Bioreaction network flux analysis for human protein producing Bacillus subtilis based on genome-scale model

Abstract

To interpret the effect of human protein gene, e.g., human growth hormone gene hGH, on the intracellular bioreaction network of Bacillus subtilis, the intracellular reaction fluxes were calculated by solving the mass-balance-based genome-scale mathematical model, at pseudo-steady state by using bioreactor data. The bioreaction network consists of 1340 reactions including 990 metabolites. Reaction fluxes in B. subtilis carrying pMK4 ::pre(subC)::hGH (rBsHGH) and B. subtilis carrying merely pMK4 (rBsP) were calculated to identify the existence of pathways and to elucidate the physiological state of the cells. Considering the characteristic cell and recombinant hGH (rhGH) concentration profiles of rBsHGH, the bioprocess was divided into five periods. The reaction network of rBsHGH started operation with 328 reactions (Period I: 0< t<4 h) and lowered to 313 in Period II (4< t<12 h), increased to 314 in Period III (12< t<16 h) by starting rhGH synthesis, and decreased to 296 reactions in Period IV (16< t<24 h) where cell and rhGH formation proceeded; further, decreased to 135 reactions in Period V (24< t<32 h) where cell formation ends and rhGH synthesis continues. However, the reaction network of rBsP started operation with 322 reactions, and lowered to 318 in Period II, and decreased to 43 reactions in Period III where cell formation ends. The flux of the reaction R1340 toward rhGH was the highest (2.0×10 −4 mmol g −1DW h −1) in Period V. The foreign protein gene stimulated and prolonged the cell synthesis (R1339) till the end of Period IV. To increase rhGH production further an exponential glucose feeding strategy between Periods III–V to maintain the specific growth rate μ within 0.02–0.08 h −1, is proposed.