Long‐term adaptation of Saccharomyces cerevisiae to the burden of recombinant insulin production

Abstract

High-level production of heterologous proteins is likely to impose a metabolic burden on the host cell and can thus affect various aspects of cellular physiology. A data-driven approach was applied to study the secretory production of a human insulin analog precursor (IAP) in Saccharomyces cerevisiae during prolonged cultivation (80 generations) in glucose-limited aerobic chemostat cultures. Physiological characterization of the recombinant cells involved a comparison with cultures of a congenic reference strain that did not produce IAP, and time-course analysis of both strains aimed at identifying the metabolic adaptation of the cells towards the burden of IAP production. All cultures were examined at high cell density conditions (30 g/L dry weight) to increase the industrial relevance of the results. The burden of heterologous protein production in the recombinant strain was explored by global transcriptome analysis and targeted metabolome analysis, including the analysis of intracellular amino acid pools, glycolytic metabolites, and TCA intermediates. The cellular re-arrangements towards IAP production were categorized in direct responses, for example, enhanced metabolism of amino acids as precursors for the formation of IAP, as well as indirect responses, for example, changes in the central carbon metabolism. As part of the long-term adaptation, a metabolic re-modeling of the IAP-expressing strain was observed, indicating an augmented negative selection pressure on glycolytic overcapacity, and the emergence of mitochondrial dysfunction. The evoked metabolic re-modeling of the cells led to less optimal conditions with respect to the expression and processing of the target protein and thus decreased the cellular expression capacity for the secretory production of IAP during prolonged cultivation.

Multi‐omic profiling ­of EPO‐producing Chinese hamster ovary cell panel reveals metabolic adaptation to heterologous protein production

“…Based on these data, we speculate that the amino acid metabolism in CHO cells may undergo adaptation in favor of the produced heterologous protein during long‐term cultivation. The adaptation of gene expression levels in amino acid metabolism in favor of heterologous protein production during prolonged chemostat cultivation has been reported before in the eukaryotic protein production host Saccharomyces cerevisiae (Kazemi Seresht et al, ).…”

Understanding the metabolic burden of recombinant antibody production in Saccharomyces cerevisiae using a quantitative metabolomics approach

“…For expression of the scFv fragment, the lag period, followed by a general decrease in growth rate, has been observed before (Kauffman et al, ). Moreover, several S. cerevisiae strains have been reported to show a decreased growth rate upon expression of recombinant proteins, for example for a recombinant insulin, xylanase and cellulases (Görgens et al, ; Kazemi Seresht et al, ; Van Rensburg et al, ). Interestingly, in this study we also observed that the decrease in growth rate showed a correlation with the size and structural complexity of the expressed protein.…”

“…This is the result of a redirection of resources from regular cellular activities towards the needs created by recombinant protein production (Glick, ). For S. cerevisiae this was shown to lead to a reduction in the maximum specific growth rate, a decreased biomass yield and a lower respiratory capacity (Glick, ; Görgens et al, ; Karim, Curran, & Alper, ; Kauffman, Pridgen, Doyle, Dhurjati, & Robinson, ; Kazemi Seresht et al, ). Noting that many cellular processes change when recombinant protein production is induced, it is to be expected that a shift in the direction of cellular resources to accommodate elevated protein production also alters the levels of intracellular metabolites.…”

“…The changes in intracellular metabolite levels caused by recombinant protein production in S. cerevisiae have often been neglected, even though such insights could lead to the identification of novel leads for metabolic engineering of recombinant strains. In one study, it was shown that long‐term cultivation of a recombinant insulin‐producing S. cerevisiae strain eventually reduced the insulin production owing to the cellular adaptation to the metabolic burden (Kazemi Seresht et al, ). This indicates that an evaluation of the intracellular metabolic alteration owing to high‐level expression of recombinant proteins provides us with new insights into cellular adaptations to the stress, as currently not much is known about how the expression of heterologous proteins affects the allocation of cellular resources in S. cerevisiae .…”

Deletion of theSaccharomyces cerevisiae ARO8gene, encoding an aromatic amino acid transaminase, enhances phenylethanol production from glucose

“…Experimental analysis and modelling of the transaminases is further complicated by the compartmentation of intracellular amino acid pools, which involves separate cytosolic, mitochondrial and vacuolar pools (Kitamoto et al , ; Szabados and Savouré, ). The size and composition of intracellular amino acid pools is highly relevant for yeast‐based processes ranging from production of yeast extracts to the expression of heterologous proteins (Kazemi Seresht et al , ). The results presented in this study identify transaminase genes as highly interesting targets for empirical optimization of the production of amino acid‐derived products by S. cerevisiae .…”