Influence of growth temperature on the production of antibody Fab fragments in different microbes: A host comparative analysis

Abstract

Microorganisms encounter diverse stress conditions in their native habitats but also during fermentation processes, which have an impact on industrial process performance. These environmental stresses and the physiological reactions they trigger, including changes in the protein folding/secretion machinery, are highly interrelated. Thus, the investigation of environmental factors, which influence protein expression and secretion is still of great importance. Among all the possible stresses, temperature appears particularly important for bioreactor cultivation of recombinant hosts, as reductions of growth temperature have been reported to increase recombinant protein production in various host organisms. Therefore, the impact of temperature on the secretion of proteins with therapeutic interest, exemplified by a model antibody Fab fragment, was analyzed in five different microbial protein production hosts growing under steady-state conditions in carbon-limited chemostat cultivations. Secretory expression of the heterodimeric antibody Fab fragment was successful in all five microbial host systems, namely Saccharomyces cerevisiae, Pichia pastoris, Trichoderma reesei, Escherichia coli and Pseudoalteromonas haloplanktis. In this comparative analysis we show that a reduction of cultivation temperature during growth at constant growth rate had a positive effect on Fab 3H6 production in three of four analyzed microorganisms, indicating common physiological responses, which favor recombinant protein production in prokaryotic as well as eukaryotic microbes.

Biomarkers to evaluate the effects of temperature and methanol on recombinant Pichia pastoris

“…This has directly affected the production of heterologous proteins that requires maintaining a constant specific growth rate. Our results shown a higher concentration when the yeast was grown at 10 °C, differing from those observed at 15 °C in such yeasts as P. pastoris and Saccharomyces cerevisiae , where the biomass was reduced (Steels et al , 1994; Pizarro et al , 2008) and where methanol could have negative effects on cell growth compared to other inductors such as glycerol and glucose (Dragosits et al , 2011). …”

The effect of hypoxia on the lipidome of recombinant Pichia pastoris

“…In this context, environmental conditions have a significant impact on the levels of recombinant proteins. For instance, lower culture temperature [12, 13], low oxygen availability [14], as well as adequate substrate feeding strategies in high cell density cultures [15, 16], and type of carbon source(s) [17, 18] resulted in positive effects on protein secretion.…”

Integral Membrane Protein Expression in Saccharomyces cerevisiae

“…Oxygen availability can effect growth rates and energy availability (7), which may alter plasmid replication or partitioning. Studies have also shown that reducing growth temperatures can improve yields if toxicity is suspected (8), but it can also be detrimental to the expression of IMPs (1) ( see Note 58 ). Several reports have shown chemical chaperones can increase protein expression levels by improving the folding of IMPs (9,10).…”