Combined ¹³C-assisted Metabolomics And Metabolic Flux Analysis Reveals The Impacts Of Glutamate On The Physiological Metabolism Of High ?-galactosidase Producing Pichia Pastoris

As a popular recombinant protein expression system, Pichia pastoris has been widely applied in the pharmaceutical industry in recent years. In previous work, it is found that the addition of glutamate can enhance the productivity of ?-galactosidase in P.pastoris G1HL. However, it is not clear how the glutamate works. In present study, the impact of glutamate added to the medium was researched by ¹³C-assisted metabolomics and metabolic flux analysis and the intracellular regulation mechanism was revealed.Firstly, the effect of glutamate on the extracelluar fluxesof G1HL was studied. The results indicated that the ?-glactosidase productivity was about 50% higher in substrate with glutamate compared to which in substrate with glucose as sole carbon source. Companied with the improvement of ?-galactosidase productivity, the specific growth rate of cell reduced by 6%, which indicated that the improvement of P-galactosidase was at the cost of cell growth. In addition, the CER and ?-galactosidase yield to oxygen increased by 34% and 14% respectively in the substrate with glutamate, indicate the higher need of energy and reducing power.The effect of glutamate on the intracelluar metabolism was analysed by the ¹³C-assisted metabolomics and metabolic flux analysis. The metabolomics analysis reveals that there was an significant increase in intracellar metabolites, specially the amino acids pool, when the cell grew on substrate with glutamate. The metabolic flux results indicate that in substrate with glutamate, the EMP flux and TCA cycle downstream flux increased by 50% and by 67% respectively, wheras the flux in PP pathway decreased by 40% and the flux in upstream of TCA cycle kept unaltered compared to which in substrate glucose.The fluxes of intracelluar energy and cofactors were estimated by the central carbon metabolic fluxes values. The results indicated that the generation fluxes of NADH and ATP all increased to meet the need of energy for higher P-galactosidase in substrate with glutamate. The intracelluar nucleotides pool size indicates the cells grown on substrate with glutamate were in a lower energy charge and redox status.In sumarry, in P. pastoris G1HL, the precurser amino acids as well as the energy may be the bottoleneck limiting the further enhencement of productivity of ?-galactosidase, and the addition of glutamate release this limitation. The impacts of glutamate on the central metabolism of P. pastoris G1HL was inferred to be modulate through the inhibitions and/or activation of the enzyme in key nodes together with the energy and redox status in order to meet the increased demands of redox cofactors and energy for higher ?-galactosidase production on substrate with glutamate.By this study, the knowledge of the modulation mechanism of glutamate addition on central metabolism of P. pastoris G1HL is enhanced, which may provide the clue for the optimization of bioprocess.