Impacts of high β-galactosidase expression on central metabolism of recombinant Pichia pastoris GS115 using glucose as sole carbon source via 13C metabolic flux analysis

Nie, Yongsheng; Huang, Mingzhi; Lu, Junjie; Qian, Jiangchao; Lin, Weilu; Chu, Ju; Zhuang, Yingping; Zhang, Siliang

doi:10.1016/j.jbiotec.2014.07.011

Cited by 33 publications

(60 citation statements)

References 53 publications

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“…The batch cultivations were performed in a 5-L bioreactor (National Engineering Research Center for Biotechnology, Shanghai, China) with the working volume of 3 L. The media and growth conditions were the same as those described in our previous study (Nie et al 2014). The fermentation medium contained 20 g/L glucose, 18.2 g/L K 2 SO 4 , 0.93 g/L CaSO 4 , 14.9 g/L MgSO 4 ·7H 2 O, 4.13 g/L KOH, 26.8 mL/L H 3 PO 4 , 12 mL/L PTM1, and 1 mL/L antifoam.…”

Section: Methodsmentioning

confidence: 99%

“…The preparation of 13 C-labeling experiments was also the same as our previous study (Nie et al 2014). A 250-mL bioreactor (National Engineering Research Center for Biotechnology, Shanghai, China) with the working volume of 150 mL was used.…”

Section: Methodsmentioning

confidence: 99%

“…The β-galactosidase assays were performed according to the protocol supplied in our previous study (Nie et al 2014). One milliliter broth of yeast cells was disrupted, and β-galactosidase was detected by Pichia pastoris expression kit (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

“…The metabolic model used was similar to our previous study (Nie et al 2014). However, the unidirectional reaction from α-ketoglutarate (αkG) to glutamate was changed to reversible reaction to acknowledge the uptake of glutamate.…”

Section: Methodsmentioning

confidence: 99%

“…The addition of glutamate was found to be related to the improvement of β-galactosidase production (Nie et al 2014). The increase of protein production through the addition of amino acids has been also observed in other studies (Görgens et al 2005a, b; Heyland et al 2011).…”

Section: Introductionmentioning

confidence: 99%

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

Liu

Huang

Guo

et al. 2016

Bioresour. Bioprocess.

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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.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Liu

Huang

Guo

et al. 2016

Bioresour. Bioprocess.

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On stability analysis of cascaded linear time varying systems in dynamic isotope experiments

et al. 2020

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The dynamic 13C labeling experiment, as an emerging experimental technique, can be utilized to quantify intracellular fluxes of the cell culture under metabolic unsteady states conditions, for example, under fed batch mode. One main disadvantage of the current dynamic isotope experiment technique is that the intracellular metabolic pools have to be at pseudo‐steady state. Furthermore, to the best of our knowledge, the stability issue of dynamic isotope experiments is not addressed in the literature. In this work, if one assumes that concentrations of intracellular metabolites are nonsteady, it is shown that dynamic cumomer balance equations for any metabolic network are uniformly bounded‐input and bounded‐output stable regardless of the existence of traps. It is valid under very weak conditions. Specifically, it requires that the intracellular flux and the size of intracellular pool are finite and strictly greater than zero at any time. The new finding paves the way to utilize the dynamic isotope experiment to approximately quantify intracellular fluxes and sizes of intracellular pools under intracellular metabolic nonsteady state with piecewise affine fluxes.

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Metabolic engineering of Pichia pastoris for malic acid production from methanol

Guo

Dai

Peng

et al. 2020

Biotech & Bioengineering

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The application of rational design in reallocating metabolic flux to accumulate desired chemicals is always restricted by the native regulatory network. In this study, recombinant Pichia pastoris was constructed for malic acid production from sole methanol through rational redistribution of metabolic flux. Different malic acid accumulation modules were systematically evaluated and optimized in P. pastoris. The recombinant PP-CM301 could produce 8.55 g/L malic acid from glucose, which showed a 3.45-fold increase compared to the parent strain. To improve the efficiency of site-directed gene knockout, NHEJ-related protein Ku70 was destroyed, whereas leading to the silencing of heterogenous genes. Hence, genes related to byproduct generation were deleted via a specially designed FRT/FLP system, which successfully reduced succinic acid and ethanol production. Furthermore, a key node in the methanol assimilation pathway, glucose-6-phosphate isomerase was knocked out to liberate metabolic fluxes trapped in the XuMP cycle, which finally enabled 2.79 g/L malic acid accumulation from sole methanol feeding with nitrogen source optimization. These results will provide guidance and reference for the metabolic engineering of P. pastoris to produce value-added chemicals from methanol.

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Impacts of high β-galactosidase expression on central metabolism of recombinant Pichia pastoris GS115 using glucose as sole carbon source via 13C metabolic flux analysis

Cited by 33 publications

References 53 publications

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

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

On stability analysis of cascaded linear time varying systems in dynamic isotope experiments

Metabolic engineering of Pichia pastoris for malic acid production from methanol

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