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

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.

Methods

To 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.

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.

Conclusion

The 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.

Keywords

Glutamate / ¹³C metabolic flux analysis / Metabolomics / P. pastoris / Recombinant protein expression

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Funding

National Basic Research Program of China(2013CB733600)

National Natural Science Foundation of China(201276081)

National Key Technology Support Program of China(2011BAF02B05)