Five-step continuous production of PHB analyzed by elementary flux, modes, yield space analysis and high structured metabolic model

• High structured metabolic mathematical model for PHB production by C. necator.
• PHB production by C. necator on glucose in 5-stage continuous reactor cascade.
• Elementary modes concept and metabolic yield analysis of multistage PHB production.
• In silico metabolic flux calculations for different metabolic states of C. necator.
• Two metabolic situations concerning glucose-6-phosphate isomerase were in silico tested.

A high structured metabolic model for PHB synthesis by Cupriavidus necator DSM 545 consisting of 43 mass balance equations related to the same number of intracellular compounds was established. The metabolic state of cells cultivated in a continuously operated five stage bioreactor cascade was analyzed by help of elementary flux modes and two-dimensional yield space. Two different C-source feeding strategies were performed. Concerning PHB and biomass yields, values of the more efficient strategy were used as the data source for elementary modes and metabolic flux calculations, respectively. Metabolic fluxes were calculated from experimental yield data using a combination of elementary modes by applying the quadratic programming approach, in which the sum of squared weighting factors was minimized. Two different metabolic situations concerning activity of glucose-6-phosphate isomerase were tested. The high structured metabolic model was validated by comparison of experimental data from 24 h batch cultivation and simulated results.