title: Impacts of high β-galactosidase expression on central metabolism of recombinant Pichia pastoris GS115 using glucose as sole carbon source via (13)C metabolic flux analysis.
abstract: The yeast Pichia pastoris GS115 is a widely used microbial cell factory for the production of heterologous protein. In order to reveal the impacts of high heterologous protein expression on the central metabolism of Pichia pastoris GS115 using glucose as sole carbon source, we engineered a high β-galactosidase expression strain P. pastoris G1HL and a low expression control strain P. pastoris GHL through controlling the initiation strength of constitutive promoter pGAP. The carbon flux distributions in these two strains were quantified via (13)C metabolic flux analysis. Compared to the control strain, G1HL showed a lower growth rate, a higher flux through glycolysis pathway, a higher flux through pentose phosphate pathway, and a lower flux through by-products secretion pathway. The metabolic flux redistribution in G1HL was thought to compensate the increased redox cofactors and energy demands caused by the high protein expression. Although the fluxes through Krebs cycle in two engineered strains were almost the same, they were significantly lower than those in wild strain. The enhanced expression of β-galactosidase by glutamate supplementation demonstrated the potential of P. pastoris GS115 to catabolize more carbon through the Krebs cycle for even higher protein expression. In conclusion, our work indicates that P. pastoris GS115 can readjusts the central metabolism for higher heterologous protein expression and provides strategies for strain development or process optimization for enhancing production of heterologous protein.
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China.
authors: Yongsheng
authors: Y
authors: Nie
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China. Electronic address: huangmz@ecust.edu.cn.
authors: Mingzhi
authors: M
authors: Huang
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China.
authors: Junjie
authors: J
authors: Lu
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China.
authors: Jiangchao
authors: J
authors: Qian
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China.
authors: Weilu
authors: W
authors: Lin
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China.
authors: Ju
authors: J
authors: Chu
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China.
authors: Yingping
authors: Y
authors: Zhuang
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China.
authors: Siliang
authors: S
authors: Zhang
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D002247:Carbon Isotopes / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D060847:Metabolic Engineering / Q000379:methods*; D064688:Metabolic Flux Analysis; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010843:Pichia / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
chemical_list: D002247:Carbon Isotopes; D011994:Recombinant Proteins; D009243:NAD; D009249:NADP; D000255:Adenosine Triphosphate; D001616:beta-Galactosidase; D005947:Glucose
keywords: (13)C metabolic flux analysis; Central metabolism; Heterologous protein expression; P. pastoris