13C-constrained metabolic flux balancing analysis of Pichia pastoris grown on different carbon sources

13C-constrained flux balancing analysis based on NMR data [1] has been applied as a method for the estimation of intracellular carbon fluxes of Pichia pastoris cells. In this approach, the underdetermined system of metabolite balances deduced from stoichiometric relations and measured extracellular rates is complemented with 13C constraints from metabolic flux ratio analysis [2, 3]. Steady state fluxes in central carbon metabolism of the methylotrophic yeast grown in chemostat cultures were estimated by 13C-constrained flux balancing method in different 13C-labeled carbon source experiments. In particular, cells were grown aerobically in chemostat cultures fed at two dilution rates (0.05 h 1, 0.16 h 1) with glucose, glycerol or varying mixtures of glycerol and methanol as carbon sources. In previous studies [4, 5], the metabolic pathways associated with the TCA cycle intermediates of P. pastoris were characterised in terms of metabolic flux ratios based on the 13C labelling pattern of proteinogenic amino acids using 13C-NMR measurements. Based on this data, analysis of metabolic flux responses to environmental modifications (carbon source and growth rate) has now been performed by 13C-constrained flux balancing analysis. A metabolic network has been reconstructed from literature data and summarized in a stoichiometric model including up to 54 internal metabolic reactions distributed into mitochondrial, cytoplasmic and biomass generation reactions, which include up to 55 metabolites. The underdetermined linear equation system was solved using flux partitioning ratios as additional constraints to obtain the global net flux solution. Overall, calculations show that the co-assimilation of methanol has a clear impact on the distribution of metabolic fluxes through the pentose phosphate pathway (PPP) and TCA cycle, as well as the distribution of methanol carbon into assimilatory and dissimilatory (direct oxidation to CO2) pathways. Also, such impact is clearly dependant on the specific growth rate of the cells. This study brings new insights on the P. pastoris' methanol metabolism complex regulation in the presence of a second carbon source, revealing important implications for biotechnological applications.