High expression of heterologous proteins by Saccharomyces cerevisiae grown on ethanol

Abstract

The production of recombinant proteins is of great importance for industrial applications in fields such as pharmaceutical ingredients and industrial enzymes. One of these products are camelid antibody fragments, produced by Saccharomyces cerevisiae in high cell density fed batch fermentation processes, using glucose as sole carbon source. To improve this production, process optimisation was performed on both biological and technical aspects. First the whole production system was analysed for possible improvements by comparing shake flask and fed-batch cultivation. It was found that not glucose, but ethanol as sole carbon source could greatly increase the productivity, which was verified in an ethanol fed batch fermentation protocol. It showed that especially under limiting conditions in the final phase, this process is much more efficient. This process was further developed to exploit the ethanol characteristics to its fullest potential. These characteristics were first determined in batch and fed-batch experiments by investigating the influence of growth rate, oxygen concentration and ethanol accumulation level on the productivity of the process. This resulted in an improved production protocol which was also shown to work for other heterologous proteins. It appeared that proteins that are normally produced in small quantities because of their structural characteristics are significantly improved in secretion in the ethanol process. This suggested a strong influence of ethanol on protein folding processes. To better understand the impact of ethanol in fed batch fermentations, the cell state was investigated by using DNA micro array analysis of the glucose and ethanol production process. Samples were taken to verify the influence of ethanol, a decreasing growth rate and a combination of these two. It was found that in ethanol fed-batch fermentations the central carbon metabolism behaves according to earlier descriptions. But nitrogen and fatty acid metabolism seem to play a much bigger role than expected by directing intermediates to the central metabolism for replenishment. Also a very strong redox and oxidative stress effect was found and protein catabolic processes were highly up regulated as well as genes that are glucose repressed. To link the cell state to increased heterologous protein production, the secretion pathway was further analysed by using the DNA micro arrays data and performing pulse chase experiments and electron microscopy. This showed that indeed glucose derepression plays an important role in the heterologous gene transcription. However, the general transcription and translation is down regulated in ethanol cultivation as a result of the changed environment. The flux of correctly folded proteins is higher on ethanol, probably due to an increase in folding capacity and a higher turnover of incorrect folded proteins. Furthermore, the changed redox potential and oxidative stress seem to have a large impact on the folding process and the post Golgi transport is strongly induced in ethanol cultivated cells. Therefore, the use of ethanol in heterologous protein production in Saccharomyces cerevisiae has a strong impact on cell state and metabolism which results in a higher secretion capacity and flux of heterologous proteins.