Different species of microorganisms including yeasts, filamentous fungi and bacteria have been used in the past 25 years for the controlled production of foreign proteins of scientific, pharmacological or industrial interest. A major obstacle for protein production processes and a limit to overall success has been the abundance of misfolded polypeptides, which fail to reach their native conformation. The presence of misfolded or folding-reluctant protein species causes considerable stress in host cells. The characterization of such adverse conditions and the elicited cell responses have permitted to better understand the physiology and molecular biology of conformational stress. Therefore, microbial cell factories for recombinant protein production are depicted here as a source of knowledge that has considerably helped to picture the extremely rich landscape of in vivo protein folding, and the main cellular players of this complex process are described for the most important cell factories used for biotechnological purposes.

Background: Yeasts are attractive expression platforms for many recombinant proteins, and there is evidence for an important interrelation between the protein secretion machinery and environmental stresses. While adaptive responses to such stresses are extensively studied in Saccharomyces cerevisiae, little is known about their impact on the physiology of Pichia pastoris. We have recently reported a beneficial effect of hypoxia on recombinant Fab secretion in P. pastoris chemostat cultivations. As a consequence, a systems biology approach was used to comprehensively identify cellular adaptations to low oxygen availability and the additional burden of protein production. Gene expression profiling was combined with proteomic analyses and the ¹³C isotope labelling based experimental determination of metabolic fluxes in the central carbon metabolism. Results: The physiological adaptation of P. pastoris to hypoxia showed distinct traits in relation to the model yeast S. cerevisiae. There was a positive correlation between the transcriptomic, proteomic and metabolic fluxes adaptation of P. pastoris core metabolism to hypoxia, yielding clear evidence of a strong transcriptional regulation component of key pathways such as glycolysis, pentose phosphate pathway and TCA cycle. In addition, the adaptation to reduced oxygen revealed important changes in lipid metabolism, stress responses, as well as protein folding and trafficking. Conclusions: This systems level study helped to understand the physiological adaptations of cellular mechanisms to low oxygen availability in a recombinant P. pastoris strain. Remarkably, the integration of data from three different levels allowed for the identification of differences in the regulation of the core metabolism between P. pastoris and S. cerevisiae. Detailed comparative analysis of the transcriptomic data also led to new insights into the gene expression profiles of several cellular processes that are not only susceptible to low oxygen concentrations, but might also contribute to enhanced protein secretion.

Proteins can be secreted from a host organism with the aid of N-terminal secretion signals. The budding yeast Pichia pastoris (Komagataella sp.) is widely employed to secrete proteins of academic and industrial interest. For this yeast, the most commonly used secretion signal is the N-terminal portion of pre-pro-α-factor from Saccharomyces cerevisiae. However, this secretion signal promotes posttranslational translocation into the endoplasmic reticulum (ER), so proteins that can fold in the cytosol may be inefficiently translocated and thus poorly secreted. In addition, if a protein self-associates, the α-factor pro region can potentially cause aggregation, thereby hampering export from the ER. This study addresses both limitations of the pre-pro-α-factor secretion signal. We engineered a hybrid secretion signal consisting of the S. cerevisiae Ost1 signal sequence, which promotes cotranslational translocation into the ER, followed by the α-factor pro region. Secretion and intracellular localization were assessed using as a model protein the tetrameric red fluorescent protein E2-Crimson. When paired with the α-factor pro region, the Ost1 signal sequence yielded much more efficient secretion than the α-factor signal sequence. Moreover, an allelic variant of the α-factor pro region reduced aggregation of the E2-Crimson construct in the ER. The resulting improved secretion signal enhanced secretion of E2-Crimson up to 20-fold compared to the levels obtained with the original α-factor secretion signal. Similar findings were obtained with the lipase BTL2, which exhibited 10-fold enhanced secretion with the improved secretion signal. The improved secretion signal confers dramatic benefits for the secretion of certain proteins from P. pastoris. These benefits are likely to be most evident for proteins that can fold in the cytosol and for oligomeric proteins.