[NMR paper] Metabolic flux analysis of recombinant Pichia pastoris growing on different glycerol/methanol mixtures by iterative fitting of NMR-derived (13)C-labelling data from proteinogenic amino acids.
Metabolic flux analysis of recombinant Pichia pastoris growing on different glycerol/methanol mixtures by iterative fitting of NMR-derived (13)C-labelling data from proteinogenic amino acids.
Metabolic flux analysis of recombinant Pichia pastoris growing on different glycerol/methanol mixtures by iterative fitting of NMR-derived (13)C-labelling data from proteinogenic amino acids.
N Biotechnol. 2013 Jul 8;
Authors: Jordà J, de Jesus SS, Peltier S, Ferrer P, Albiol J
Abstract
The yeast P. pastoris has emerged as one of the most promising yeast cell factories for the production of heterologous proteins. The readily available genetic tools and the ease of high-cell density cultivations using methanol or glycerol/methanol mixtures are among the key factors for this development. Previous studies have shown that the use of mixed feeds of glycerol and methanol seem to alleviate the metabolic burden derived from protein production, allowing for higher specific and volumetric process productivities. However, initial studies of glycerol/methanol co-metabolism in P. pastoris by classical metabolic flux analyses using (13)C-derived Metabolic Flux Ratio (METAFoR) constraints were hampered by the reduced labelling information obtained when using C3:C1 substrate mixtures in relation to the conventional C6 substrate, i.e. glucose. In this study, carbon flux distributions through the central metabolic pathways in glycerol/methanol co-assimilation conditions have been further characterised using biosynthetically directed fractional (13)C labelling. In particular, metabolic flux distributions were obtained under 3 different glycerol/methanol ratios and growth rates by iterative fitting of NMR-derived (13)C-labelling data from proteinogenic amino acids using the software tool (13)CFlux2. Specifically, cells were grown aerobically in chemostat cultures fed with 80:20, 60:40 and 40:60 (w:w) glycerol/methanol mixtures at two dilutions rates (0.05h(-1) and 0.16h(-1)), allowing to obtain additional data (biomass composition and extracellular fluxes) to complement pre-existing datasets. The performed (13)C-MFA reveals a significant redistribution of carbon fluxes in the central carbon metabolism as a result of the shift in the dilution rate, while the ratio of carbon sources have a lower impact on carbon flux distribution in cells growing at the same dilution rate. At low growth rate, the percentage of methanol directly dissimilated to CO2 ranges between 50 -70%. At high growth rate the methanol is completely dissimilated to CO2 by the direct pathway, in the two conditions of highest methanol content.
PMID: 23845285 [PubMed - as supplied by publisher]
Metabolic flux profiling of recombinant protein secreting Pichia pastoris ... - 7thSpace Interactive (press release)
Metabolic flux profiling of recombinant protein secreting Pichia pastoris ... - 7thSpace Interactive (press release)
<img alt="" height="1" width="1" />
Metabolic flux profiling of recombinant protein secreting Pichia pastoris ...
7thSpace Interactive (press release)
... for improved protein production. Results: The metabolic flux distribution in the central metabolism of P. pastoris growing on a mixed feed of glucose and methanol was analyzed by Metabolic Flux Analysis (MFA) using 13C-NMR-derived constraints.
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05-08-2012 06:09 PM
VITAL NMR: using chemical shift derived secondary structure information for a limited set of amino acids to assess homology model accuracy
VITAL NMR: using chemical shift derived secondary structure information for a limited set of amino acids to assess homology model accuracy
Abstract Homology modeling is a powerful tool for predicting protein structures, whose success depends on obtaining a reasonable alignment between a given structural template and the protein sequence being analyzed. In order to leverage greater predictive power for proteins with few structural templates, we have developed a method to rank homology models based upon their compliance to secondary structure derived from experimental solid-state NMR...
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12-22-2011 06:50 AM
Glycerol and Glycerol Carbonate as ultraviscous solvents for mixture analysis by NMR
Glycerol and Glycerol Carbonate as ultraviscous solvents for mixture analysis by NMR
Publication year: 2011
Source: Journal of Magnetic Resonance, In Press, Accepted Manuscript, Available online 1 July 2011</br>
Pedro, Lameiras , Leslie, Boudesocque , Zéphirin, Mouloungui , Jean-Hugues, Renault , Jean-Michel, Wieruszeski , ...</br>
NMR of weakly polar analytes in an apolar ultraviscous solvent has recently been proposed for mixture analysis as a pertinent alternative to the DOSY experiment. The present article reports the first use of glycerol and glycerol carbonate as polar...
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07-03-2011 05:40 AM
Overproduction of 15N-labeled r-RGD-hirudin in pichia pastoris for NMR studies.
Overproduction of 15N-labeled r-RGD-hirudin in pichia pastoris for NMR studies.
Overproduction of 15N-labeled r-RGD-hirudin in pichia pastoris for NMR studies.
Protein Pept Lett. 2010;17(10):1228-33
Authors: Wang J, Zhang Y, Li S, Liu X, Yan X, Song H, Yu M, Dai L, Mo W
The novel recombinant hirudin, r-RGD-hirudin, inhibits thrombin and platelet aggregation. Here, we reported over-expression of (15)N-labeled r-RGD-hirudin by Pichia pastoris in minimal medium. After extensive optimization, the yield of active r-RGD-hirudin reached ?600 mg/L when...
[NMR paper] Recombinant expression of Ole e 6, a Cys-enriched pollen allergen, in Pichia pastoris
Recombinant expression of Ole e 6, a Cys-enriched pollen allergen, in Pichia pastoris yeast: detection of partial oxidation of methionine by NMR.
Related Articles Recombinant expression of Ole e 6, a Cys-enriched pollen allergen, in Pichia pastoris yeast: detection of partial oxidation of methionine by NMR.
Protein Expr Purif. 2004 Oct;37(2):336-43
Authors: Barral P, Tejera ML, Treviño MA, Batanero E, Villalba M, Bruix M, Rodríguez R
Olive pollen is one of the main causes of allergy in Mediterranean countries. Ole e 6, an olive pollen...
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11-24-2010 10:01 PM
[NMR paper] Expression in Pichia pastoris and characterization by circular dichroism and NMR of r
Expression in Pichia pastoris and characterization by circular dichroism and NMR of rhodostomin.
Related Articles Expression in Pichia pastoris and characterization by circular dichroism and NMR of rhodostomin.
Proteins. 2001 Jun 1;43(4):499-508
Authors: Guo RT, Chou LJ, Chen YC, Chen CY, Pari K, Jen CJ, Lo SJ, Huang SL, Lee CY, Chang TW, Chaung WJ
Rhodostomin (Rho) is a snake venom protein isolated from Calloselasma rhodostoma. Rho is a disintegrin that inhibits platelet aggregation by blocking the binding of fibrinogen to the integrin...
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11-19-2010 08:32 PM
[NMR analysis blog] Mnova 6.0, at last! GSD, Line Fitting, Data Analysis, handling of LC/GC/MS data and m
Mnova 6.0, at last! GSD, Line Fitting, Data Analysis, handling of LC/GC/MS data and much more!
It's been over 6 weeks since my last post on this blog but don’t worry, I haven’t been idle. On the contrary, I have a very good excuse for this lack of posts: We all at Mestrelab have been working very hard trying to get version 6.0 of Mnova finished. Now I’m delighted to announce that we have done it and version 6.0 is finally available for download from our Web site. This is certainly a major upgrade of the software in which we have put a lot of work and passion. It brings a number of...