Metabolic flux analysis of recombinant Pichia pastoris growing on different glycerol/methanol mixtures by iterative fitting of NMR-derived 13C-labelling data from proteinogenic amino acids

Increased dosage of AOX1 promoter-regulated expression cassettes leads to transcription attenuation of the methanol metabolism in Pichia pastoris

“…At the end of batch phase (reaching a final OD 600 of 50) the chemostat culture was started at a dilution rate (D) of 0.1 h −1 , by feeding a defined growth medium (controlled by weighing scales) containing 25 g L −1 of glycerol/methanol mixture (60% glycerol/40% methanol, w/w) as a carbon source. This choice was based on the interest and potential of mixed substrate feeds for improvement of high cell density fed-batch cultivation processes for heterologous protein production purposes3233 as well as to facilitate the comparison of datasets of this study with those previously reported mixed-carbon chemostat studies using the same reference Rol-producing strains163234. Culture media for the batch and continuous culture phases were the same as previously described16.…”

Genome‐scale metabolic model of Pichia pastoris with native and humanized glycosylation of recombinant proteins

“…The updated model (ihGlycopastoris) was validated for its ability to predict growth rates. Experimental data on exchanges fluxes were set as constraints in ihGlycopastoris while biomass production was set as the objective function, and the resulting growth predictions were compared to reported experimentally measured growth rate (Dragosits et al, 2009;Jord a et al, 2014;Jungo et al, 2006Jungo et al, , 2007aSol a et al, 2004Sol a et al, , 2007Tortajada Serra, 2012;Zhang et al, 2004). Additional to growth data, the model's ability to predict recombinant protein production rates in the reported experimental conditions were also compared to experimental yields (Dragosits et al, 2009;Jungo et al, 2007aJungo et al, , 2007bSol a et al, 2007;Zhang et al, 2004).…”

13C metabolic flux profiling of Pichia pastoris grown in aerobic batch cultures on glucose revealed high relative anabolic use of TCA cycle and limited incorporation of provided precursors of branched‐chain amino acids

“…In spite of its outstanding importance for biotechnology and life sciences in general, however, the incomplete annotation of its genome [4,5] and the lack of a model organism database, as they exist for Escherichia coli and Saccharomyces cerevisiae, impede research progress [6]. Experimental studies of P. pastoris' central carbon metabolism are likewise comparatively rare [7][8][9][10][11][12][13][14][15][16].…”