title: Combined13C-assisted metabolomics and metabolic flux analysis reveals the impacts of glutamate on the central metabolism of high β-galactosidase-producingPichia pastoris.
abstract: BACKGROUND
  Pichia pastorisis a popular recombinant protein expression system for its accessibility of efficient gene manipulation and high protein production. Sufficient supply of precursors, energy, and redox cofactors is crucial for high recombinant protein production. In our present work, we found that the addition of glutamate improved the recombinant β-galactosidase (β-gal) production byP. pastorisG1HL.
  
  
  METHODS
  To elucidate the impacts of glutamate on the central metabolism in detail, a combined13C-assisted metabolomics and13C metabolic flux analysis was conducted based on LC-MS/MS and GC-MS data.
  
  
  RESULTS
  The pool sizes of intracellular amino acids were obviously higher on glucose/glutamate (Glc/Glu). The fluxes in EMP entry reaction and in downstream TCA cycle were 50 and 67% higher on Glc/Glu than on Glc, respectively. While the fluxes in upstream TCA cycle kept almost unaltered, the fluxes in PPP oxidative branch decreased.
  
  
  CONCLUSIONS
  The addition of glutamate leads to a remarkable change on the central metabolism of high β-galactosidase-producingP. pastorisG1HL. To meet the increased demands of redox cofactors and energy for higher β-galactosidase production on Glc/Glu,P. pastorisG1HL redistributes the fluxes in central metabolism through the inhibitions and/or activation of the enzymes in key nodes together with the energy and redox status.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, No.130, Meilong Road, Shanghai, 200237 China.
authors.forename: Ping
authors.initials: P
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, No.130, Meilong Road, Shanghai, 200237 China.
authors.forename: Mingzhi
authors.initials: M
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, No.130, Meilong Road, Shanghai, 200237 China.
authors.forename: Menglei
authors.initials: M
authors.lastname: Guo
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, No.130, Meilong Road, Shanghai, 200237 China.
authors.forename: Jiangchao
authors.initials: J
authors.lastname: Qian
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, No.130, Meilong Road, Shanghai, 200237 China.
authors.forename: Weilu
authors.initials: W
authors.lastname: Lin
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, No.130, Meilong Road, Shanghai, 200237 China.
authors.forename: Ju
authors.initials: J
authors.lastname: Chu
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, No.130, Meilong Road, Shanghai, 200237 China.
authors.forename: Yingping
authors.initials: Y
authors.lastname: Zhuang
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, No.130, Meilong Road, Shanghai, 200237 China.
authors.forename: Siliang
authors.initials: S
authors.lastname: Zhang
keywords: 13C metabolic flux analysis; Glutamate; Metabolomics; P. pastoris; Recombinant protein expression