase for much more active with ProCoA comparing to AcCoA; b) amplifying PHB synthase; c) fully or partially blocking isocitrate dehydrogenase enzyme; d) fully or partially blocking methylcitrate lyase; e) amplifying glucose-6-phosphate dehydrogenase enzyme to produce more NADPH; and f) increasing intracellular ProCoA by feeding additional ProCoA-yielding substrate and/or introducing an additional ProCoA formation route.