Abstract
BackgroundEnvironmental and intrinsic stress factors can result in the global alteration of yeast physiology, as evidenced by several transcriptional studies. Hypoxia has been shown to have a beneficial effect on the expression of recombinant proteins in Pichia pastoris growing on glucose. Furthermore, transcriptional profiling analyses revealed that oxygen availability was strongly affecting ergosterol biosynthesis, central carbon metabolism and stress responses, in particular the unfolded protein response. To contribute to the better understanding of the effect and interplay of oxygen availability and foreign protein secretion on central metabolism, a first quantitative metabolomic analysis of free amino acids pools in a recombinant P. pastoris strain growing under different oxygen availability conditions has been performed.ResultsThe values obtained indicate significant variations in the intracellular amino acid pools due to different oxygen availability conditions, showing an overall increase of their size under oxygen limitation. Notably, even while foreign protein productivities were relatively low (about 40–80 μg Fab/gDCW·h), recombinant protein production was found to have a limited but significant impact on the intracellular amino acid pools, which were generally decreased in the producing strain compared with the reference strain. However, observed changes in individual amino acids pools were not correlated with their corresponding relative abundance in the recombinant protein sequence, but to the overall cell protein amino acid compositional variations.ConclusionsOverall, the results obtained, combined with previous transcriptomic and proteomic analyses provide a systematic metabolic fingerprint of the oxygen availability impact on recombinant protein production in P. pastoris.
Highlights
Environmental and intrinsic stress factors can result in the global alteration of yeast physiology, as evidenced by several transcriptional studies
We have previously reported the beneficial impact of hypoxia conditions on recombinant protein production in P. pastoris [29]
Pastoris The burden caused by recombinant protein production in yeast and, in particular, P. pastoris, has been recently suggested to impact the central metabolism even at relatively low expression levels, i.e. where increased precursor demands for recombinant protein production may be negligible [10,23,24]
Summary
Environmental and intrinsic stress factors can result in the global alteration of yeast physiology, as evidenced by several transcriptional studies. Production of recombinant proteins may cause cellular stress due to unfolded proteins and unsuitable or inefficient secretion [19], which, in turn, may negatively affect cell growth, even at relatively low expression levels [15,20], that is, at the product yields range where effects derived from increased energy and precursor demands for protein synthesis on cell growth should be theoretically negligible In this context, limited but significant alterations in the carbon flux distribution over the central metabolism have been recently reported [21,22,23,24]. Glutamic and Aspartic acid for instance, are components of the aspartate/malate redox shuttle [28] and their concentrations may indirectly impact on the rate of oxidation of glycolytic NADH
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