Combined 13C-assisted metabolomics and metabolic flux analysis reveals the impacts of glutamate on the central metabolism of high β-galactosidase-producing Pichia pastoris.

Weilu Lin,Mingzhi Huang,Jiangchao Qian,Ju Chu,Ping Liu,Yingping Zhuang,Siliang Zhang,Menglei Guo

doi:10.1186/s40643-016-0124-6

Abstract

Background Pichia pastoris is 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 by P. pastoris G1HL.MethodsTo elucidate the impacts of glutamate on the central metabolism in detail, a combined 13C-assisted metabolomics and 13C metabolic flux analysis was conducted based on LC–MS/MS and GC–MS data.ResultsThe 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.ConclusionThe addition of glutamate leads to a remarkable change on the central metabolism of high β-galactosidase-producing P. pastoris G1HL. To meet the increased demands of redox cofactors and energy for higher β-galactosidase production on Glc/Glu, P. pastoris G1HL 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s40643-016-0124-6) contains supplementary material, which is available to authorized users.

Highlights

Pichia pastoris is a popular recombinant protein expression system for its accessibility of efficient gene manipulation and high protein production
Physiology of G1HL on Glc P value of T test (Glc)/Glu and on Glc The addition of glutamate resulted in remarkable impacts on the physiology of the high β-galactosidase-producing strain P. pastoris G1HL (Fig. 1; Additional file 2)
The reduced specific growth rate indicated the presence of metabolic burden on Glc/Glu

Summary

Introduction

Pichia pastoris is a popular recombinant protein expression system for its accessibility of efficient gene manipulation and high protein production. As a popular recombinant protein expression system, P. pastoris has been widely applied in pharmaceutical and chemical industries. This can be credited to its fine characteristics including the ability to perform post-translational modification (De Schutter et al 2009; Hamilton et al 2006), the accessibility for genetic manipulation (Cregg et al 1993), the ability to obtain high cell density on simple and defined media (Cereghino et al 2002), and the simple downstream harvesting process due to the. The high expression of recombinant proteins generates extra demands for precursors, energy, and redox cofactors, which are reflected in a reduced substrate uptake rate, lower specific growth (Cos et al 2005), and decreased cell viability (Glick 1995).

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