¹³C Metabolic Flux Analysis Of The Impacts Of High β-Galactosidase Expression On Physiological Metabolism Of P.Pastoris

The yeast P. pastoris is a widely used microbial cell factory for the production of heterologous protein due to its efficiency on eukaryotic proteins expression, the readiness of gene manipulation tools and the ease of high cell density cultivation on simple medium. Although the methyltrophic P. pastoris using methanol-induced pAOX as promoter is most commonly used, the inflammable and explosive feature limits its applications in large-scale industrial processes. A recombinant P. pastoris under the control of constitutive pGAP was successfully engineered in our previous study, and the heterologous protein was expressed using glucose as sole carbon source. In order to reveal the impacts of heterologous protein expression on the physiological metabolism profoundly and comprehensively, a13C metabolic flux analysis was conducted and the impacts of different heterologous protein expression levels on metabolism was elucidated.13C metabolic flux analysis is a reliable method to obtain accurate metabolic flux distribution. The high price of isotopic tracer substrate makes shake flask the commonly used reactor for labeling experiment. But the difficult in process control and parameter measurement and the lower oxygen transfer ability in shake flask limit its application in the13C metabolic flux analysis of P. pastoris. In this study, a250mL miniature bioreactor system was developed based on process scale-down principle. The system can be controlled precisely and meets the high oxygen demand of P. pastoris.In this study, a P. pastoris GS115derived strain P. pastoris G1HL with high β-galactosidase expression and a low expression control strain P. pastoris GHL were engineered using the promoter with different initiation strength. The macroscopic metabolic characteristics of the two strains were compared during batch culture using glucose as sole carbon source. G1HL experienced significantly decreased specific growth rate and specific glucose uptake rate, as well as by-products secretion. By contrast, G1HL showed higher normalized respiration rate and biomass yield.The microscopic metabolism of these two strains was studied using the250mL miniature bioreactor system. We found that G1HL showed a higher flux through glycolysis pathway, a higher flux through pentose phosphate pathway and a higher flux through TCA cycle than GHL. The reduction equivalents and energy metabolism were further analyzed. The metabolic fluxes redistribution was thought to compensate the increased NADPH and energy demands caused by the high protein expression. Otherwise, the fluxes through TCA cycle in the two engineered strains were significantly lower than those in wild strain. It suggests the potentials of P. pastoris GS115to catabolize more carbon through the TCA cycle for even higher protein expression.Therefore, the metabolism adaption of P. pastoris GS115using glucose as sole carbon source to high heterologous protein expression was elucidated from the perspective of metabolic fluxome. The strategies for strain development or process optimization for enhancing the production of heterologous protein were proposed based on our discovery. The present study will provide clues for safe and efficient production of heterologous protein in P. pastoris platform.